Tumor Suppressor SMAR1 Regulates PKM Alternative Splicing by HDAC6 Mediated Deacetylation of PTBP1

Tumor Suppressor SMAR1 Regulates PKM Alternative Splicing by HDAC6 Mediated Deacetylation of PTBP1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Tumor Suppressor SMAR1 Regulates PKM Alternative Splicing by HDAC6 Mediated Deacetylation of PTBP1 Arpankumar Choksi, Apoorva Parulekar, Richa Pant, Vibhuti Kumar Shah, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-151525/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Apr, 2021 Read the published version in Cancer & Metabolism → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Highly proliferating cancer cells exhibit the Warburg effect by regulation of PKM alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of PKM alternative splicing and inhibition of the Warburg effect by SMAR1. Methods: Immunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on PKM pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by in vitro enzymatic assays and functional assays upon SMAR1 knockdown. Besides, in vivo tumor formation by injecting adeno-SMAR1 transduced MDA-MB-231 cells in NOD/SCID mice was performed. Results: The expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells and in vivo tumor generation in PKM2 dependent manner. Conclusions: SMAR1 regulates PKM alternative splicing by causing HDAC6 dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of PKM alternative splicing. Cancer Biology Cellular Metabolism SMAR1 PKM1 PKM2 HDAC6 PTBP1 alternative splicing Warburg effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Reprogramming cellular metabolism is one of the key hallmarks of cancer cells. In the presence of oxygen, somatic cells convert glucose to pyruvate by glycolysis and then redirect this pyruvate to the TCA cycle in mitochondria for ATP production. In the absence of oxygen, these cells convert pyruvate to lactate which is an energetically less efficient but relatively fast reaction. Whereas highly proliferating cancer cells utilize more glucose and produce more lactate compared to normal cells, independent of the presence or absence of oxygen. This phenomenon is known as the Warburg effect or aerobic glycolysis 1 , 2 . Cancer cells reprogram their cellular metabolism by regulating alternative splicing of Pyruvate Kinase Muscle ( PKM ) isoforms 3 . Pyruvate kinase is one of the rate-determining enzymes of glycolysis and facilitates the conversion of phosphoenolpyruvate to pyruvate. There are total four isoforms of pyruvate kinase enzyme: PKL (Pyruvate kinase liver), PKR (Pyruvate kinase Red blood cells), PKM1 and PKM2. PKL is expressed in the liver, PKR is expressed in the erythrocytes, PKM1 is expressed predominantly in terminally differentiated tissues and PKM2 is highly expressed in the proliferating cells such as cancer cells and stem cells 3 , 4 . PKM gene contains 12 exons, wherein the incorporation of exon 9 and exon 10 are regulated by mutually exclusive alternative splicing resulting in the expression of PKM1 and PKM2 isoforms, respectively 5 , 6 . Exon 9 and exon 10 both codes for 56 amino acids attributing to the distinctive regulatory and enzymatic activities of PKM isoforms. PKM1 forms a tetramer and is constitutively active, whereas PKM2 forms a tetramer (enzymatically efficient), as well as a dimer (enzymatically less efficient). Enzymatic activity of PKM2 dimer is influenced by allosteric regulation due to fructose-1,6-bisphosphate levels and interaction with phosphotyrosine-binding proteins 3 , 4 , 7 . Higher expression of PKM2 provides cancer cells with a metabolic advantage over normal cells. Due to the lower enzymatic activity of PKM2 as compared to that of PKM1, the major amount of glucose present in the cell remains as glycolytic intermediates which provide building blocks, such as amino acids, nucleotides and fatty acids to the highly proliferating cancer cells 8 . The remaining glucose gets converted to pyruvate and this pyruvate instead of entering the TCA cycle gets converted to lactate. To meet the high energy and carbon demands these cancer cells utilize more and more glucose compared to normal cells 2 . Apart from its role in glucose metabolism, PKM2 also regulates other cellular processes by getting translocated to the nucleus from the cytoplasm and affecting a variety of signaling pathways leading to oncogenesis 9 , 10 . Cancer cells achieve a metabolic edge over normal cells by regulating PKM alternative splicing and promoting the expression of PKM2 3,8 . hnRNPs (heterogeneous ribonucleoproteins); hnRNP A1, hnRNP A2 and PTBP1 (hnRNP I) are the key regulators of PKM alternative splicing 11 , 12 . c-Myc binds to promoters of these three genes and regulates their expression 12 . In majority of cancers, high expression of c-Myc promotes the higher expression of these three hnRNPs. When present in abundance, these three proteins bind on the intronic region flanking exon 9 and inhibit the incorporation of exon 9 and as an effect, there is the inclusion of exon 10 of PKM gene 13 . Molecular targeting of regulation of PKM alternative splicing by these three proteins might prove to be an effective strategy in eradicating the cancer cells 14 . The nuclear matrix is the site for many important nuclear events such as DNA replication, DNA repair, transcription and post-transcriptional modifications 15 , 16 . Post-transcriptional modifications include RNA splicing, RNA capping and poly-A tail addition. The nuclear matrix is a scaffold around which chromatin folds and many nuclear matrix binding proteins are involved in chromatin organization 15 . Apart from chromatin compaction, nuclear matrix binding proteins which are also known as the matrix-associated region (MAR) binding proteins (MARBPs), actively participate in the regulation of DNA replication, DNA repair, transcription and post-transcriptional modifications 15 , 16 . One such MARBP is SMAR1 (Scaffold/Matrix attachment region binding protein 1) 17 . The human homolog of SMAR1 is also known as BANP (BTG3 Associated Nuclear Protein). SMAR1 has been reported to play an important role as a tumor suppressor protein and in the majority of higher grades of cancer, SMAR1 has been reported to be dysregulated 18 – 24 . SMAR1 is located on human chromosome 16q24 and loss of heterozygosity (LOH) has been reported for this chromosomal region in various cancers 25 – 27 . SMAR1 has been reported to colocalize with SC35 which is a marker of nuclear splicing speckles and an important regulator of alternative splicing 28 . Moreover, SMAR1 has an RS domain that facilitates the RNA binding and it interacts with snRNAs which are the core components of splicing machinery 28 . SMAR1 has been reported to regulate splicing of CD44 variants by deacetylation of Sam68 with help of HDAC6 in breast cancer cell lines. In addition to CD44 variants, SMAR1 also regulates alternative splicing of FAS ligand 28 . ChIP-sequencing study of SMAR1 in HCT116 cells suggests that there are several global gene targets of SMAR1 which are involved in the regulation of RNA processing and alternative splicing 29 . This suggests that SMAR1 might be involved in alternative splicing regulation of other cancer-associated genes. This study focuses on the role of SMAR1 in the regulation of PKM alternative splicing via HDAC6 dependent deacetylation of PTBP1 and its implication in inhibition of the Warburg effect and tumorigenesis. Our study thus demonstrates the inhibition of cancer cell metabolism and breast cancer progression by SMAR1 via suppression of oncogenic isoform PKM2. Materials And Methods Cell culture MCF7, MDA-MB-231, MDA-MB-468 and T47D cells were obtained from the NCCS cell repository, Pune, India. MCF7, MDA-MB-231 and MDA-MB-468 were cultured in DMEM (Gibco) and T47D was cultured in RPMI (Gibco). All the cell lines were supplemented with 10% Fetal Bovine Serum (FBS) (Gibco) and 100 units/ml Penicillin and Streptomycin (Gibco) and incubated in a humidified 5% CO 2 incubator at 37ºC. Plasmids, siRNAs and shRNA constructs 3xFlag26-SMAR1 was used to over-express SMAR1 and 3xFlag26-Vector was used as a control. Silencer ™ Select Negative Control No. 1 siRNA (4390843) and si-RNA against human-SMAR1 (BANP) (s29889) were obtained from Thermo Fischer Scientific Silencer® select siRNA range. shSMAR1-eGFP (ULTRA-3344235) was obtained from TransOMIC and used for shRNA-mediated SMAR1 knockdown. shNon translated-1-eGFP (shNT1) (TLNSU1420) was also obtained from TransOMIC and used as a control for all shRNA-mediated knockdown studies. shHDAC6 clone TRCN0000004839 was used for HDAC6 knockdown 28 . shPKM2 was cloned in pLKO.1-TRC vector and the sequence targeting only PKM2 were obtained from Cortés-Cros et al. 30 . The sequence of shPKM2 used: 5′CCGGCTACCACTTGCAATTATTTGACTCGAGTCAAATAATTGCAAGTGGTAGTTTTTG3′. Transfections and treatments Transfections of various plasmids were done by the use of Polyethylenimine (PEI MAX 40000) (Polysciences, Inc.). In MCF7, transfection was performed at 70-80% confluency and the DNA:PEI ratio used for transfection was 1:3. In MDA-MB-231, reverse transfection was performed in which DNA: PEI mix was added before cell seeding. Tubacin (Sigma), a selective HDAC6 inhibitor, was used at 5 μM concentration for 5 hrs for optimum HDAC6 inhibition and an equal amount of DMSO was used as vehicle control. Cloning of dual reporter PKM minigene system To develop a dual chromatic PKM minigene system, eGFP was cloned into mCherry-N1 vector between SalI (NEB) and AgeI (NEB) restriction sites in such a way that both eGFP and mCherry remain in two different frames due to difference in one base pair and eGFP contains stop-codon at the end when in the frame. Further, Exon 8 - Exon 11 of the PKM gene along with introns was amplified from PKM minigene construct 31 (a kind gift from Dr. Adrian R. Krainer) using Platinum™ SuperFi™ DNA Polymerase (Invitrogen) and cloned into this vector between XhoI (NEB) and NdeI (NEB) sites by Infusion cloning (NEB). One base pair insertion was further introduced in the cloned plasmid in Exon 10 by Infusion cloning. Due to one base pair insertion, the incorporation of Exon 9 resulted in the expression of mCherry and the incorporation of Exon 10 led to the expression of eGFP. The expressions of mCherry and eGFP were analyzed by confocal microscopy. Dual reporter PKM minigene assay MCF7 cells were seeded onto a glass coverslip and after 24 hrs PKM minigene along with control or Flag-SMAR1 in a 1:1 ratio was introduced in these cells by PEI mediated transfection. After 48 hrs of transfection, cells were fixed with 4% paraformaldehyde for 10 mins at room temperature. Subsequently, cells were washed with 1X PBS thrice and the coverslips were mounted in fluoroshield media (Sigma) with DAPI. Cells were observed at 60X magnification using Nikon A1plus confocal microscope and images were acquired using Nikon’s NIS-elements imaging software. For eGFP and mCherry fluorescent intensity quantification, ImageJ software was used. Five independent regions of interest (ROIs) were selected per image covering cells expressing eGFP and mCherry and fluorescence intensity density was measured. Average relative fluorescence densities were calculated for all five ROIs per field and the fold change in eGFP/mCherry ratio was calculated. For statistical significance, three random fields per sample were selected and three independent experiments were performed. Mean ± SD fold change in eGFP/mCherry ratio was calculated for each sample and compared with the control sample. Immunohistochemistry (IHC) Paraffinized human breast cancer patient samples along with surrounding normal breast tissue were obtained from Ruby Hall Clinic, Pune, India. SMAR1, PKM1 and PKM2 expression were detected using standard immunohistochemical staining procedure. Briefly, after deparaffinization, endogenous peroxidase blockage and rehydration with decreasing concentrations of ethanol (100%, 95% and 70%), antigen retrieval was performed by heating the slides in 10 mM sodium citrate buffer (pH 6-7). Further samples were incubated with the antibody of SMAR1 (Bethyl) at 1: 100 dilution, PKM1 (CST) at 1: 100 dilution and PKM2 (CST) 1: 300 dilution overnight at 4ºC. After incubation with HRP-conjugated secondary antibodies for 1 hr at room temperature, sections were then treated in DAB (3,3'-Diaminobenzidine) for 15 mins to allow the development of brown precipitate corresponding to the sites of HRP-bound antibodies. Samples were washed and counterstained with Hematoxylin for nuclei staining. Tissue sections were observed at 20X magnification using a Nikon microscope (Eclipse E600) and images were acquired using Nikon’s NIS-elements imaging software. Quantitative RT PCR Total RNA was extracted from cultured MCF7 cells and MDA-MB-231 cells by TRizol TM (Invitrogen) according to the manufacturer’s instruction. RNA was reverse transcribed by MMLV-RT (Invitrogen) as per the manufacturer’s instructions. Amplification reactions were prepared in triplicate using iQTaq SYBR green (Biorad) and amplification was performed on Eppendorf realplex 2.0 according to the manufacturer’s instruction. The average cycle thresholds from three independent biological replicate samples were calculated as described in Singh et al. with slight modifications 32 . Briefly, the average cycle thresholds from three independent biological replicate samples were normalized to housekeeping control gene 18S rRNA . Normalization was performed using 18S rRNA as a normalization control using the formula: [2^ (Ct control – Ct target) ]. Along with control gene normalization, constitutive exon (exon 11) normalization was performed for PKM1 (Exon 8-9/9) and PKM2 (Exon 10-11/11) expression analysis. The student’s t-test was used to compare expression between two different groups. A list of primers used is given in Table S1. Western blotting Cells were incubated in TNN buffer [50 mM Tris-Cl pH 7.5, 5 mM EDTA, 0.5% NP40, 50 mM NaF, 1 mM DTT, 0.2 mM sodium orthovanadate, 0.5 mM PMSF, 150 mM NaCl and 1X Protease inhibitor cocktail (Thermo Scientific)] for cell lysis and lysates containing equal concentration of proteins were resolved using SDS-PAGE and transferred onto PVDF membrane. The membranes were incubated with primary antibodies such as anti-SMAR1 (Bethyl - A300-279A), Anti-PKM1 (CST- 7067), Anti-PKM2 (CST- 4053), Anti-hnRNP A1(CST- 8443), Anti-hnRNP A2 (Abcam- ab6102), Anti-PTBP1 (Thermo Scientific- 32-4800), Anti-HDAC6 (CST- 7558), Anti-HDAC1 (CST- 5356), Anti-GFP (Proteintech- 66002-1-Ig), Anti-mCherry (Proteintech- 26765-1-AP), Anti-β-actin (Sigma- A2228) and Anti-Flag tag (CST- 14793). This was followed by three washes and incubation with appropriate HRP conjugated secondary antibodies. Visualization was achieved with ECL substrate (Pierce) and exposure to X-ray films or imaging in Syngene G:BOX Chemi XRQ. Antibody cross-linking, Co-Immunoprecipitation and sequential Co-Immunoprecipitation The majority of Immunoprecipitation (IP) experiments were done via covalently cross-linking the antibody with Protein G Dynabeads (Pierce) to avoid non-specific binding and contamination of immunoglobulin in the immunoprecipitated protein eluates. Approximately 1 µg of antibody was cross-linked with 10 µl of beads. Beads were washed thrice with ice-cold 1X PBS and further incubated with the desired antibody in IP buffer (1X PBS with 0.1% NP-40) containing protease inhibitor cocktail (Pierce). The antibody-bead mixture was incubated overnight at 4ºC. The unbound antibody was removed by three wash of the antibody-bead complex with IP buffer. Further, the antibody-bead complex was incubated with 500 μL of 10 mg/ml of Dimethyl pimelimidate (DMP) (Sigma) for 60 mins at room temperature with rotation. 50 μL of 1M Tris-Cl pH 8 was added to quench the reaction and incubated for 30 mins at room temperature with rotation. Unbound antibody was removed by washing it with 0.2 M Glycine pH 3 followed by three washes with IP buffer. The antibody-bead complex was further equilibrated with the IP buffer. These beads were either used immediately or stored at 4ºC for 2-3 days. Protein-bound to the antibody-bead complex was eluted at 95ºC by using SDS loading dye. For IP experiments, 500 µg of nuclear extracts were pre-cleared with control normal IgG (Sigma) bound dynabeads G and subsequently incubated for 12 hrs at 4ºC with dynabeads G crosslinked with the desired primary antibody. The protein associated bead complexes were washed thrice with IP buffer and then further eluted with SDS loading dye. The eluates were probed with indicated antibodies. For sequential IP experiments, nuclear extracts (1 mg) were immunoprecipitated first with 3 µg of anti-SMAR1 antibody. Before proceeding for the second IP, a minor fraction of the eluates was examined for the presence of HDAC6. Subsequently, the eluate was immunoprecipitated with 2 µg of anti-HDAC6. The final eluates were probed with anti-PTBP1 to evaluate the association. Anti-Acetyl-Lysine Acetylation Assay For assessing the acetylation status of protein, 1 mg of nuclear extract was incubated with 2 μg of anti-acetyl-lysine antibody (CST- 9441) crosslinked with dynabeads G at 4ºC with slight mixing for 12 hrs. Immunocomplexes were washed thrice with 500 μL of IP buffer and further eluted at 95ºC with SDS loading dye. The eluates were loaded onto the SDS-PAGE and immunoblotted with indicated antibodies. UV-crosslinking and RNA immunoprecipitation (CLIP) CLIP experiment was done via covalently cross-linking the anti-PTBP1 antibody with Protein G Dynabeads (Thermo Scientific) as described in antibody cross-linking. Normal mouse IgG crosslinked beads were used as a negative control. Antibody cross-linked beads were further blocked for 1 hr at 4ºC with 100 nM yeast tRNA to avoid non-specific binding with RNA. Cells were washed with ice-cold 1X PBS, UV-irradiated (150 mJ/cm 2 ) and harvested and lysed with lysis buffer (100 mM KCl, 5 mM MgCl 2 , 10 mM HEPES - pH 7.0, 0.5% NP40, 1 mM DTT, 100 units/ml RNase Out and Protease inhibitor cocktail) for 10 mins on ice. RNase A (1:1000 dilution) and DNase I (Invitrogen) was added to the lysate and incubated at 37ºC for 3 mins. 5% lysate was taken as input and TRizol LS TM (Invitrogen) was added for RNA extraction. An equal amount of protein (10 mg) was taken for control and SMAR1 knockdown sample and anti-PTBP1 and normal IgG conjugated beads were added for immunoprecipitation and incubated on rotation at 4ºC for 3 hrs. After two washes with wash buffer (50 mM Tris-HCl - pH 7.4, 150 mM NaCl, 1 mM MgCl 2, 0.05% NP40) supplemented with RNase inhibitor, an aliquot (10%) of beads was kept as control of immunoprecipitation while the rest was treated with 30 μg of Proteinase K and incubated for 1 hr at 55ºC. RNA was then extracted by TRizol LS TM (Invitrogen) and RNA was reverse transcribed by MMLV-RT (Invitrogen) as per the manufacturer’s instructions. Immunoprecipitated fractions and 5% input were analyzed by quantitative real-time PCR in duplicate using iQTaq SYBR green (Biorad) and amplification was performed on Eppendorf realplex 2.0 and specific primers for PTBP1 binding site on Intron 8 of PKM were used (sequences mentioned in Table S1). Primer sequence for PKM Intron 8 PTBP1 binding site was obtained from Chen et al. 13 . The experiment was performed three times and normalization was performed to input using the formula: [2^ (Ct input – Ct immunoprecipitation) ]. Fold enrichment was calculated relative to normal mouse IgG control. Resultant fold enrichment was further normalized with densitometry measurement of a western blot for immunoprecipitated PTBP1 protein samples. The student’s t-test was used to identify the significance between two different groups. Glucose Assay MCF7 cells were transfected with respective shRNA and control shRNA. shRNA transfected cells were selected with puromycin and an equal number of cells were plated in a 6 well plate. After 24 hrs, the cells were replenished with 10% FBS containing high glucose DMEM (without sodium pyruvate). After 24 hrs of media replenishment, media was collected and the amount of glucose was calculated with the use of a Glucose Assay Kit (Abcam, ab65333) as per the manufacturer’s protocol by colorimetric method. The amount of glucose present was normalized with the total amount of protein. The glucose utilization was calculated by subtracting the glucose level of samples from that of cell-free media. The percentage of glucose utilization was calculated compared to the control. Lactate assay MCF7 cells were transfected with respective shRNA and control shRNA. shRNA transfected cells were selected with puromycin and an equal number of cells were plated in a 6 well plate. After 24 hrs, the cells were replenished with 10% FBS containing high glucose DMEM (without sodium pyruvate). After 24 hrs of media replenishment, media was collected and the amount of lactate was calculated with the use of a Lactate Assay Kit (Abcam, ab65331) as per the manufacturer’s protocol. The amount of lactate present in each sample was normalized with the total amount of protein. The percentage of lactate production was calculated for each sample compared to the control. Glucose (2-NBDG) uptake assay MCF7 cells were transfected with SMAR1 siRNA and control siRNA by Lipofectamine RNAiMax TM (Ambion) according to the manufacturer’s protocol. After 24 hrs of transfection, media was removed and replenished with 10% FBS containing DMEM (Without glucose and Sodium Pyruvate) and incubated at 37ºC for 1 hr. 10 μM fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) (Invitrogen) was added to culture media and cells were incubated for 1 hr at 37ºC. The 2-NBDG uptake reaction was stopped by removing the incubation medium and the cells were washed with ice-cold 1X PBS. 1 μg/ml Propidium Iodide (PI) was added to distinguish the viable cell population. For each measurement, data from 10,000 single-cell events were collected using FACS Canto II (BD Bioscience). The percentage of 2-NBDG uptake was calculated from mean fluorescence intensity (MFI) compared to the control. Cell viability assay MCF7 cells were transfected with BORIS shRNA and shNT1 was used as a control in six-well culture plates. After the selection of transfected cells with puromycin, cells (4x10 3 ) were seeded in 96 well culture plates and were cultured for 24 hrs, 48 hrs, and 72 hrs. Cell growth was determined by measuring the conversion of MTT Tetrazolium salt (Sigma) to MTT formazan. In brief, 50 µl of MTT stock solution (5 mg/ml) was added to each well along with 50 µl of 10% FBS containing DMEM and incubated for 4 hrs. After the incubation time, formazan crystals formed in the cells were solubilized in Isopropanol. The cell viability was measured by Spectramax M5 (Molecular Devices) at an optical density of 570 nm. The percentage proliferation was calculated and cell viability at 0 hr for each sample was considered as 100%. Compared to 0 hr, the percentage of proliferation was calculated for other time points. Colony formation assay MCF7 cells were transfected with respective shRNAs and shNT1 was used as a control. The cells were selected with puromycin and 1x10 3 cells were seeded in the new 6-well plate. After 10 days, cells were fixed using methanol and acetic acid (3:1) for 5 mins. After fixation, cells were washed with 1X PBS thrice. After washing cells were stained with 0.05% crystal violet stain, images were taken and colonies formed were counted manually with help of ImageJ software. The percentage of colony formation was calculated for each sample compared to the control. Transwell migration and invasion assay Transwell chamber: 24-well, 8.0-μm pore membranes (Corning USA) were used according to the manufacturer’s protocol. MCF7 cells were transfected with respective shRNAs and shNT1 was used as a control. After the selection of transfected cells with puromycin, 1x10 5 cells per well were seeded in the upper chamber in a serum-free medium, and DMEM with 5% FBS was added to the lower chamber as a chemoattractant at the same time. After incubation of 24 hrs at 37ºC, the cells remaining at the upper surface of the membrane were removed with cotton swabs, and the cells on the lower surface of the membrane are the migrated cells. After fixation with 4% paraformaldehyde and staining with 0.5% crystal violet solution, the cells were observed at 10X magnification using the Nikon microscope (Eclipse Ti2) and images were acquired using Nikon’s NIS-elements imaging software. The transwell invasion assay was carried out as described above, except that a transwell chamber with Matrigel (Corning, USA) was used and they were pre-incubated at 37ºC after hydration with 100 μL of serum-free medium for 2 hrs before the cells were seeded onto the membrane, followed by incubation of 48 hrs at 37ºC. Five fields were randomly captured and the number of migrating/invading cells were quantified manually with the help of Image J software. The percentage of migration/invasion was calculated for each sample compared to the control. Wound healing assay MCF7 cells were transfected with respective shRNAs and shNT1 was used as a control. The cells were selected with puromycin and 1.5x10 5 cells were seeded in each well of 12 well plate and allowed to grow at 37ºC to form a monolayer. Cells were synchronized by serum starvation for 12 hrs and scratch was introduced in the middle of the monolayer by sterile pipette tip, generating a cell-free area of approximately 1 mm in width and cell debris was removed by washing twice with 1X PBS. Three fields per well were imaged in the area where the wound was introduced at 0 hr 24 hrs. The area of the wound was measured by ImageJ for 0 hr and 24 hrs and the percentage of wound migration was calculated compared to that of the control. All images were taken at 10X magnification using the Nikon microscope (Eclipse Ti2) with help of Nikon’s NIS-elements imaging software. In vivo tumor generation All mice used in this experiment were bred at the animal resource facility of NCCS, Pune, India. Standard protocols approved and monitored by the Institutional Animal Ethical Committee were followed for this experiment. The MDA-MB-231 cells were transduced with SMAR1-adenovirus and control-adenovirus. 1 million cells were injected subcutaneously in 6-8 weeks old NOD/SCID mice. After 1 month of injection, mice were sacrificed and tumors were dissected. The volume and weight of tumors were measured and parts of the tumor were utilized for western blot and immunohistochemistry as described in the above section. Statistical analysis All statistical analysis was performed using Microsoft Excel and graphs were plotted using GraphPad Prism7. Data has been represented as mean ± SD. The student’s t-test was used to determine the statistical significance of the difference between the groups. The p-value of < 0.05 was considered significant. * p <0.05, ** p <0.01 and *** p <0.001. Images were analyzed and quantified using ImageJ software. Results SMAR1 exhibit a negative correlation with PKM2 and a positive correlation with PKM1 expression in breast cancer cells Dysregulation of various tumor suppressor proteins is one of the key features of various cancers 33 . SMAR1 being an important tumor suppressor protein is known to get downregulated in higher grades of breast and colon cancer 21 , 23 , 24 . LOH of SMAR1 locus (human chromosome 16q24) has been reported in various cancer 25 , 26 . Moreover, Cdc20 mediated proteasomal degradation of SMAR1 has been studied in breast cancer cells 23 . In contrast, upregulation of key oncogenes such as PKM2 gives metabolic as well as tumorigenic advantage to the cancer cells. The majority of normal differentiated cells have a lower expression of PKM2 and in cancer cells, there is an enhanced expression of PKM2 34 . Expression of SMAR1, PKM2 and PKM1 was measured in breast cancer patient samples and compared with surrounding non-cancerous tissue by immunohistochemistry. IHC staining of SMAR1 revealed that its expression was diminished in tumor samples compared to that of normal control tissue (Fig. 1 A). These results correspond with the earlier studies, confirming the downregulation of SMAR1 in breast cancer samples 21 , 23 . Moreover, IHC staining of PKM isoforms revealed that PKM2 expression was elevated and PKM1 expression was significantly low in tumor samples compared to that of normal control tissue. These results further comply with earlier reports suggesting a switch in PKM isoform expression between normal tissue and cancer tissue leading to higher expression of oncogenic isoform PKM2 and suppression of PKM1 3,32 . Human breast cancer cells such as MDA-MB-231, MDA-MB-468 and T47D harbor LOH for SMAR1 locus, whereas MCF7 cells lack LOH for this locus 25 , 27 . LOH harboring cells have significant downregulation of SMAR1 expression compared to that of MCF7 28 . Expression profile of SMAR1, PKM1 and PKM2 in these breast cancer cell lines MCF7, MDA-MB-231, MDA-MB-468 and T47D were found to be correlating with that of the patient samples (Fig. 1 B). These results suggest that SMAR1 has an inverse correlation with PKM2 expression whereas it has a direct correlation with PKM1 expression. This indicates that SMAR1 might be promoting the expression of PKM1 and suppressing the expression of oncogenic isoform PKM2 in the non-cancerous tissue. SMAR1 regulates PKM alternative splicing by promoting the incorporation of exon 9 and suppressing the incorporation of exon 10 SMAR1 is known to colocalize with one of the key splicing regulators SC35 in nuclear splicing speckles. It also interacts with snRNAs which are the core components of splicing machinery. Moreover, SMAR1 has been reported to regulate alternative splicing of CD44 variants and FAS ligand 28 . On the basis of an inverse correlation between SMAR1 and PKM2 expression as well as a positive correlation between SMAR1 and PKM1 observed in breast cancer, we hypothesized that SMAR1 might play a crucial role in regulating alternative splicing of the PKM gene. Based on the expression profile of SMAR1 in various breast cancer cell lines, MCF7 and MDA-MB-231 were used for further experiments. To investigate the role of SMAR1 in the regulation of PKM alternative splicing, PKM isoform expression was measured upon shRNA-mediated depletion of SMAR1 expression in MCF7 cells at the RNA level by performing quantitative RT-PCR and at the protein level by western blot. Knockdown of SMAR1 in MCF7 resulted in PKM1 downregulation and increased PKM2 expression at the transcript level (Fig. 2 A). Relative fold change in SMAR1 expression upon shRNA-mediated SMAR1 knockdown in MCF7 has been represented in Figure S1A. Further analysis of PKM isoform expression in SMAR1 depleted cells at the protein level suggests diminished expression of PKM1 and upregulation of PKM2 which was further in coherence with RNA expression analysis (Fig. 2 B). Moreover, PKM isoform expression was quantified upon ectopic expression of SMAR1 in LOH-containing MDA-MB-231 at the RNA level by qRT-PCR and at the protein level by western blot. Upon Flag-SMAR1 overexpression in MDA-MB-231, PKM1 was observed to be increased and PKM2 was downregulated at the transcript level (Fig. 2 C). Flag-SMAR1 mediated overexpression in MDA-MB-231 at transcript level has been represented in Figure S1B. Moreover, PKM isoform expression at the protein level in SMAR1 overexpressed cells was in concordance with that of transcript level analysis (Fig. 2 D). These results suggest that SMAR1 being a tumor suppressor protein, promotes the expression of PKM1 isoform over oncogenic isoform PKM2. To further validate the role of SMAR1 in PKM alternative splicing regulation, a dual reporter PKM minigene system has been generated. Schematic representation of dual reporter PKM minigene system has been described in Fig. 2 E. In this dual reporter PKM minigene assay, incorporation of exon 10 leads to eGFP expression whereas incorporation of exon 9 leads to mCherry expression. Fluorescence imaging was done upon ectopic expression of SMAR1 along with the PKM minigene system by confocal microscopy to measure eGFP and mCherry expression. Further relative fluorescence intensity was calculated and the fold change of eGFP/mCherry ratio has been calculated. Expression of SMAR1, eGFP and mCherry was confirmed by western blot (Figure S1C). The fold difference in eGFP/mCherry ratio in SMAR1 overexpression condition was reduced by 2-fold as compared to that of control which further validates its role in PKM alternative splicing regulation (Fig. 2 F). These results suggest that SMAR1 actively promotes the incorporation of exon 9 and the exclusion of exon 10 leading to higher expression of PKM1 and repression of PKM2 expression in breast cancer cells. SMAR1 interacts with PTBP1 and SMAR1-HDAC6 makes a triple complex with PTBP1 hnRNP A1, hnRNP A2 and PTBP1 are the three key regulators of PKM alternative splicing 11 , 12 . When these three proteins are present in higher concentration, they bind to intronic regions flanking exon 9 of PKM which leads to the inhibition of incorporation of exon 9 while promoting the incorporation of exon 10, thereby resulting in higher expression of PKM2 and diminished expression of PKM1 13 . To delineate the detailed molecular mechanism of SMAR1 mediated regulation of PKM alternative splicing, expression of hnRNP A1, hnRNP A2 and PTBP1 was measured upon shRNA-mediated knockdown of SMAR1 in MCF7. It was observed that there was no change in the expression of these three splicing regulators upon SMAR1 knockdown (Fig. 3 A). This observation eliminates the possibility of transcriptional repression of hnRNP A1, hnRNP A2 and PTBP1 by SMAR1. To determine the role of SMAR1 in the regulation of post-translational modification, the interaction of SMAR1 with these hnRNPs was checked by co-immunoprecipitation (co-IP) and observed that SMAR1 interacts with PTBP1 (Fig. 3 B). Interaction of SMAR1 with PTBP1 was further confirmed by reverse co-IP of PTBP1 with SMAR1 (Fig. 3 C). SMAR1 is known to regulate various cellular processes with the assistance of HDACs such as HDAC1 and HDAC6 20,22,28,35 . To identify which HDAC is involved in SMAR1 mediated regulation of PKM alternative splicing, the interaction of PTBP1 with HDAC1 and HDAC6 was checked by co-IP which revealed that HDAC6 interacts with PTBP1 (Fig. 3 C). Interaction of HDAC6 with PTBP1 and SMAR1 was further confirmed by reverse co-IP of HDAC6 (Fig. 3 D). To further investigate the molecular interplay of SMAR1-HDAC6 interaction with PTBP1, sequential IP of SMAR1 and HDAC6 with PTBP1 was performed. Sequential IP experiment revealed that SMAR1-HDAC6 forms a triple complex (Fig. 3 E). These results demonstrate that SMAR1 directly interacts with PTBP1 along with HDAC6 and the coexistence of SMAR1-HDAC6-PTBP1 as a ternary complex. This trimeric complex formation further indicates molecular dynamics between these proteins in SMAR1 mediated regulation of PKM alternative splicing. SMAR1 mediated regulation of PKM alternative splicing is HDAC6 dependent To delineate the role of HDAC6 in SMAR1 mediated regulation of PKM alternative splicing, expression of PKM isoforms was checked upon shRNA-mediated knockdown of HDAC6 by Western blot. Upon depletion of HDAC6, there was a downregulation in PKM1 expression and an increase in PKM2 expression (Fig. 4 A). This indicates the involvement of HDAC6 in PKM alternative splicing regulation. To further determine the role of HDAC6 in SMAR1 mediated regulation of PKM alternative splicing, SMAR1 overexpressed cells were treated with a specific HDAC6 inhibitor (Tubacin) and PKM isoform expression was checked by western blot. Expression of PKM1 was high and PKM2 was downregulated in the SMAR1 overexpression condition compared to that of control. However, upon Tubacin treatment in SMAR1 overexpressed cells, the expression of PKM1 was reduced and the expression of PKM2 was increased compared to that of only SMAR1 overexpressed cells (Fig. 4 B). This confirms that SMAR1 mediated regulation of PKM alternative splicing is HDAC6 dependent process. Further validation of the role of HDAC6 in SMAR1 mediated regulation of PKM alternative splicing was done by dual reporter PKM minigene assay. Fold change in eGFP/mCherry ratio was measured upon SMAR1 overexpression along with Tubacin treatment in cells transfected with PKM minigene system compared to the control condition. Fold change in eGFP/mCherry ratio was decreased upon ectopic expression of SMAR1 compared to the control. However, upon tubacin treatment in SMAR1 overexpressed cells resulted in a further increase in the fold change of eGFP/mCherry ratio (Fig. 4 C). These observations confirm the role of HDAC6 in SMAR1 mediated regulation of PKM alternative splicing. This demonstrates that HDAC6 is actively involved in PKM alternative splicing regulation orchestrated by SMAR1. SMAR1-HDAC6 deacetylates PTBP1 and modulates its affinity to PKM pre-mRNA Post-translational modifications such as phosphorylation, acetylation, ubiquitination, sumoylation of splicing factors are known to regulate various alternative splicing events 36 – 39 . SMAR1 is known to regulate the alternative splicing of CD44 variants by HDAC6 assisted deacetylation of Sam68 28 . Moreover, sirtuin-mediated deacetylation of hnRNP A1 has been reported to regulate alternative splicing of the PKM gene 40 . To further delineate the role of SMAR1-HDAC6 in the deacetylation of PTBP1, the acetylation status of PTBP1 was checked upon shRNA-mediated depletion of SMAR1 by immunoprecipitation with an anti-acetyl-lysine antibody. In the SMAR1 knockdown condition, there was an increase in the acetylation status of PTBP1 compared to control (Fig. 5 A). This observation indicates that the SMAR1 level dictates the acetylation status of PTBP1 and it maintains PTBP1 in a deacetylated state. The acetylation status of PTBP1 was further estimated after SMAR1 overexpression along with Tubacin treatment. Ectopic expression of SMAR1 resulted in a decrease in acetylation status of PTBP1 but upon Tubacin treatment in SMAR1 overexpressed cells, the acetylation status of PTBP1 was restored (Fig. 5 B). This suggests that SMAR1 maintains PTBP1 in the deacetylated state in HDAC6 dependent manner. To inspect the effect of SMAR1-HDAC6 mediated deacetylation of PTBP1 on its affinity for PKM pre-mRNA, UV-crosslinking and RNA immunoprecipitation (CLIP) of PTBP1 was performed upon shRNA-mediated depletion of SMAR1 in MCF7. Enrichment of PTBP1 on Intron 8 of PKM pre-mRNA was enhanced by 2-fold in the case of SMAR1 knockdown condition as compared to that of control (Fig. 5 C). Immunoprecipitation of PTBP1 in the CLIP experiment was confirmed by western blot (Fig. 5 D). Increased enrichment of PTBP1 on PKM pre-mRNA in SMAR1 knockdown condition suggests that SMAR1-HDAC6 mediated deacetylation of PTBP1 leads to a reduction in its affinity for PKM pre-mRNA and ultimately modulates PKM alternative splicing. SMAR1 regulates the Warburg effect and breast cancer growth via regulation of PKM2 expression Higher expression of PKM2 compared to PKM1 is one of the key factors for cancer cells in achieving the metabolic advantage of the Warburg effect compared to normal cells 3 . Reduction in PKM2 expression might lead to inhibition of the Warburg effect and ultimately suppress the tumorigenic potential of cancer cells. SMAR1 mediated regulation of PKM alternative splicing leads to suppression of PKM2 and increased expression of PKM1. Based on this observation, we hypothesized that SMAR1 might be playing important role in the regulation of cancer cell metabolism. To determine the role of SMAR1 in the regulation of tumor metabolism, glucose utilization was estimated upon shRNA-mediated decrease of SMAR1 expression along with PKM2 in MCF7. Depletion of PKM2 by shRNA was confirmed by western blot (Figure S2A). A reduction in SMAR1 expression resulted in increased glucose utilization. Further depletion of PKM2 in SMAR1 knockdown cells resulted in decreased glucose utilization (Fig. 6 A). Expression of SMAR1 and PKM isoforms upon shRNA-mediated knockdown of SMAR1 along with PKM2 depletion was confirmed by western blot (Figure S2B). 2-NBDG mediated glucose uptake upon siRNA-mediated knockdown of SMAR1 validates its role in the regulation of glucose metabolism (Figure S2C). siRNA-mediated knockdown of SMAR1 in MCF7 was confirmed by western blot (Figure S2D). To further decipher the role of SMAR1 in the reversal of the Warburg effect, lactate production was measured in SMAR1 knockdown condition along with PKM2 depletion. SMAR1 depletion condition revealed enhanced lactate production compared to control. Moreover, PKM2 knockdown in SMAR1 depleted cells were having decreased lactate production compared to SMAR1 depleted cells (Fig. 6 B). This suggests that SMAR1 mediated regulation of PKM alternative splicing inhibits the Warburg effect via suppression of PKM2. These observations reveal the important regulatory role of SMAR1 in keeping cellular metabolism in check via regulation of PKM isoform expression. PKM2 mediated increase in glucose uptake and lactate production provides a metabolic advantage to cancer cells leading to increased tumor growth 3 . For tumor cells, reprogramming of glucose metabolism and higher glucose utilization are essential for their proliferation and survival 2 , 41 . The proliferative potential of cancer cells upon shRNA-mediated depletion of SMAR1 along with PKM2 in MCF7 was assessed by proliferation assay and colony formation assay. In the SMAR1 knockdown condition proliferation rate and colony formation potential were increased which were further reduced due to PKM2 depletion (Fig. 6 C and D). These results suggest that SMAR1 inhibits breast cancer growth via reversal of the Warburg effect by suppressing PKM2 expression. Increased lactate production by cancer cells creates acidic surroundings which contribute to the tumorigenic properties of cancer cells such as migration, invasion and metastasis 42 . Further, the tumorigenic potential of cancer cells upon depletion of SMAR1 along with PKM2 knockdown was measured by in vitro functional assays such as transwell migration, invasion and wound healing assays. SMAR1 knockdown resulted in an increase in migration, invasion and wound healing properties of MCF7. Knockdown of PKM2 in SMAR1 depleted cells showed a decrease in migration, invasion and wound healing ability of cancer cells (Fig. 6 E-G). These observations demonstrate that SMAR1 inhibits the tumorigenic potential of cancer cells due to the inhibition of the cancer cell metabolism through the downregulation of PKM2. SMAR1 regulates in vivo tumor formation via regulation of PKM2 expression PKM2 contributes to in vivo tumor generation and its progression via regulation of cancer cell metabolism 3 . To identify the role of SMAR1 in tumor formation, tumor xenografts of SMAR1 overexpressed cells were generated by injection of SMAR1-adenovirus transduced MDA-MB-231 cells in NOD/SCID mice. The tumor burden was compared among SMAR1 overexpressing MDA-MB-231 tumors to that of normal MDA-MB-231 control tumors. In SMAR1 overexpressing group, tumor burden was less compared to that of the control group (Fig. 7 A). Moreover, in the SMAR1 overexpression condition, there was a significant reduction in the weight and volume of the tumor compared to control (Fig. 7 B and C). These observations indicate that SMAR1 suppresses in vivo tumor generation. A quantitative expression of PKM1 and PKM2 in these xenograft tumor samples was checked by western blot. In adeno-SMAR1 over-expressed tumor samples expression of tumorigenic isoform PKM2 was reduced and PKM1 expression was increased compared to control (Fig. 7 D). IHC staining of PKM isoforms in these in vivo tumor samples were observed to be correlating with western blot results suggesting higher expression of PKM1 isoform and depleted expression of PKM2 isoform in SMAR1 overexpressing tumors compared to that of control tumors (Fig. 7 E). These observations substantiate that SMAR1 suppresses in vivo tumor formation via the regulation of PKM isoform expression. Discussion Here we describe the detailed mechanism of PKM alternative splicing regulation by one of the key tumor suppressor proteins, SMAR1 and its implications in inhibition of cancer cell metabolism and tumorigenesis. SMAR1 has been reported to co-localize with key splicing factor SC35 and it interacts with splicing regulator Sam68 28 . SMAR1 also contains the RS domain which facilitates its interactions with various snRNAs which are the core components of splicing machinery 28 . Furthermore, SMAR1 regulates alternative splicing of CD44 variants and FAS ligand 28 . This indicates the active involvement of SMAR1 in various alternative splicing events. Our results demonstrate that SMAR1 follows a positive correlation with PKM1 and a negative correlation with oncogenic isoform PKM2 in breast cancer patient samples as well as in breast cancer cell lines. Further experimental approaches have revealed that SMAR1 is actively involved in the regulation of PKM alternative splicing. SMAR1 promotes the inclusion of exon 9 and exclusion of exon 10 thus inhibiting the expression of PKM2 which is a tumorigenic isoform of the PKM gene. These observations were validated by dual chromatic PKM minigene assay, which further confirms the regulatory role of SMAR1 in dictating the outcome of PKM alternative splicing. The downregulation of SMAR1 in breast cancer is one of the reasons for enhanced PKM2 expression that leads to altered glucose metabolism and contributes to cancer cell growth. Nuclear matrix binding protein, SMAR1 is known to perform various biological functions with help of regulatory proteins such as HDACs 20 , 22 , 28 , 35 . Additionally, various HDACs have been reported to participate in the regulation of alternative splicing through modulation of the acetylation status of histone and non-histone proteins including splicing factors 43 . Earlier studies suggest that SMAR1 inhibits the expression of various genes such as Cyclin D1 , BAX and PUMA by recruiting the repressor complex of HDAC1-mSin3a on the promoter and keeping it in a repressed state by epigenetic modulations 20 , 22 . Moreover, SMAR1 also plays a crucial role in deacetylating target proteins such as Ku70 and Sam68 with the help of HDAC6 28,35 . This points to the crucial role of SMAR1 in bringing about the post-translational regulation of its target proteins and ultimately dictating their role in various molecular and biological processes. SMAR1-HDAC6 mediated deacetylation of Ku70 dictates cell fate upon exposure to ionizing radiation via regulation of DNA repair and apoptosis 35 . SMAR1 further inhibits metastasis via regulation of CD44 variants alternative splicing via HDAC6 mediated deacetylation of Sam68 in breast cancer cells 28 . Many post-translational modifications such as phosphorylation, acetylation, ubiquitination, and sumoylation of splicing regulators have been associated with the regulation of various alternative splicing events 28,36−40 . Previous studies involving PKM alternative splicing suggest that three important splicing inhibitors hnRNP A1, hnRNP A2 and PTBP1 regulate PKM alternative splicing 11 , 12 . Moreover, Sirtuin mediated deacetylation of hnRNP A1 has been reported to regulate alternative splicing of PKM gene 40 . This suggests that the acetylation status of splicing factors plays an important role in the regulation of alternative splicing events 28 , 40 . Herein, we showed that SMAR1 mediated PKM alternative splicing regulation is HDAC6 dependent and SMAR1-HDAC6 forms a triple complex with PTBP1 and maintains it in the deacetylated form. CLIP experiment of PTBP1 in SMAR1 depleted condition resulted in the increased binding of PTBP1 on Intron 8 of PKM pre-mRNA. This indicates that the binding affinity of PTBP1 is more on PKM pre-mRNA in absence of SMAR1. Based on this observation it can be further implied that the deacetylation of PTBP1 by SMAR1-HDAC6 results in the lower affinity of PTBP1 for PKM pre-mRNA and thus leading to the inclusion of exon 9 and exclusion of exon 10. This leads to higher expression of PKM1 and lower expression of tumorigenic isoform PKM2. Our findings suggest that SMAR1 brings about HDAC6 mediated deacetylation of PTBP1 and thus maintains PTBP1 in deacetylated form. This deacetylated PTBP1 ultimately leads to the regulation of PKM alternative splicing promoting expression of PKM1 and repressing oncogenic isoform PKM2. Highly proliferating cancer cells gain a metabolic advantage over normal cells via higher expression of PKM2 and exhibits peculiar metabolic properties known as the Warburg effect 2 . Glucose is a primary source of energy and anabolic demands for highly proliferating cancer cells 2 . Moreover, a higher amount of lactate production creates an acidic micro-environment in tumors which favors invasion and metastasis 42 . Our study demonstrates that SMAR1 mediated suppression of PKM2 isoform and upregulation of PKM1 isoform results in inhibition of cancer cell metabolism. SMAR1 inhibits glucose utilization and lactate production via regulation of PKM alternative splicing. Our results demonstrate that SMAR1 mediated reversal of the Warburg effect leads to inhibition of cancer cell proliferation, migration and invasion. Moreover, SMAR1 suppresses in vivo tumor formation via the regulation of PKM isoform expression. This demonstrates the tumor suppressor function of key nuclear matrix binding protein SMAR1 and its importance in the maintenance of metabolic equilibrium. Future studies on the role of SMAR1 in global alternative splicing regulation might give more insight into the active involvement of tumor suppressor proteins in cancer-associated alternative splicing regulation and its implication in various cellular processes. In higher grades of cancer, downregulation of SMAR1 expression has been correlated with an increase in tumorigenic potential 21 , 23 , 24 . SMAR1 expression and functions are also known to be modulated by various cancer-associated signaling pathways including JNK signaling, Wnt signaling, ERK-MAPK pathway 23 , 24 , 28 . SMAR1 gets highly dysregulated in Wnt signaling associated colorectal cancer (CRC) 24 . Moreover, activation of ERK-MAPK signaling leads to translocation of SMAR1 to the cytoplasm, resulting in increased CD44 variants alternative splicing and metastasis in breast cancer cells 28 . A study in the breast cancer model revealed that SMAR1 undergoes proteasomal degradation by Cdc20 in a JNK kinase-dependent manner 23 . Furthermore, microbial peptides that promote SMAR1 stabilization have been correlated with inhibition of Wnt/β-catenin activities in CRC 24 . A study involving stabilization of SMAR1 expression by treatment with isothiocyanate derivative has been co-related with anti-HIV activity 44 . Our findings reveal that SMAR1 knockdown promotes the Warburg effect and tumorigenic potential of breast cancer cells by modulating PKM isoform expression indicating an important regulatory role of tumor suppressor protein in cellular homeostasis. This indicates that future studies targeting cancer cell metabolism and cancer progression via stabilization of SMAR1 expression might lead to better therapeutic efficacy in cancer treatment. Conclusion Our study in the breast cancer model highlights that nuclear matrix binding protein SMAR1 regulates PKM alternative splicing and inhibits the expression of PKM2 and promotes the expression of PKM1. SMAR1 regulates PKM alternative splicing via deacetylation of a key alternative splicing inhibitor PTBP1 in an HDAC6 dependent manner. Further, in vitro enzymatic assays, functional assays and in vivo tumor model suggest that SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells by suppressing the expression of tumor-associated isoform PKM2 and promoting the expression of enzymatically more efficient PKM1 isoform. Abbreviations ATP: Adenosine triphosphate CLIP: UV-crosslinking and RNA immunoprecipitation CRC: colorectal cancer HDAC6: Histone deacetylase 6 hnRNP: Heterogeneous nuclear ribonucleoprotein IP: Immunoprecipitation MAR: Matrix associated region MARBP: Matrix associated region binding protein PKM: Pyruvate Kinase Muscle PTBP1: Polypyrimidine tract-binding protein 1 SMAR1: Scaffold-matrix associated region binding protein 1 TCA: Tricarboxylic acid Declarations Ethics approval All the experiments involving the usage of animals were conducted following CPCSEA guidelines approved by the Institutional Animal Ethics Committee. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Funding The authors are thankful to the Council of Scientific & Industrial Research (CSIR), India; University Grants Commission (UGC), India; National Centre for Cell Science (NCCS), Pune, India and Department of Biotechnology (DBT), India for their fellowships. We would also like to acknowledge the DBT, India; Department of Science and Technology (DST), India and J. C. Bose fellowship-SERB (Science and Engineering Research Board), India to SC, for financial support. Authors’ contributions AC and SC conceptualized, planned and designed the experiments. AC, AP, RP, VS, RN, PF performed experiments. AC wrote the manuscript. AC, AP, RP, VS, RN, PF, SmS, SaS, GCK and SC provided intellectual inputs and helped in preparing the manuscript. GCK helped in obtaining patient samples. SaS and SmS conceptualized the dual chromatic PKM minigene system and AC cloned the dual chromatic PKM minigene system. Acknowledgment We are thankful to the National Centre for Cell Science (NCCS), Pune, India for providing infrastructure and experimental facilities. We would like to acknowledge Dr. Marta Cortés-Cros for providing us sequence information of shPKM2. We would like to thank Dr. Adrian R. Krainer for providing the PKM minigene construct which we used to design dual reporter PKM minigene. Conflict of interest The authors declare that there is no conflict of interest. References O. Warburg. On the origin of cancer cells. Science 123 , 309–314 (1956). Heiden, M. G. V., Cantley, L. C. & Thompson, C. B. Understanding the warburg effect: The metabolic requirements of cell proliferation. Science 324 , 1029–1033 (2009). Christofk, H. R. et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452 , 230–233 (2008). Jurica, M. S. et al. The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6 , 195–210 (1998). 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Differences were considered statistically significant with *p < 0.05, **p < 0.01 and ***p 0.05). FigureS2.tif Additional File 2: Figure S2: (A) shRNA-mediated PKM2 knockdown in MCF7. (B) Expression of PKM isoforms upon SMAR1 and PKM2 knockdown in MCF7. (C) % Glucose uptake (2-NBDG) upon siRNA-mediated knockdown of SMAR1 in MCF7 by FACS. (D) Expression of PKM isoforms upon siRNA-mediated knockdown of SMAR1 in MCF7 by western blot. Error bars show mean values ± SD. Differences were considered statistically significant with *p < 0.05, **p < 0.01 and ***p 0.05). TableS1.pdf Additional File 3: Table S1: List of primer sequences utilized for qRT-PCR. Cite Share Download PDF Status: Published Journal Publication published 16 Apr, 2021 Read the published version in Cancer & Metabolism → Version 1 posted Review # 2 received at journal 08 Feb, 2021 Editorial decision: Minor revision 08 Feb, 2021 Review # 1 received at journal 07 Feb, 2021 Reviewer # 2 agreed at journal 30 Jan, 2021 Reviewer # 1 agreed at journal 26 Jan, 2021 Reviewers invited by journal 25 Jan, 2021 Editor assigned by journal 18 Jan, 2021 Submission checks completed at journal 18 Jan, 2021 Editor invited by journal 18 Jan, 2021 First submitted to journal 17 Jan, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-151525","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":9767805,"identity":"c05a5d8c-4468-453c-9e36-0b838be9b520","order_by":0,"name":"Arpankumar Choksi","email":"","orcid":"","institution":"NCCS: National Centre For Cell Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Arpankumar","middleName":"","lastName":"Choksi","suffix":""},{"id":9767806,"identity":"cc4875b6-a3ec-4932-871c-e05427cd8677","order_by":1,"name":"Apoorva Parulekar","email":"","orcid":"","institution":"NCCS: National Centre For Cell Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Apoorva","middleName":"","lastName":"Parulekar","suffix":""},{"id":9767807,"identity":"000ffddd-cfe2-4429-b856-9ec5ad2f3704","order_by":2,"name":"Richa Pant","email":"","orcid":"","institution":"NCCS: National Centre For Cell Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Richa","middleName":"","lastName":"Pant","suffix":""},{"id":9767808,"identity":"e3ee29d8-11c3-4850-b25c-bc744d5c2f32","order_by":3,"name":"Vibhuti Kumar Shah","email":"","orcid":"","institution":"NCCS: National Centre For Cell Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vibhuti","middleName":"Kumar","lastName":"Shah","suffix":""},{"id":9767809,"identity":"8f96d2d7-4a9e-4725-93ff-a3d51906976f","order_by":4,"name":"Ramakrishna Nimma","email":"","orcid":"","institution":"NCCS: National Centre For Cell Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ramakrishna","middleName":"","lastName":"Nimma","suffix":""},{"id":9767810,"identity":"04bf6b2c-394f-4e47-bbf2-88a1e3265e22","order_by":5,"name":"Priyanka Firmal","email":"","orcid":"","institution":"NCCS: National Centre For Cell Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Priyanka","middleName":"","lastName":"Firmal","suffix":""},{"id":9767811,"identity":"5957d80d-555c-40c1-a134-61d5b083a03a","order_by":6,"name":"Smriti Singh","email":"","orcid":"","institution":"IISER Bhopal: Indian Institute of Science Education and Research Bhopal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Smriti","middleName":"","lastName":"Singh","suffix":""},{"id":9767812,"identity":"3b344d08-5cec-4e56-8fa7-4b3819327864","order_by":7,"name":"Gopal C. Kundu","email":"","orcid":"","institution":"Kalinga Institute of Industrial Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gopal","middleName":"C.","lastName":"Kundu","suffix":""},{"id":9767813,"identity":"cc2f8d9c-4cec-4b7c-ac62-940c8e365cea","order_by":8,"name":"Sanjeev Shukla","email":"","orcid":"","institution":"IISER Bhopal: Indian Institute of Science Education and Research Bhopal","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sanjeev","middleName":"","lastName":"Shukla","suffix":""},{"id":9767814,"identity":"ebb1dbb6-b8f9-4cd2-99c2-7c1e8d7732b1","order_by":9,"name":"Samit Chattopadhyay","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYJCCA2AEAhIGNkCCGC0HgFp42MBa0ojTwgDXwsBwmLAW+fbTiYc/1NyRt5dvPvbBouB8Yv/s5oMPGGpsonFpMTiTu+HAgWPPDHvY2JJnSBjcTpxx51iyAcOxtNwGXFoYgFoONhxm7GHjMWYAaWm4kWMmwdhwGKcW+f63YC32PWz8n4FaziXOJ6SF4QbElkSgLcxALQcSNxDSYnADaMuZY4eTe46lgRyWbLzxRlqyQQIev8j3527+UFFz2La9+fBjZok/drLzbiQffPChxga3w5ABMzBKHMEqE4hRDgKMHxgY7IlVPApGwSgYBSMHAADKS2UN3Hn8LwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3581-9625","institution":"Birla Institute of Technology and Science Pilani - K K Birla Goa Campus","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Samit","middleName":"","lastName":"Chattopadhyay","suffix":""}],"badges":[],"createdAt":"2021-01-20 11:11:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-151525/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-151525/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40170-021-00252-x","type":"published","date":"2021-04-16T19:06:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":5510974,"identity":"19ad9680-7b15-4204-b323-dce5db13502b","added_by":"auto","created_at":"2021-02-01 18:03:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":555635,"visible":true,"origin":"","legend":"Expression of SMAR1 and PKM isoforms in breast cancer. (A) Expression of SMAR1, PKM1 and PKM2 in paraffinized breast tumor samples compared to normal tissue by immunohistochemistry (n=3). (B) Expression profile of SMAR1, PKM1 and PKM2 in various breast cancer cell lines by western blot.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/9bc6153af1e7368e3a676fa6.jpg"},{"id":5511369,"identity":"8444f629-0fa1-4fb3-a4ce-6b73a25dbc0c","added_by":"auto","created_at":"2021-02-01 18:09:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":335841,"visible":true,"origin":"","legend":"SMAR1 regulates PKM alternative splicing. (A) qRT-PCR of PKM isoforms normalized to 18S rRNA and further normalized to constitutive PKM exon (Exon 11) upon shRNA-mediated knockdown of SMAR1 in MCF7 (n=3). (B) Expression of PKM isoforms upon shRNA-mediated knockdown of SMAR1 in MCF7 by western blot. (C) qRT-PCR of PKM isoforms normalized to 18S rRNA and further normalized to constitutive PKM exon (Exon 11) upon Flag-SMAR1 mediated overexpression of SMAR1 in MDA-MB-231 (n=3). (D) Expression of PKM isoforms upon Flag-SMAR1 mediated overexpression of SMAR1 in MDA-MB-231 by western blot. (E) Schematic representation of dual chromatic PKM minigene system. (F) Confocal microscopy to check the expression of eGFP and mCherry in MCF7 cells transfected with PKM minigene along with Flag-SMAR1. Relative fluorescence intensity of eGFP and mCherry was quantified with the help of ImageJ and fold change in eGFP/mCherry ratio was calculated for control and Flag-SMAR1 overexpression (n=3). Error bars show mean values ± SD. Differences were considered statistically significant with *p \u003c 0.05, **p \u003c 0.01 and ***p \u003c 0.001, ns non-significant difference (p \u003e 0.05).","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/93732de7b5b8b9ef9e5595fa.jpg"},{"id":5511175,"identity":"b02397d8-d13b-43f9-ba02-38e064cf8d16","added_by":"auto","created_at":"2021-02-01 18:06:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":290812,"visible":true,"origin":"","legend":"SMAR1 makes a triple complex with PTBP1 and HDAC6. (A) Expression of hnRNP A1, hnRNP A2 and PTBP1 upon shRNA-mediated knockdown of SMAR1 in MCF7. (B) Co-IP of SMAR1 with hnRNP A1, hnRNP A2, PTBP1 and HDAC6 in MCF7 suggest that SMAR1 interacts with PTBP1 and HDAC6. (C) Co-IP of PTBP1 with SMAR1, HDAC1 and HDAC6 in MCF7 suggest that PTBP1 interacts with SMAR1 and HDAC6. (D) Co-IP of HADC6 with hnRNP A1, hnRNP A2, PTBP1 and SMAR1 in MCF7 confirms that HDAC6 interacts with PTBP1 and SMAR1. (E) Sequential Co-IP of SMAR1 with HDAC6 and PTBP1 in MCF7 suggest that SMAR1-HDAC6 makes a triple complex with PTBP1.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/f329324accc6d6db351c2c43.jpg"},{"id":5510978,"identity":"ae5d5294-c9e1-4700-b496-adef771da210","added_by":"auto","created_at":"2021-02-01 18:03:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":322631,"visible":true,"origin":"","legend":"SMAR1 mediated PKM alternative splicing is HDAC6 dependent. (A) Expression of PKM isoforms upon shRNA-mediated knockdown of HDAC6 in MCF7 by western blot. (B) Expression of PKM isoforms upon SMAR1 overexpression followed by Tubacin (5 μM) treatment in MCF7 by western blot. (C) Confocal microscopy to check the expression of eGFP and mCherry in MCF7 cells transfected with PKM minigene along with Flag-SMAR1. Relative fluorescence intensity of eGFP and mCherry was quantified with the help of ImageJ and fold change in eGFP/mCherry ratio was calculated for control, control + Tubacin (5 μM), Flag-SMAR1 and Flag-SMAR1 + Tubacin (5 μM). Fold change in eGFP/mCherry ratio for SMAR1 overexpression and Tubacin treatment validates the role of HDAC6 in SMAR1 mediated regulation of PKM alternative splicing (n=3). Error bars show mean values ± SD. Differences were considered statistically significant with *p \u003c 0.05, **p \u003c 0.01 and ***p \u003c 0.001, ns non-significant difference (p \u003e 0.05).","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/03319cacf7e43ec93d22348e.jpg"},{"id":5510983,"identity":"7535e05b-e9a6-4f2d-8b16-7701a0ae7054","added_by":"auto","created_at":"2021-02-01 18:03:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":219772,"visible":true,"origin":"","legend":"SMAR1-HDAC6 deacetylates PTBP1 and inhibits its binding to PKM pre-mRNA. (A) The acetylation status of PTBP1 upon SMAR1 knockdown in MCF7 was checked by IP with an anti-Acetyl-Lysine antibody. (B) The acetylation status of PTBP1 upon SMAR1 overexpression followed by Tubacin (5 μM) treatment in MCF7 was checked by IP with the anti-Acetyl-Lysine antibody. (C) CLIP experiment of PTBP1 on Intron 8 of PKM pre-mRNA in SMAR1 knockdown condition (n=3). (D) IP of PTBP1 to confirm pull down in CLIP experiment upon SMAR1 knockdown in MCF7. Error bars show mean values ± SD. Differences were considered statistically significant with *p \u003c 0.05, **p \u003c 0.01 and ***p \u003c 0.001, ns non-significant difference (p \u003e 0.05).","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/9b427619694ae5375b0f37eb.jpg"},{"id":5511454,"identity":"641a3feb-b650-4f71-9540-cd8eb41b19f4","added_by":"auto","created_at":"2021-02-01 18:12:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":733945,"visible":true,"origin":"","legend":"SMAR1 inhibits Warburg effect and tumorigenesis. (A) % Glucose utilization was measured in shRNA-mediated knockdown of SMAR1 along with PKM2 in MCF7 by enzymatic assay (n=3). (B) % Lactate formation was measured in shRNA-mediated knockdown of SMAR1 along with PKM2 in MCF7 by enzymatic assay (n=3). (C) % Cell proliferation was measured in shRNA-mediated knockdown of SMAR1 along with PKM2 in MCF7 by MTT assay (n=3). (D) % Colony formation was measured in shRNA-mediated knockdown of SMAR1 along with PKM2 in MCF7 (n=3). (E) % Transwell cell migration was measured in shRNA-mediated knockdown of SMAR1 along with PKM2 in MCF7 (n=3). (F) % Transwell cell invasion was measured in shRNA-mediated knockdown of SMAR1 along with PKM2 in MCF7 (n=3). (G) % Wound migration was measured in shRNA-mediated knockdown of SMAR1 along with PKM2 in MCF7 (n=3). Error bars show mean values ± SD. Differences were considered statistically significant with *p \u003c 0.05, **p \u003c 0.01 and ***p \u003c 0.001, ns non-significant difference (p \u003e 0.05).","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/3e021e95c35f17622fe136dc.jpg"},{"id":5511500,"identity":"8f5354b6-6ed9-442b-aa05-a55f8a6dbd6c","added_by":"auto","created_at":"2021-02-01 18:15:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":361096,"visible":true,"origin":"","legend":"SMAR1 inhibits in vivo tumor formation. (A) Tumors generated in NOD/SCID mice upon injection of adeno-SMAR1 transduced MDA-MB-231 cells compared to control (n=4). (B and C) graphs representing the Tumor weight and Tumor volume. (D and E) Expression of PKM isoforms was checked in xenograft samples by western blot and immunohistochemistry. Error bars show mean values ± SD. Differences were considered statistically significant with *p \u003c 0.05, **p \u003c 0.01 and ***p \u003c 0.001, ns non-significant difference (p \u003e 0.05).","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/57fccd474b1a161c72cfed63.jpg"},{"id":13653708,"identity":"0f46caaa-25e5-4376-917a-ca2b84412097","added_by":"auto","created_at":"2021-09-17 09:54:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1391950,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/ac075581-b2a9-47d2-bcb7-bc6e07180d56.pdf"},{"id":5511174,"identity":"337c9ec6-c42e-483d-a677-43706eefb86c","added_by":"auto","created_at":"2021-02-01 18:06:19","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":276110,"visible":true,"origin":"","legend":"Additional File 1: Figure S1: (A) shRNA-mediated knockdown of SMAR1 in MCF7 by qRT-PCR. (B) Flag-SMAR1 mediated overexpression of SMAR1 in MDA-MB-231 by qRT-PCR. (C) Expression of eGFP/mCherry upon SMAR1 overexpression in MCF7. Error bars show mean values ± SD. Differences were considered statistically significant with *p \u003c 0.05, **p \u003c 0.01 and ***p \u003c 0.001, ns non-significant difference (p \u003e 0.05).","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/2111a1e482f68d1a502a37aa.tif"},{"id":5511370,"identity":"88ce9ce5-2685-4a40-85fc-933d7c5d0725","added_by":"auto","created_at":"2021-02-01 18:09:19","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":388344,"visible":true,"origin":"","legend":"Additional File 2: Figure S2: (A) shRNA-mediated PKM2 knockdown in MCF7. (B) Expression of PKM isoforms upon SMAR1 and PKM2 knockdown in MCF7. (C) % Glucose uptake (2-NBDG) upon siRNA-mediated knockdown of SMAR1 in MCF7 by FACS. (D) Expression of PKM isoforms upon siRNA-mediated knockdown of SMAR1 in MCF7 by western blot. Error bars show mean values ± SD. Differences were considered statistically significant with *p \u003c 0.05, **p \u003c 0.01 and ***p \u003c 0.001, ns non-significant difference (p \u003e 0.05).","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/9288262c5831a1760a92b66b.tif"},{"id":5511371,"identity":"099787a1-22c7-447e-8427-9b1d278223d3","added_by":"auto","created_at":"2021-02-01 18:09:19","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":68393,"visible":true,"origin":"","legend":"Additional File 3: Table S1: List of primer sequences utilized for qRT-PCR.","description":"","filename":"TableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-151525/v1/ee5040ff8825954de14c6561.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTumor Suppressor SMAR1 Regulates PKM Alternative Splicing by HDAC6 Mediated Deacetylation of PTBP1\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eReprogramming cellular metabolism is one of the key hallmarks of cancer cells. In the presence of oxygen, somatic cells convert glucose to pyruvate by glycolysis and then redirect this pyruvate to the TCA cycle in mitochondria for ATP production. In the absence of oxygen, these cells convert pyruvate to lactate which is an energetically less efficient but relatively fast reaction. Whereas highly proliferating cancer cells utilize more glucose and produce more lactate compared to normal cells, independent of the presence or absence of oxygen. This phenomenon is known as the Warburg effect or aerobic glycolysis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Cancer cells reprogram their cellular metabolism by regulating alternative splicing of Pyruvate Kinase Muscle (\u003cem\u003ePKM\u003c/em\u003e) isoforms\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Pyruvate kinase is one of the rate-determining enzymes of glycolysis and facilitates the conversion of phosphoenolpyruvate to pyruvate. There are total four isoforms of pyruvate kinase enzyme: PKL (Pyruvate kinase liver), PKR (Pyruvate kinase Red blood cells), PKM1 and PKM2. PKL is expressed in the liver, PKR is expressed in the erythrocytes, PKM1 is expressed predominantly in terminally differentiated tissues and PKM2 is highly expressed in the proliferating cells such as cancer cells and stem cells\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePKM\u003c/em\u003e gene contains 12 exons, wherein the incorporation of exon 9 and exon 10 are regulated by mutually exclusive alternative splicing resulting in the expression of PKM1 and PKM2 isoforms, respectively\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Exon 9 and exon 10 both codes for 56 amino acids attributing to the distinctive regulatory and enzymatic activities of PKM isoforms. PKM1 forms a tetramer and is constitutively active, whereas PKM2 forms a tetramer (enzymatically efficient), as well as a dimer (enzymatically less efficient). Enzymatic activity of PKM2 dimer is influenced by allosteric regulation due to fructose-1,6-bisphosphate levels and interaction with phosphotyrosine-binding proteins\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHigher expression of PKM2 provides cancer cells with a metabolic advantage over normal cells. Due to the lower enzymatic activity of PKM2 as compared to that of PKM1, the major amount of glucose present in the cell remains as glycolytic intermediates which provide building blocks, such as amino acids, nucleotides and fatty acids to the highly proliferating cancer cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The remaining glucose gets converted to pyruvate and this pyruvate instead of entering the TCA cycle gets converted to lactate. To meet the high energy and carbon demands these cancer cells utilize more and more glucose compared to normal cells\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Apart from its role in glucose metabolism, PKM2 also regulates other cellular processes by getting translocated to the nucleus from the cytoplasm and affecting a variety of signaling pathways leading to oncogenesis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCancer cells achieve a metabolic edge over normal cells by regulating \u003cem\u003ePKM\u003c/em\u003e alternative splicing and promoting the expression of PKM2\u003csup\u003e3,8\u003c/sup\u003e. hnRNPs (heterogeneous ribonucleoproteins); hnRNP A1, hnRNP A2 and PTBP1 (hnRNP I) are the key regulators of \u003cem\u003ePKM\u003c/em\u003e alternative splicing\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. c-Myc binds to promoters of these three genes and regulates their expression\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In majority of cancers, high expression of c-Myc promotes the higher expression of these three hnRNPs. When present in abundance, these three proteins bind on the intronic region flanking exon 9 and inhibit the incorporation of exon 9 and as an effect, there is the inclusion of exon 10 of \u003cem\u003ePKM\u003c/em\u003e gene\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Molecular targeting of regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing by these three proteins might prove to be an effective strategy in eradicating the cancer cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe nuclear matrix is the site for many important nuclear events such as DNA replication, DNA repair, transcription and post-transcriptional modifications\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Post-transcriptional modifications include RNA splicing, RNA capping and poly-A tail addition. The nuclear matrix is a scaffold around which chromatin folds and many nuclear matrix binding proteins are involved in chromatin organization\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Apart from chromatin compaction, nuclear matrix binding proteins which are also known as the matrix-associated region (MAR) binding proteins (MARBPs), actively participate in the regulation of DNA replication, DNA repair, transcription and post-transcriptional modifications\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. One such MARBP is SMAR1 (Scaffold/Matrix attachment region binding protein 1)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The human homolog of SMAR1 is also known as BANP (BTG3 Associated Nuclear Protein). SMAR1 has been reported to play an important role as a tumor suppressor protein and in the majority of higher grades of cancer, SMAR1 has been reported to be dysregulated\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eSMAR1\u003c/em\u003e is located on human chromosome 16q24 and loss of heterozygosity (LOH) has been reported for this chromosomal region in various cancers\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSMAR1 has been reported to colocalize with SC35 which is a marker of nuclear splicing speckles and an important regulator of alternative splicing\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, SMAR1 has an RS domain that facilitates the RNA binding and it interacts with snRNAs which are the core components of splicing machinery\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. SMAR1 has been reported to regulate splicing of \u003cem\u003eCD44\u003c/em\u003e variants by deacetylation of Sam68 with help of HDAC6 in breast cancer cell lines. In addition to \u003cem\u003eCD44\u003c/em\u003e variants, SMAR1 also regulates alternative splicing of \u003cem\u003eFAS\u003c/em\u003e ligand\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. ChIP-sequencing study of SMAR1 in HCT116 cells suggests that there are several global gene targets of SMAR1 which are involved in the regulation of RNA processing and alternative splicing\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. This suggests that SMAR1 might be involved in alternative splicing regulation of other cancer-associated genes. This study focuses on the role of SMAR1 in the regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing via HDAC6 dependent deacetylation of PTBP1 and its implication in inhibition of the Warburg effect and tumorigenesis. Our study thus demonstrates the inhibition of cancer cell metabolism and breast cancer progression by SMAR1 via suppression of oncogenic isoform PKM2.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003ch2\u003eCell culture\u003c/h2\u003e\n\u003cp\u003eMCF7, MDA-MB-231, MDA-MB-468 and T47D cells were obtained from the NCCS cell repository, Pune, India. MCF7, MDA-MB-231 and MDA-MB-468 were cultured in DMEM (Gibco) and T47D was cultured in RPMI (Gibco). All the cell lines were supplemented with 10% Fetal Bovine Serum (FBS) (Gibco) and 100 units/ml Penicillin and Streptomycin (Gibco) and incubated in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026ordm;C.\u003c/p\u003e\n\u003ch2\u003ePlasmids, siRNAs and shRNA constructs\u003c/h2\u003e\n\u003cp\u003e3xFlag26-SMAR1 was used to over-express SMAR1 and 3xFlag26-Vector was used as a control. \u003cem\u003eSilencer\u003c/em\u003e\u0026trade; Select Negative Control No. 1 siRNA (4390843) and si-RNA against human-SMAR1 (BANP) (s29889) were obtained from Thermo Fischer Scientific Silencer\u0026reg; select siRNA range. shSMAR1-eGFP (ULTRA-3344235) was obtained from TransOMIC and used for shRNA-mediated SMAR1 knockdown. shNon translated-1-eGFP (shNT1) (TLNSU1420) was also obtained from TransOMIC and used as a control for all shRNA-mediated knockdown studies. shHDAC6 clone TRCN0000004839 was used for HDAC6 knockdown\u003csup\u003e28\u003c/sup\u003e. shPKM2 was cloned in pLKO.1-TRC vector and the sequence targeting only PKM2 were obtained from Cort\u0026eacute;s-Cros et al.\u003csup\u003e30\u003c/sup\u003e. The sequence of shPKM2 used:\u003c/p\u003e\n\u003cp\u003e5\u0026prime;CCGGCTACCACTTGCAATTATTTGACTCGAGTCAAATAATTGCAAGTGGTAGTTTTTG3\u0026prime;.\u003c/p\u003e\n\u003ch2\u003eTransfections and treatments\u003c/h2\u003e\n\u003cp\u003eTransfections of various plasmids were done by the use of Polyethylenimine (PEI MAX 40000) (Polysciences, Inc.). In MCF7, transfection was performed at 70-80% confluency and the DNA:PEI ratio used for transfection was 1:3. In MDA-MB-231, reverse transfection was performed in which DNA: PEI mix was added before cell seeding. Tubacin (Sigma), a selective HDAC6 inhibitor, was used at 5 \u0026mu;M concentration for 5 hrs for optimum HDAC6 inhibition and an equal amount of DMSO was used as vehicle control.\u003c/p\u003e\n\u003ch2\u003eCloning of dual reporter \u003cem\u003ePKM\u003c/em\u003e minigene system\u003c/h2\u003e\n\u003cp\u003eTo develop a dual chromatic \u003cem\u003ePKM\u003c/em\u003e minigene system, eGFP was cloned into mCherry-N1 vector between SalI (NEB) and AgeI (NEB) restriction sites in such a way that both eGFP and mCherry remain in two different frames due to difference in one base pair and eGFP contains stop-codon at the end when in the frame. Further, Exon 8 - Exon 11 of the \u003cem\u003ePKM\u003c/em\u003e gene along with introns was amplified from \u003cem\u003ePKM\u003c/em\u003e minigene construct\u003csup\u003e31\u003c/sup\u003e (a kind gift from Dr. Adrian R. Krainer) using Platinum\u0026trade; SuperFi\u0026trade; DNA Polymerase (Invitrogen) and cloned into this vector between XhoI (NEB) and NdeI (NEB) sites by Infusion cloning (NEB). One base pair insertion was further introduced in the cloned plasmid in Exon 10 by Infusion cloning. Due to one base pair insertion, the incorporation of Exon 9 resulted in the expression of mCherry and the incorporation of Exon 10 led to the expression of eGFP. The expressions of mCherry and eGFP were analyzed by confocal microscopy.\u003c/p\u003e\n\u003ch2\u003eDual reporter\u003cem\u003e PKM\u003c/em\u003e minigene assay\u003c/h2\u003e\n\u003cp\u003eMCF7 cells were seeded onto a glass coverslip and after 24 hrs \u003cem\u003ePKM\u003c/em\u003e minigene along with control or Flag-SMAR1 in a 1:1 ratio was introduced in these cells by PEI mediated transfection. After 48 hrs of transfection, cells were fixed with 4% paraformaldehyde for 10 mins at room temperature. Subsequently, cells were washed with 1X PBS thrice and the coverslips were mounted in fluoroshield media (Sigma) with DAPI. Cells were observed at 60X magnification using Nikon A1plus confocal microscope and images were acquired using Nikon\u0026rsquo;s NIS-elements imaging software. For eGFP and mCherry fluorescent intensity quantification, ImageJ software was used. Five independent regions of interest (ROIs) were selected per image covering cells expressing eGFP and mCherry and fluorescence intensity density was measured. Average relative fluorescence densities were calculated for all five ROIs per field and the fold change in eGFP/mCherry ratio was calculated. For statistical significance, three random fields per sample were selected and three independent experiments were performed. Mean \u0026plusmn; SD fold change in eGFP/mCherry ratio was calculated for each sample and compared with the control sample.\u003c/p\u003e\n\u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e\n\u003cp\u003eParaffinized human breast cancer patient samples along with surrounding normal breast tissue were obtained from Ruby Hall Clinic, Pune, India. SMAR1, PKM1 and PKM2 expression were detected using standard immunohistochemical staining procedure. Briefly, after deparaffinization, endogenous peroxidase blockage and rehydration with decreasing concentrations of ethanol (100%, 95% and 70%), antigen retrieval was performed by heating the slides in 10 mM sodium citrate buffer (pH 6-7). Further samples were incubated with the antibody of SMAR1 (Bethyl) at 1: 100 dilution, PKM1 (CST) at 1: 100 dilution and PKM2 (CST) 1: 300 dilution overnight at 4\u0026ordm;C. After incubation with HRP-conjugated secondary antibodies for 1 hr at room temperature, sections were then treated in DAB (3,3'-Diaminobenzidine) for 15 mins to allow the development of brown precipitate corresponding to the sites of HRP-bound antibodies. Samples were washed and counterstained with Hematoxylin for nuclei staining. Tissue sections were observed at 20X magnification using a Nikon microscope (Eclipse E600) and images were acquired using Nikon\u0026rsquo;s NIS-elements imaging software.\u003c/p\u003e\n\u003ch2\u003eQuantitative RT PCR\u003c/h2\u003e\n\u003cp\u003eTotal RNA was extracted from cultured MCF7 cells and MDA-MB-231 cells by TRizol\u003csup\u003eTM\u003c/sup\u003e (Invitrogen) according to the manufacturer\u0026rsquo;s instruction. RNA was reverse transcribed by MMLV-RT (Invitrogen) as per the manufacturer\u0026rsquo;s instructions. Amplification reactions were prepared in triplicate using iQTaq SYBR green (Biorad) and amplification was performed on Eppendorf realplex 2.0 according to the manufacturer\u0026rsquo;s instruction. The average cycle thresholds from three independent biological replicate samples were calculated as described in Singh et al. with slight modifications\u003csup\u003e32\u003c/sup\u003e. Briefly, the average cycle thresholds from three independent biological replicate samples were normalized to housekeeping control gene \u003cem\u003e18S rRNA\u003c/em\u003e. Normalization was performed using \u003cem\u003e18S rRNA\u003c/em\u003e as a normalization control using the formula: [2^ \u003csup\u003e(Ct control \u0026ndash; Ct target)\u003c/sup\u003e]. Along with control gene normalization, constitutive exon (exon 11) normalization was performed for \u003cem\u003ePKM1\u003c/em\u003e (Exon 8-9/9) and \u003cem\u003ePKM2\u003c/em\u003e (Exon 10-11/11) expression analysis. The student\u0026rsquo;s t-test was used to compare expression between two different groups. A list of primers used is given in Table S1.\u003c/p\u003e\n\u003ch2\u003eWestern blotting\u003c/h2\u003e\n\u003cp\u003eCells were incubated in TNN buffer [50 mM Tris-Cl pH 7.5, 5 mM EDTA, 0.5% NP40, 50 mM NaF, 1 mM DTT, 0.2 mM sodium orthovanadate, 0.5 mM PMSF, 150 mM NaCl and 1X Protease inhibitor cocktail (Thermo Scientific)] for cell lysis and lysates containing equal concentration of proteins were resolved using SDS-PAGE and transferred onto PVDF membrane. The membranes were incubated with primary antibodies such as anti-SMAR1 (Bethyl - A300-279A), Anti-PKM1 (CST- 7067), Anti-PKM2 (CST- 4053), Anti-hnRNP A1(CST- 8443), Anti-hnRNP A2 (Abcam- ab6102), Anti-PTBP1 (Thermo Scientific- 32-4800), Anti-HDAC6 (CST- 7558), Anti-HDAC1 (CST- 5356), Anti-GFP (Proteintech- 66002-1-Ig), Anti-mCherry (Proteintech- 26765-1-AP), Anti-\u0026beta;-actin (Sigma- A2228) and Anti-Flag tag (CST- 14793). This was followed by three washes and incubation with appropriate HRP conjugated secondary antibodies. Visualization was achieved with ECL substrate (Pierce) and exposure to X-ray films or imaging in Syngene G:BOX Chemi XRQ.\u003c/p\u003e\n\u003ch2\u003eAntibody cross-linking, Co-Immunoprecipitation and sequential Co-Immunoprecipitation\u003c/h2\u003e\n\u003cp\u003eThe majority of Immunoprecipitation (IP) experiments were done via covalently cross-linking the antibody with Protein G Dynabeads (Pierce) to avoid non-specific binding and contamination of immunoglobulin in the immunoprecipitated protein eluates. Approximately 1 \u0026micro;g of antibody was cross-linked with 10 \u0026micro;l of beads. Beads were washed thrice with ice-cold 1X PBS and further incubated with the desired antibody in IP buffer (1X PBS with 0.1% NP-40) containing protease inhibitor cocktail (Pierce). The antibody-bead mixture was incubated overnight at 4\u0026ordm;C. The unbound antibody was removed by three wash of the antibody-bead complex with IP buffer. Further, the antibody-bead complex was incubated with 500 \u0026mu;L of 10 mg/ml of Dimethyl pimelimidate (DMP) (Sigma) for 60 mins at room temperature with rotation. 50 \u0026mu;L of 1M Tris-Cl pH 8 was added to quench the reaction and incubated for 30 mins at room temperature with rotation. Unbound antibody was removed by washing it with 0.2 M Glycine pH 3 followed by three washes with IP buffer. The antibody-bead complex was further equilibrated with the IP buffer. These beads were either used immediately or stored at 4\u0026ordm;C for 2-3 days. Protein-bound to the antibody-bead complex was eluted at 95\u0026ordm;C by using SDS loading dye.\u003c/p\u003e\n\u003cp\u003eFor IP experiments, 500 \u0026micro;g of nuclear extracts were pre-cleared with control normal IgG (Sigma) bound dynabeads G and subsequently incubated for 12 hrs at 4\u0026ordm;C with dynabeads G crosslinked with the desired primary antibody. The protein associated bead complexes were washed thrice with IP buffer and then further eluted with SDS loading dye. The eluates were probed with indicated antibodies.\u003c/p\u003e\n\u003cp\u003eFor sequential IP experiments, nuclear extracts (1 mg) were immunoprecipitated first with 3 \u0026micro;g of anti-SMAR1 antibody. Before proceeding for the second IP, a minor fraction of the eluates was examined for the presence of HDAC6. Subsequently, the eluate was immunoprecipitated with 2 \u0026micro;g of anti-HDAC6. The final eluates were probed with anti-PTBP1 to evaluate the association.\u003c/p\u003e\n\u003ch2\u003eAnti-Acetyl-Lysine Acetylation Assay\u003c/h2\u003e\n\u003cp\u003eFor assessing the acetylation status of protein, 1 mg of nuclear extract was incubated with 2 \u0026mu;g of anti-acetyl-lysine antibody (CST- 9441) crosslinked with dynabeads G at 4\u0026ordm;C with slight mixing for 12 hrs. Immunocomplexes were washed thrice with 500 \u0026mu;L of IP buffer and further eluted at 95\u0026ordm;C with SDS loading dye. The eluates were loaded onto the SDS-PAGE and immunoblotted with indicated antibodies.\u003c/p\u003e\n\u003ch2\u003eUV-crosslinking and RNA immunoprecipitation (CLIP)\u003c/h2\u003e\n\u003cp\u003eCLIP experiment was done via covalently cross-linking the anti-PTBP1 antibody with Protein G Dynabeads (Thermo Scientific) as described in antibody cross-linking. Normal mouse IgG crosslinked beads were used as a negative control. Antibody cross-linked beads were further blocked for 1 hr at 4\u0026ordm;C with 100 nM yeast tRNA to avoid non-specific binding with RNA. Cells were washed with ice-cold 1X PBS, UV-irradiated (150 mJ/cm\u003csup\u003e2\u003c/sup\u003e) and harvested and lysed with lysis buffer (100 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM HEPES - pH 7.0, 0.5% NP40, 1 mM DTT, 100 units/ml RNase Out and Protease inhibitor cocktail) for 10 mins on ice. RNase A (1:1000 dilution) and DNase I (Invitrogen) was added to the lysate and incubated at 37\u0026ordm;C for 3 mins. 5% lysate was taken as input and TRizol LS\u003csup\u003eTM\u003c/sup\u003e (Invitrogen) was added for RNA extraction. An equal amount of protein (10 mg) was taken for control and SMAR1 knockdown sample and anti-PTBP1 and normal IgG conjugated beads were added for immunoprecipitation and incubated on rotation at 4\u0026ordm;C for 3 hrs. After two washes with wash buffer (50 mM Tris-HCl - pH 7.4, 150 mM NaCl, 1 mM MgCl\u003csub\u003e2, \u003c/sub\u003e0.05% NP40) supplemented with RNase inhibitor, an aliquot (10%) of beads was kept as control of immunoprecipitation while the rest was treated with 30 \u0026mu;g of Proteinase K and incubated for 1 hr at 55\u0026ordm;C. RNA was then extracted by TRizol LS\u003csup\u003eTM\u003c/sup\u003e (Invitrogen) and RNA was reverse transcribed by MMLV-RT (Invitrogen) as per the manufacturer\u0026rsquo;s instructions. Immunoprecipitated fractions and 5% input were analyzed by quantitative real-time PCR in duplicate using iQTaq SYBR green (Biorad) and amplification was performed on Eppendorf realplex 2.0 and specific primers for PTBP1 binding site on Intron 8 of \u003cem\u003ePKM\u003c/em\u003e were used (sequences mentioned in Table S1). Primer sequence for \u003cem\u003ePKM\u003c/em\u003e Intron 8 PTBP1 binding site was obtained from Chen et al.\u003csup\u003e13\u003c/sup\u003e. The experiment was performed three times and normalization was performed to input using the formula: [2^\u003csup\u003e (Ct input \u0026ndash; Ct immunoprecipitation)\u003c/sup\u003e]. Fold enrichment was calculated relative to normal mouse IgG control. Resultant fold enrichment was further normalized with densitometry measurement of a western blot for immunoprecipitated PTBP1 protein samples. The student\u0026rsquo;s t-test was used to identify the significance between two different groups.\u003c/p\u003e\n\u003ch2\u003eGlucose Assay\u003c/h2\u003e\n\u003cp\u003eMCF7 cells were transfected with respective shRNA and control shRNA. shRNA transfected cells were selected with puromycin and an equal number of cells were plated in a 6 well plate. After 24 hrs, the cells were replenished with 10% FBS containing high glucose DMEM (without sodium pyruvate). After 24 hrs of media replenishment, media was collected and the amount of glucose was calculated with the use of a Glucose Assay Kit (Abcam, ab65333) as per the manufacturer\u0026rsquo;s protocol by colorimetric method. The amount of glucose present was normalized with the total amount of protein. The glucose utilization was calculated by subtracting the glucose level of samples from that of cell-free media. The percentage of glucose utilization was calculated compared to the control.\u003c/p\u003e\n\u003ch2\u003eLactate assay\u003c/h2\u003e\n\u003cp\u003eMCF7 cells were transfected with respective shRNA and control shRNA. shRNA transfected cells were selected with puromycin and an equal number of cells were plated in a 6 well plate. After 24 hrs, the cells were replenished with 10% FBS containing high glucose DMEM (without sodium pyruvate). After 24 hrs of media replenishment, media was collected and the amount of lactate was calculated with the use of a Lactate Assay Kit (Abcam, ab65331) as per the manufacturer\u0026rsquo;s protocol. The amount of lactate present in each sample was normalized with the total amount of protein. The percentage of lactate production was calculated for each sample compared to the control.\u003c/p\u003e\n\u003ch2\u003eGlucose (2-NBDG) uptake assay\u003c/h2\u003e\n\u003cp\u003eMCF7 cells were transfected with SMAR1 siRNA and control siRNA by Lipofectamine RNAiMax\u003csup\u003eTM\u003c/sup\u003e (Ambion) according to the manufacturer\u0026rsquo;s protocol. After 24 hrs of transfection, media was removed and replenished with 10% FBS containing DMEM (Without glucose and Sodium Pyruvate) and incubated at 37\u0026ordm;C for 1 hr. 10 \u0026mu;M fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) (Invitrogen) was added to culture media and cells were incubated for 1 hr at 37\u0026ordm;C. The 2-NBDG uptake reaction was stopped by removing the incubation medium and the cells were washed with ice-cold 1X PBS. 1 \u0026mu;g/ml Propidium Iodide (PI) was added to distinguish the viable cell population. For each measurement, data from 10,000 single-cell events were collected using FACS Canto II (BD Bioscience). The percentage of 2-NBDG uptake was calculated from mean fluorescence intensity (MFI) compared to the control.\u003c/p\u003e\n\u003ch2\u003eCell viability assay\u003c/h2\u003e\n\u003cp\u003eMCF7 cells were transfected with BORIS shRNA and shNT1 was used as a control in six-well culture plates. After the selection of transfected cells with puromycin, cells (4x10\u003csup\u003e3\u003c/sup\u003e) were seeded in 96 well culture plates and were cultured for 24 hrs, 48 hrs, and 72 hrs. Cell growth was determined by measuring the conversion of MTT Tetrazolium salt (Sigma) to MTT formazan. In brief, 50 \u0026micro;l of MTT stock solution (5 mg/ml) was added to each well along with 50 \u0026micro;l of 10% FBS containing DMEM and incubated for 4 hrs. After the incubation time, formazan crystals formed in the cells were solubilized in Isopropanol. The cell viability was measured by Spectramax M5 (Molecular Devices) at an optical density of 570 nm. The percentage proliferation was calculated and cell viability at 0 hr for each sample was considered as 100%. Compared to 0 hr, the percentage of proliferation was calculated for other time points.\u003c/p\u003e\n\u003ch2\u003eColony formation assay\u003c/h2\u003e\n\u003cp\u003eMCF7 cells were transfected with respective shRNAs and shNT1 was used as a control. The cells were selected with puromycin and 1x10\u003csup\u003e3\u003c/sup\u003e cells were seeded in the new 6-well plate. After 10 days, cells were fixed using methanol and acetic acid (3:1) for 5 mins. After fixation, cells were washed with 1X PBS thrice. After washing cells were stained with 0.05% crystal violet stain, images were taken and colonies formed were counted manually with help of ImageJ software. The percentage of colony formation was calculated for each sample compared to the control.\u003c/p\u003e\n\u003ch2\u003eTranswell migration and invasion assay\u003c/h2\u003e\n\u003cp\u003eTranswell chamber: 24-well, 8.0-\u0026mu;m pore membranes (Corning USA) were used according to the manufacturer\u0026rsquo;s protocol. MCF7 cells were transfected with respective shRNAs and shNT1 was used as a control. After the selection of transfected cells with puromycin, 1x10\u003csup\u003e5\u003c/sup\u003e cells per well were seeded in the upper chamber in a serum-free medium, and DMEM with 5% FBS was added to the lower chamber as a chemoattractant at the same time. After incubation of 24 hrs at 37\u0026ordm;C, the cells remaining at the upper surface of the membrane were removed with cotton swabs, and the cells on the lower surface of the membrane are the migrated cells. After fixation with 4% paraformaldehyde and staining with 0.5% crystal violet solution, the cells were observed at 10X magnification using the Nikon microscope (Eclipse Ti2) and images were acquired using Nikon\u0026rsquo;s NIS-elements imaging software.\u003c/p\u003e\n\u003cp\u003eThe transwell invasion assay was carried out as described above, except that a transwell chamber with Matrigel (Corning, USA) was used and they were pre-incubated at 37\u0026ordm;C after hydration with 100 \u0026mu;L of serum-free medium for 2 hrs before the cells were seeded onto the membrane, followed by incubation of 48 hrs at 37\u0026ordm;C. Five fields were randomly captured and the number of migrating/invading cells were quantified manually with the help of Image J software. The percentage of migration/invasion was calculated for each sample compared to the control.\u003c/p\u003e\n\u003ch2\u003eWound healing assay\u003c/h2\u003e\n\u003cp\u003eMCF7 cells were transfected with respective shRNAs and shNT1 was used as a control. The cells were selected with puromycin and 1.5x10\u003csup\u003e5\u003c/sup\u003e cells were seeded in each well of 12 well plate and allowed to grow at 37\u0026ordm;C to form a monolayer. Cells were synchronized by serum starvation for 12 hrs and scratch was introduced in the middle of the monolayer by sterile pipette tip, generating a cell-free area of approximately 1 mm in width and cell debris was removed by washing twice with 1X PBS. Three fields per well were imaged in the area where the wound was introduced at 0 hr 24 hrs. The area of the wound was measured by ImageJ for 0 hr and 24 hrs and the percentage of wound migration was calculated compared to that of the control. All images were taken at 10X magnification using the Nikon microscope (Eclipse Ti2) with help of Nikon\u0026rsquo;s NIS-elements imaging software.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eIn vivo \u003c/em\u003etumor generation\u003c/h2\u003e\n\u003cp\u003eAll mice used in this experiment were bred at the animal resource facility of NCCS, Pune, India. Standard protocols approved and monitored by the Institutional Animal Ethical Committee were followed for this experiment. The MDA-MB-231 cells were transduced with SMAR1-adenovirus and control-adenovirus. 1 million cells were injected subcutaneously in 6-8 weeks old NOD/SCID mice. After 1 month of injection, mice were sacrificed and tumors were dissected. The volume and weight of tumors were measured and parts of the tumor were utilized for western blot and immunohistochemistry as described in the above section.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eAll statistical analysis was performed using Microsoft Excel and graphs were plotted using GraphPad Prism7. Data has been represented as mean \u0026plusmn; SD. The student\u0026rsquo;s t-test was used to determine the statistical significance of the difference between the groups. The p-value of \u0026lt; 0.05 was considered significant. * p \u0026lt;0.05, ** p \u0026lt;0.01 and *** p \u0026lt;0.001. Images were analyzed and quantified using ImageJ software.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eSMAR1 exhibit a negative correlation with PKM2 and a positive correlation with PKM1 expression in breast cancer cells\u003c/h2\u003e\n\u003cp\u003eDysregulation of various tumor suppressor proteins is one of the key features of various cancers\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. SMAR1 being an important tumor suppressor protein is known to get downregulated in higher grades of breast and colon cancer\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. LOH of \u003cem\u003eSMAR1\u003c/em\u003e locus (human chromosome 16q24) has been reported in various cancer\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Moreover, Cdc20 mediated proteasomal degradation of SMAR1 has been studied in breast cancer cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In contrast, upregulation of key oncogenes such as PKM2 gives metabolic as well as tumorigenic advantage to the cancer cells. The majority of normal differentiated cells have a lower expression of PKM2 and in cancer cells, there is an enhanced expression of PKM2\u003csup\u003e34\u003c/sup\u003e. Expression of SMAR1, PKM2 and PKM1 was measured in breast cancer patient samples and compared with surrounding non-cancerous tissue by immunohistochemistry. IHC staining of SMAR1 revealed that its expression was diminished in tumor samples compared to that of normal control tissue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). These results correspond with the earlier studies, confirming the downregulation of SMAR1 in breast cancer samples\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, IHC staining of PKM isoforms revealed that PKM2 expression was elevated and PKM1 expression was significantly low in tumor samples compared to that of normal control tissue. These results further comply with earlier reports suggesting a switch in PKM isoform expression between normal tissue and cancer tissue leading to higher expression of oncogenic isoform PKM2 and suppression of PKM1\u003csup\u003e3,32\u003c/sup\u003e. Human breast cancer cells such as MDA-MB-231, MDA-MB-468 and T47D harbor LOH for \u003cem\u003eSMAR1\u003c/em\u003e locus, whereas MCF7 cells lack LOH for this locus\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. LOH harboring cells have significant downregulation of SMAR1 expression compared to that of MCF7\u003csup\u003e28\u003c/sup\u003e. Expression profile of SMAR1, PKM1 and PKM2 in these breast cancer cell lines MCF7, MDA-MB-231, MDA-MB-468 and T47D were found to be correlating with that of the patient samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results suggest that SMAR1 has an inverse correlation with PKM2 expression whereas it has a direct correlation with PKM1 expression. This indicates that SMAR1 might be promoting the expression of PKM1 and suppressing the expression of oncogenic isoform PKM2 in the non-cancerous tissue.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eSMAR1 regulates \u003cspan class=\"BoldItalic\"\u003ePKM\u003c/span\u003e alternative splicing by promoting the incorporation of exon 9 and suppressing the incorporation of exon 10\u003c/h2\u003e\n\u003cp\u003eSMAR1 is known to colocalize with one of the key splicing regulators SC35 in nuclear splicing speckles. It also interacts with snRNAs which are the core components of splicing machinery. Moreover, SMAR1 has been reported to regulate alternative splicing of \u003cem\u003eCD44\u003c/em\u003e variants and \u003cem\u003eFAS\u003c/em\u003e ligand\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. On the basis of an inverse correlation between SMAR1 and PKM2 expression as well as a positive correlation between SMAR1 and PKM1 observed in breast cancer, we hypothesized that SMAR1 might play a crucial role in regulating alternative splicing of the \u003cem\u003ePKM\u003c/em\u003e gene. Based on the expression profile of SMAR1 in various breast cancer cell lines, MCF7 and MDA-MB-231 were used for further experiments. To investigate the role of SMAR1 in the regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing, PKM isoform expression was measured upon shRNA-mediated depletion of SMAR1 expression in MCF7 cells at the RNA level by performing quantitative RT-PCR and at the protein level by western blot. Knockdown of SMAR1 in MCF7 resulted in PKM1 downregulation and increased PKM2 expression at the transcript level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Relative fold change in SMAR1 expression upon shRNA-mediated SMAR1 knockdown in MCF7 has been represented in Figure S1A. Further analysis of PKM isoform expression in SMAR1 depleted cells at the protein level suggests diminished expression of PKM1 and upregulation of PKM2 which was further in coherence with RNA expression analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, PKM isoform expression was quantified upon ectopic expression of SMAR1 in LOH-containing MDA-MB-231\u0026nbsp;at the RNA level by qRT-PCR and at the protein level by western blot. Upon Flag-SMAR1 overexpression in MDA-MB-231, PKM1 was observed to be increased and PKM2 was downregulated at the transcript level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Flag-SMAR1 mediated overexpression in MDA-MB-231\u0026nbsp;at transcript level has been represented in Figure S1B. Moreover, PKM isoform expression at the protein level in SMAR1 overexpressed cells was in concordance with that of transcript level analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results suggest that SMAR1 being a tumor suppressor protein, promotes the expression of PKM1 isoform over oncogenic isoform PKM2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further validate the role of SMAR1 in \u003cem\u003ePKM\u003c/em\u003e alternative splicing regulation, a dual reporter \u003cem\u003ePKM\u003c/em\u003e minigene system has been generated. Schematic representation of dual reporter \u003cem\u003ePKM\u003c/em\u003e minigene system has been described in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE. In this dual reporter \u003cem\u003ePKM\u003c/em\u003e minigene assay, incorporation of exon 10 leads to eGFP expression whereas incorporation of exon 9 leads to mCherry expression. Fluorescence imaging was done upon ectopic expression of SMAR1 along with the \u003cem\u003ePKM\u003c/em\u003e minigene system by confocal microscopy to measure eGFP and mCherry expression. Further relative fluorescence intensity was calculated and the fold change of eGFP/mCherry ratio has been calculated. Expression of SMAR1, eGFP and mCherry was confirmed by western blot (Figure S1C). The fold difference in eGFP/mCherry ratio in SMAR1 overexpression condition was reduced by 2-fold as compared to that of control which further validates its role in \u003cem\u003ePKM\u003c/em\u003e alternative splicing regulation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results suggest that SMAR1 actively promotes the incorporation of exon 9 and the exclusion of exon 10 leading to higher expression of PKM1 and repression of PKM2 expression in breast cancer cells.\u003c/p\u003e\n\u003ch2\u003eSMAR1 interacts with PTBP1 and SMAR1-HDAC6 makes a triple complex with PTBP1\u003c/h2\u003e\n\u003cp\u003ehnRNP A1, hnRNP A2 and PTBP1 are the three key regulators of \u003cem\u003ePKM\u003c/em\u003e alternative splicing\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. When these three proteins are present in higher concentration, they bind to intronic regions flanking exon 9 of \u003cem\u003ePKM\u003c/em\u003e which leads to the inhibition of incorporation of exon 9 while promoting the incorporation of exon 10, thereby resulting in higher expression of PKM2 and diminished expression of PKM1\u003csup\u003e13\u003c/sup\u003e. To delineate the detailed molecular mechanism of SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing, expression of hnRNP A1, hnRNP A2 and PTBP1 was measured upon shRNA-mediated knockdown of SMAR1 in MCF7. It was observed that there was no change in the expression of these three splicing regulators upon SMAR1 knockdown (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). This observation eliminates the possibility of transcriptional repression of hnRNP A1, hnRNP A2 and PTBP1 by SMAR1. To determine the role of SMAR1 in the regulation of post-translational modification, the interaction of SMAR1 with these hnRNPs was checked by co-immunoprecipitation (co-IP) and observed that SMAR1 interacts with PTBP1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Interaction of SMAR1 with PTBP1 was further confirmed by reverse co-IP of PTBP1 with SMAR1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). SMAR1 is known to regulate various cellular processes with the assistance of HDACs such as HDAC1 and HDAC6\u003csup\u003e20,22,28,35\u003c/sup\u003e. To identify which HDAC is involved in SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing, the interaction of PTBP1 with HDAC1 and HDAC6 was checked by co-IP which revealed that HDAC6 interacts with PTBP1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Interaction of HDAC6 with PTBP1 and SMAR1 was further confirmed by reverse co-IP of HDAC6 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). To further investigate the molecular interplay of SMAR1-HDAC6 interaction with PTBP1, sequential IP of SMAR1 and HDAC6 with PTBP1 was performed. Sequential IP experiment revealed that SMAR1-HDAC6 forms a triple complex (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results demonstrate that SMAR1 directly interacts with PTBP1 along with HDAC6 and the coexistence of SMAR1-HDAC6-PTBP1 as a ternary complex. This trimeric complex formation further indicates molecular dynamics between these proteins in SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eSMAR1 mediated regulation of \u003cspan class=\"BoldItalic\"\u003ePKM\u003c/span\u003e alternative splicing is HDAC6 dependent\u003c/h2\u003e\n\u003cp\u003eTo delineate the role of HDAC6 in SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing, expression of PKM isoforms was checked upon shRNA-mediated knockdown of HDAC6 by Western blot. Upon depletion of HDAC6, there was a downregulation in PKM1 expression and an increase in PKM2 expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). This indicates the involvement of HDAC6 in \u003cem\u003ePKM\u003c/em\u003e alternative splicing regulation. To further determine the role of HDAC6 in SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing, SMAR1 overexpressed cells were treated with a specific HDAC6 inhibitor (Tubacin) and PKM isoform expression was checked by western blot. Expression of PKM1 was high and PKM2 was downregulated in the SMAR1 overexpression condition compared to that of control. However, upon Tubacin treatment in SMAR1 overexpressed cells, the expression of PKM1 was reduced and the expression of PKM2 was increased compared to that of only SMAR1 overexpressed cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). This confirms that SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing is HDAC6 dependent process. Further validation of the role of HDAC6 in SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing was done by dual reporter \u003cem\u003ePKM\u003c/em\u003e minigene assay. Fold change in eGFP/mCherry ratio was measured upon SMAR1 overexpression along with Tubacin treatment in cells transfected with \u003cem\u003ePKM\u003c/em\u003e minigene system compared to the control condition. Fold change in eGFP/mCherry ratio was decreased upon ectopic expression of SMAR1 compared to the control. However, upon tubacin treatment in SMAR1 overexpressed cells resulted in a further increase in the fold change of eGFP/mCherry ratio (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). These observations confirm the role of HDAC6 in SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing. This demonstrates that HDAC6 is actively involved in \u003cem\u003ePKM\u003c/em\u003e alternative splicing regulation orchestrated by SMAR1.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eSMAR1-HDAC6 deacetylates PTBP1 and modulates its affinity to \u003cspan class=\"BoldItalic\"\u003ePKM\u003c/span\u003e pre-mRNA\u003c/h2\u003e\n\u003cp\u003ePost-translational modifications such as phosphorylation, acetylation, ubiquitination, sumoylation of splicing factors are known to regulate various alternative splicing events\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. SMAR1 is known to regulate the alternative splicing of \u003cem\u003eCD44\u003c/em\u003e variants by HDAC6 assisted deacetylation of Sam68\u003csup\u003e28\u003c/sup\u003e. Moreover, sirtuin-mediated deacetylation of hnRNP A1 has been reported to regulate alternative splicing of the \u003cem\u003ePKM\u003c/em\u003e gene\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To further delineate the role of SMAR1-HDAC6 in the deacetylation of PTBP1, the acetylation status of PTBP1 was checked upon shRNA-mediated depletion of SMAR1 by immunoprecipitation with an anti-acetyl-lysine antibody. In the SMAR1 knockdown condition, there was an increase in the acetylation status of PTBP1 compared to control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). This observation indicates that the SMAR1 level dictates the acetylation status of PTBP1 and it maintains PTBP1 in a deacetylated state. The acetylation status of PTBP1 was further estimated after SMAR1 overexpression along with Tubacin treatment. Ectopic expression of SMAR1 resulted in a decrease in acetylation status of PTBP1 but upon Tubacin treatment in SMAR1 overexpressed cells, the acetylation status of PTBP1 was restored (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). This suggests that SMAR1 maintains PTBP1 in the deacetylated state in HDAC6 dependent manner.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo inspect the effect of SMAR1-HDAC6 mediated deacetylation of PTBP1 on its affinity for \u003cem\u003ePKM\u003c/em\u003e pre-mRNA, UV-crosslinking and RNA immunoprecipitation (CLIP) of PTBP1 was performed upon shRNA-mediated depletion of SMAR1 in MCF7. Enrichment of PTBP1 on Intron 8 of \u003cem\u003ePKM\u003c/em\u003e pre-mRNA was enhanced by 2-fold in the case of SMAR1 knockdown condition as compared to that of control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Immunoprecipitation of PTBP1 in the CLIP experiment was confirmed by western blot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Increased enrichment of PTBP1 on \u003cem\u003ePKM\u003c/em\u003e pre-mRNA in SMAR1 knockdown condition suggests that SMAR1-HDAC6 mediated deacetylation of PTBP1 leads to a reduction in its affinity for \u003cem\u003ePKM\u003c/em\u003e pre-mRNA and ultimately modulates \u003cem\u003ePKM\u003c/em\u003e alternative splicing.\u003c/p\u003e\n\u003ch2\u003eSMAR1 regulates the Warburg effect and breast cancer growth via regulation of PKM2 expression\u003c/h2\u003e\n\u003cp\u003eHigher expression of PKM2 compared to PKM1 is one of the key factors for cancer cells in achieving the metabolic advantage of the Warburg effect compared to normal cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Reduction in PKM2 expression might lead to inhibition of the Warburg effect and ultimately suppress the tumorigenic potential of cancer cells. SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing leads to suppression of PKM2 and increased expression of PKM1. Based on this observation, we hypothesized that SMAR1 might be playing important role in the regulation of cancer cell metabolism. To determine the role of SMAR1 in the regulation of tumor metabolism, glucose utilization was estimated upon shRNA-mediated decrease of SMAR1 expression along with PKM2 in MCF7. Depletion of PKM2 by shRNA was confirmed by western blot (Figure S2A). A reduction in SMAR1 expression resulted in increased glucose utilization. Further depletion of PKM2 in SMAR1 knockdown cells resulted in decreased glucose utilization (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Expression of SMAR1 and PKM isoforms upon shRNA-mediated knockdown of SMAR1 along with PKM2 depletion was confirmed by western blot (Figure S2B). 2-NBDG mediated glucose uptake upon siRNA-mediated knockdown of SMAR1 validates its role in the regulation of glucose metabolism (Figure S2C). siRNA-mediated knockdown of SMAR1 in MCF7 was confirmed by western blot (Figure S2D). To further decipher the role of SMAR1 in the reversal of the Warburg effect, lactate production was measured in SMAR1 knockdown condition along with PKM2 depletion. SMAR1 depletion condition revealed enhanced lactate production compared to control. Moreover, PKM2 knockdown in SMAR1 depleted cells were having decreased lactate production compared to SMAR1 depleted cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). This suggests that SMAR1 mediated regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing inhibits the Warburg effect via suppression of PKM2. These observations reveal the important regulatory role of SMAR1 in keeping cellular metabolism in check via regulation of PKM isoform expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePKM2 mediated increase in glucose uptake and lactate production provides a metabolic advantage to cancer cells leading to increased tumor growth\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For tumor cells, reprogramming of glucose metabolism and higher glucose utilization are essential for their proliferation and survival\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The proliferative potential of cancer cells upon shRNA-mediated depletion of SMAR1 along with PKM2 in MCF7 was assessed by proliferation assay and colony formation assay. In the SMAR1 knockdown condition proliferation rate and colony formation potential were increased which were further reduced due to PKM2 depletion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC and D). These results suggest that SMAR1 inhibits breast cancer growth via reversal of the Warburg effect by suppressing PKM2 expression. Increased lactate production by cancer cells creates acidic surroundings which contribute to the tumorigenic properties of cancer cells such as migration, invasion and metastasis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Further, the tumorigenic potential of cancer cells upon depletion of SMAR1 along with PKM2 knockdown was measured by \u003cem\u003ein vitro\u003c/em\u003e functional assays such as transwell migration, invasion and wound healing assays. SMAR1 knockdown resulted in an increase in migration, invasion and wound healing properties of MCF7. Knockdown of PKM2 in SMAR1 depleted cells showed a decrease in migration, invasion and wound healing ability of cancer cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE-G). These observations demonstrate that SMAR1 inhibits the tumorigenic potential of cancer cells due to the inhibition of the cancer cell metabolism through the downregulation of PKM2.\u003c/p\u003e\n\u003ch2\u003eSMAR1 regulates \u003cspan class=\"BoldItalic\"\u003ein vivo\u003c/span\u003e tumor formation via regulation of PKM2 expression\u003c/h2\u003e\n\u003cp\u003ePKM2 contributes to \u003cem\u003ein vivo\u003c/em\u003e tumor generation and its progression via regulation of cancer cell metabolism\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. To identify the role of SMAR1 in tumor formation, tumor xenografts of SMAR1 overexpressed cells were generated by injection of SMAR1-adenovirus transduced MDA-MB-231 cells in NOD/SCID mice. The tumor burden was compared among SMAR1 overexpressing MDA-MB-231 tumors to that of normal MDA-MB-231 control tumors. In SMAR1 overexpressing group, tumor burden was less compared to that of the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Moreover, in the SMAR1 overexpression condition, there was a significant reduction in the weight and volume of the tumor compared to control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB and C). These observations indicate that SMAR1 suppresses \u003cem\u003ein vivo\u003c/em\u003e tumor generation. A quantitative expression of PKM1 and PKM2 in these xenograft tumor samples was checked by western blot. In adeno-SMAR1 over-expressed tumor samples expression of tumorigenic isoform PKM2 was reduced and PKM1 expression was increased compared to control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). IHC staining of PKM isoforms in these \u003cem\u003ein vivo\u003c/em\u003e tumor samples were observed to be correlating with western blot results suggesting higher expression of PKM1 isoform and depleted expression of PKM2 isoform in SMAR1 overexpressing tumors compared to that of control tumors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE). These observations substantiate that SMAR1 suppresses \u003cem\u003ein vivo\u003c/em\u003e tumor formation via the regulation of PKM isoform expression.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eHere we describe the detailed mechanism of \u003cem\u003ePKM\u003c/em\u003e alternative splicing regulation by one of the key tumor suppressor proteins, SMAR1 and its implications in inhibition of cancer cell metabolism and tumorigenesis. SMAR1 has been reported to co-localize with key splicing factor SC35 and it interacts with splicing regulator Sam68\u003csup\u003e28\u003c/sup\u003e. SMAR1 also contains the RS domain which facilitates its interactions with various snRNAs which are the core components of splicing machinery\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Furthermore, SMAR1 regulates alternative splicing of \u003cem\u003eCD44\u003c/em\u003e variants and \u003cem\u003eFAS\u003c/em\u003e ligand\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This indicates the active involvement of SMAR1 in various alternative splicing events. Our results demonstrate that SMAR1 follows a positive correlation with PKM1 and a negative correlation with oncogenic isoform PKM2 in breast cancer patient samples as well as in breast cancer cell lines. Further experimental approaches have revealed that SMAR1 is actively involved in the regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing. SMAR1 promotes the inclusion of exon 9 and exclusion of exon 10 thus inhibiting the expression of PKM2 which is a tumorigenic isoform of the \u003cem\u003ePKM\u003c/em\u003e gene. These observations were validated by dual chromatic \u003cem\u003ePKM\u003c/em\u003e minigene assay, which further confirms the regulatory role of SMAR1 in dictating the outcome of \u003cem\u003ePKM\u003c/em\u003e alternative splicing. The downregulation of SMAR1 in breast cancer is one of the reasons for enhanced PKM2 expression that leads to altered glucose metabolism and contributes to cancer cell growth.\u003c/p\u003e \u003cp\u003eNuclear matrix binding protein, SMAR1 is known to perform various biological functions with help of regulatory proteins such as HDACs\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, various HDACs have been reported to participate in the regulation of alternative splicing through modulation of the acetylation status of histone and non-histone proteins including splicing factors\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Earlier studies suggest that SMAR1 inhibits the expression of various genes such as \u003cem\u003eCyclin D1\u003c/em\u003e, \u003cem\u003eBAX\u003c/em\u003e and \u003cem\u003ePUMA\u003c/em\u003e by recruiting the repressor complex of HDAC1-mSin3a on the promoter and keeping it in a repressed state by epigenetic modulations\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Moreover, SMAR1 also plays a crucial role in deacetylating target proteins such as Ku70 and Sam68 with the help of HDAC6\u003csup\u003e28,35\u003c/sup\u003e. This points to the crucial role of SMAR1 in bringing about the post-translational regulation of its target proteins and ultimately dictating their role in various molecular and biological processes. SMAR1-HDAC6 mediated deacetylation of Ku70 dictates cell fate upon exposure to ionizing radiation via regulation of DNA repair and apoptosis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. SMAR1 further inhibits metastasis via regulation of \u003cem\u003eCD44\u003c/em\u003e variants alternative splicing via HDAC6 mediated deacetylation of Sam68 in breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMany post-translational modifications such as phosphorylation, acetylation, ubiquitination, and sumoylation of splicing regulators have been associated with the regulation of various alternative splicing events\u003csup\u003e28,36\u0026minus;40\u003c/sup\u003e. Previous studies involving \u003cem\u003ePKM\u003c/em\u003e alternative splicing suggest that three important splicing inhibitors hnRNP A1, hnRNP A2 and PTBP1 regulate \u003cem\u003ePKM\u003c/em\u003e alternative splicing\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Moreover, Sirtuin mediated deacetylation of hnRNP A1 has been reported to regulate alternative splicing of \u003cem\u003ePKM\u003c/em\u003e gene\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This suggests that the acetylation status of splicing factors plays an important role in the regulation of alternative splicing events\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Herein, we showed that SMAR1 mediated \u003cem\u003ePKM\u003c/em\u003e alternative splicing regulation is HDAC6 dependent and SMAR1-HDAC6 forms a triple complex with PTBP1 and maintains it in the deacetylated form. CLIP experiment of PTBP1 in SMAR1 depleted condition resulted in the increased binding of PTBP1 on Intron 8 of \u003cem\u003ePKM\u003c/em\u003e pre-mRNA. This indicates that the binding affinity of PTBP1 is more on \u003cem\u003ePKM\u003c/em\u003e pre-mRNA in absence of SMAR1. Based on this observation it can be further implied that the deacetylation of PTBP1 by SMAR1-HDAC6 results in the lower affinity of PTBP1 for \u003cem\u003ePKM\u003c/em\u003e pre-mRNA and thus leading to the inclusion of exon 9 and exclusion of exon 10. This leads to higher expression of PKM1 and lower expression of tumorigenic isoform PKM2. Our findings suggest that SMAR1 brings about HDAC6 mediated deacetylation of PTBP1 and thus maintains PTBP1 in deacetylated form. This deacetylated PTBP1 ultimately leads to the regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing promoting expression of PKM1 and repressing oncogenic isoform PKM2.\u003c/p\u003e \u003cp\u003eHighly proliferating cancer cells gain a metabolic advantage over normal cells via higher expression of PKM2 and exhibits peculiar metabolic properties known as the Warburg effect\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Glucose is a primary source of energy and anabolic demands for highly proliferating cancer cells\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Moreover, a higher amount of lactate production creates an acidic micro-environment in tumors which favors invasion and metastasis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Our study demonstrates that SMAR1 mediated suppression of PKM2 isoform and upregulation of PKM1 isoform results in inhibition of cancer cell metabolism. SMAR1 inhibits glucose utilization and lactate production via regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing. Our results demonstrate that SMAR1 mediated reversal of the Warburg effect leads to inhibition of cancer cell proliferation, migration and invasion. Moreover, SMAR1 suppresses \u003cem\u003ein vivo\u003c/em\u003e tumor formation via the regulation of PKM isoform expression. This demonstrates the tumor suppressor function of key nuclear matrix binding protein SMAR1 and its importance in the maintenance of metabolic equilibrium. Future studies on the role of SMAR1 in global alternative splicing regulation might give more insight into the active involvement of tumor suppressor proteins in cancer-associated alternative splicing regulation and its implication in various cellular processes.\u003c/p\u003e \u003cp\u003eIn higher grades of cancer, downregulation of SMAR1 expression has been correlated with an increase in tumorigenic potential\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. SMAR1 expression and functions are also known to be modulated by various cancer-associated signaling pathways including JNK signaling, Wnt signaling, ERK-MAPK pathway\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. SMAR1 gets highly dysregulated in Wnt signaling associated colorectal cancer (CRC)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Moreover, activation of ERK-MAPK signaling leads to translocation of SMAR1 to the cytoplasm, resulting in increased \u003cem\u003eCD44\u003c/em\u003e variants alternative splicing and metastasis in breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. A study in the breast cancer model revealed that SMAR1 undergoes proteasomal degradation by Cdc20 in a JNK kinase-dependent manner\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Furthermore, microbial peptides that promote SMAR1 stabilization have been correlated with inhibition of Wnt/β-catenin activities in CRC\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. A study involving stabilization of SMAR1 expression by treatment with isothiocyanate derivative has been co-related with anti-HIV activity\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Our findings reveal that SMAR1 knockdown promotes the Warburg effect and tumorigenic potential of breast cancer cells by modulating PKM isoform expression indicating an important regulatory role of tumor suppressor protein in cellular homeostasis. This indicates that future studies targeting cancer cell metabolism and cancer progression via stabilization of SMAR1 expression might lead to better therapeutic efficacy in cancer treatment.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eOur study in the breast cancer model highlights that nuclear matrix binding protein SMAR1 regulates \u003cem\u003ePKM\u003c/em\u003e alternative splicing and inhibits the expression of PKM2 and promotes the expression of PKM1. SMAR1 regulates \u003cem\u003ePKM\u003c/em\u003e alternative splicing via deacetylation of a key alternative splicing inhibitor PTBP1 in an HDAC6 dependent manner. Further, \u003cem\u003ein vitro\u003c/em\u003e enzymatic assays, functional assays and \u003cem\u003ein vivo\u003c/em\u003e tumor model suggest that SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells by suppressing the expression of tumor-associated isoform PKM2 and promoting the expression of enzymatically more efficient PKM1 isoform.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eATP: Adenosine triphosphate\u003c/p\u003e\n\u003cp\u003eCLIP: UV-crosslinking and RNA immunoprecipitation\u003c/p\u003e\n\u003cp\u003eCRC: colorectal cancer\u003c/p\u003e\n\u003cp\u003eHDAC6: Histone deacetylase 6\u003c/p\u003e\n\u003cp\u003ehnRNP: Heterogeneous nuclear ribonucleoprotein\u003c/p\u003e\n\u003cp\u003eIP: Immunoprecipitation\u003c/p\u003e\n\u003cp\u003eMAR: Matrix associated region\u003c/p\u003e\n\u003cp\u003eMARBP: Matrix associated region binding protein\u003c/p\u003e\n\u003cp\u003ePKM: Pyruvate Kinase Muscle\u003c/p\u003e\n\u003cp\u003ePTBP1: Polypyrimidine tract-binding protein 1\u003c/p\u003e\n\u003cp\u003eSMAR1: Scaffold-matrix associated region binding protein 1\u003c/p\u003e\n\u003cp\u003eTCA: Tricarboxylic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eAll the experiments involving the usage of animals were conducted following CPCSEA guidelines approved by the Institutional Animal Ethics Committee.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe authors are thankful to the Council of Scientific \u0026amp; Industrial Research (CSIR), India; University Grants Commission (UGC), India; National Centre for Cell Science (NCCS), Pune, India and Department of Biotechnology (DBT), India for their fellowships. We would also like to acknowledge the DBT, India; Department of Science and Technology (DST), India and J. C. Bose fellowship-SERB (Science and Engineering Research Board), India to SC, for financial support.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eAC and SC conceptualized, planned and designed the experiments. AC, AP, RP, VS, RN, PF performed experiments. AC wrote the manuscript. AC, AP, RP, VS, RN, PF, SmS, SaS, GCK and SC provided intellectual inputs and helped in preparing the manuscript. GCK helped in obtaining patient samples. SaS and SmS conceptualized the dual chromatic \u003cem\u003ePKM\u003c/em\u003e minigene system and AC cloned the dual chromatic \u003cem\u003ePKM\u003c/em\u003e minigene system.\u003c/p\u003e\n\u003ch2\u003eAcknowledgment\u003c/h2\u003e\n\u003cp\u003eWe are thankful to the National Centre for Cell Science (NCCS), Pune, India for providing infrastructure and experimental facilities. We would like to acknowledge Dr. Marta Cort\u0026eacute;s-Cros for providing us sequence information of shPKM2. We would like to thank Dr. Adrian R. Krainer for providing the \u003cem\u003ePKM\u003c/em\u003e minigene construct which we used to design dual reporter \u003cem\u003ePKM\u003c/em\u003e minigene.\u003c/p\u003e\n\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eO. Warburg. On the origin of cancer cells. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e123\u003c/strong\u003e, 309\u0026ndash;314 (1956).\u003c/li\u003e\n\u003cli\u003eHeiden, M. G. V., Cantley, L. C. \u0026amp; Thompson, C. B. 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Emerging roles of histone modifications and HDACs in RNA splicing. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 4911\u0026ndash;4926 (2019).\u003c/li\u003e\n\u003cli\u003eTrivedi, J. \u003cem\u003eet al.\u003c/em\u003e A novel isothiocyanate derivative inhibits HIV-1 gene expression and replication by modulating the nuclear matrix associated protein SMAR1. \u003cem\u003eAntiviral Res.\u003c/em\u003e \u003cstrong\u003e173\u003c/strong\u003e, 104648 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-and-metabolism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmet","sideBox":"Learn more about [Cancer \u0026 Metabolism](http://cancerandmetabolism.biomedcentral.com/)","snPcode":"40170","submissionUrl":"https://submission.nature.com/new-submission/40170/3","title":"Cancer \u0026 Metabolism","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SMAR1, PKM1, PKM2, HDAC6, PTBP1, alternative splicing, Warburg effect","lastPublishedDoi":"10.21203/rs.3.rs-151525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-151525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground:\u003c/p\u003e\u003cp\u003eHighly proliferating cancer cells exhibit the Warburg effect by regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing and inhibition of the Warburg effect by SMAR1.\u003c/p\u003e\u003cp\u003eMethods:\u003c/p\u003e\u003cp\u003eImmunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on \u003cem\u003ePKM\u003c/em\u003e pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by \u003cem\u003ein vitro\u003c/em\u003e enzymatic assays and functional assays upon SMAR1 knockdown. Besides, \u003cem\u003ein vivo \u003c/em\u003etumor formation by injecting adeno-SMAR1 transduced MDA-MB-231 cells in NOD/SCID mice was performed. \u003c/p\u003e\u003cp\u003eResults:\u003c/p\u003e\u003cp\u003eThe expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on \u003cem\u003ePKM\u003c/em\u003e pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells and \u003cem\u003ein vivo \u003c/em\u003etumor generation in PKM2 dependent manner.\u003c/p\u003e\u003cp\u003eConclusions:\u003c/p\u003e\u003cp\u003eSMAR1 regulates \u003cem\u003ePKM\u003c/em\u003e alternative splicing by causing HDAC6 dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on \u003cem\u003ePKM\u003c/em\u003e pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of \u003cem\u003ePKM\u003c/em\u003e alternative splicing.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Tumor Suppressor SMAR1 Regulates PKM Alternative Splicing by HDAC6 Mediated Deacetylation of PTBP1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-01 18:03:17","doi":"10.21203/rs.3.rs-151525/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-02-09T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Minor revision","date":"2021-02-09T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-08T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-01-31T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-01-27T00:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-01-26T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-01-19T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-01-18T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-01-18T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2021-01-18T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-and-metabolism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmet","sideBox":"Learn more about [Cancer \u0026 Metabolism](http://cancerandmetabolism.biomedcentral.com/)","snPcode":"40170","submissionUrl":"https://submission.nature.com/new-submission/40170/3","title":"Cancer \u0026 Metabolism","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b06c34e3-92f6-4663-b26d-4d5d183774cb","owner":[],"postedDate":"February 1st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":2174513,"name":"Cancer Biology"},{"id":2174514,"name":"Cellular Metabolism"}],"tags":[],"updatedAt":"2021-08-18T19:34:35+00:00","versionOfRecord":{"articleIdentity":"rs-151525","link":"https://doi.org/10.1186/s40170-021-00252-x","journal":{"identity":"cancer-and-metabolism","isVorOnly":false,"title":"Cancer \u0026 Metabolism"},"publishedOn":"2021-04-16 19:06:31","publishedOnDateReadable":"April 16th, 2021"},"versionCreatedAt":"2021-02-01 18:03:17","video":"","vorDoi":"10.1186/s40170-021-00252-x","vorDoiUrl":"https://doi.org/10.1186/s40170-021-00252-x","workflowStages":[]},"version":"v1","identity":"rs-151525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-151525","identity":"rs-151525","version":["v1"]},"buildId":"369fNeqWncA4NS6XSWjrt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}