LncRNA PVT-1 promotes osteosarcoma cancer stem-like properties through direct interaction with TRIM28 and TSC2 ubiquitination

LncRNA PVT-1 promotes osteosarcoma cancer stem-like properties through direct interaction with TRIM28 and TSC2 ubiquitination | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article LncRNA PVT-1 promotes osteosarcoma cancer stem-like properties through direct interaction with TRIM28 and TSC2 ubiquitination Jason Yustein, Susan Tsang, Nino Rainusso, Motonari Nomura, Tajhal Patel, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1813153/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Nov, 2022 Read the published version in Oncogene → Version 1 posted 7 You are reading this latest preprint version Abstract Osteosarcoma, the most common pediatric bone tumor, is an aggressive heterogeneous malignancy defined by complex chromosomal aberrations. Overall survival rates remain at approximately 70%, but patients with chemoresistant or metastatic disease have extremely poor outcomes of less than 30%. A subgroup of tumors harbor amplification of chromosome 8q24.2 and increased expression of the oncogenic long noncoding RNA (lncRNA) Plasmacytoma Variant Translocation-1 (PVT-1), which is associated with an extremely poor clinical prognosis. Through in vitro assays, this study demonstrated that PVT-1 is critical for osteosarcoma tumor-initiation potential. Chromatin Hybridization by RNA Purification analysis identified Tripartite-Motif Containing Family 28 (TRIM28) as a novel PVT-1 binding partner. Mechanistically, co-immunoprecipitation studies showed the PVT-1/TRIM28 complex binds and increases SUMOylation of phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34), which leads to enhanced ubiquitination and degradation of tumor suppressor complex 2 (TSC2), thus contributing to stem cell phenotypes. Importantly, osteosarcoma cells with increased PVT-1 have enhanced sensitivity to a SUMOylation inhibitor, TAK-981. Altogether, this study elucidated a role for PVT-1 in the enhancement of cancer stem-like behaviors, including migration and invasion, in osteosarcoma, and identified the novel PVT-1/TRIM28 axis signaling cascade as a potential therapeutic target for osteosarcoma treatment. lncRNA PVT-1 cancer-stem cells osteosarcoma and TRIM28 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Osteosarcoma is the most common primary bone tumor in pediatric patients (1). Like many malignancies, osteosarcoma is comprised of a heterogeneous population of cells, including a subpopulation of tumor-initiating cells, also known as cancer stem cells, which have the ability to self-renew and drive tumorigenesis (2, 3). In the osteosarcoma field, no well-established cell surface markers exist to identify osteosarcoma stem cells; however, the field strongly relies on the use of characterization through stem cell marker profiling (i.e., Sox2, Oct4, Nanog, and MYC) and the ability of stem cells to form sarcospheres under specific cancer stem cell culture conditions (2, 4, 5). As cancer stem cells play an important role in sustaining cancer growth, therapy resistance and recurrence, there is a critical need to identify cancer stem cell drivers with the goal of developing more effective treatments for osteosarcoma patients (2, 6). A genetic anomaly of osteosarcoma is its high extent of recurrent chromosomal aneuploidy (7, 8). Approximately 30% of osteosarcoma patients present with 8q24.2 amplification, which is associated with poor clinical prognosis (9). Within the 8q24.2 amplicon resides c-MYC (from here on referred to as MYC ) oncogene and PVT-1 (10, 11). As lncRNAs are involved in tumor proliferation and metastasis in other cancer types, the aim of this study was to characterize the role of PVT-1 in osteosarcoma. LncRNAs ability to control different forms of gene regulation impacts multiple tumorigenic behaviors (12,13). For instance, oncogenic lncRNA CDKN2B-AS1 promotes renal cell carcinoma progression by its interaction with insulin-like growth factor 2 mRNA-binding protein 3, which leads to the epigenetic activation of the Ndc80 kinetochore complex (14). While lncRNA, MALAT1 can enhance breast cancer through the regulation of alternative mRNA splicing (14). Previous reports revealed that PVT-1 promotes tumorigenesis and interacts with microRNAs in several cancers (10,15-18). However, studies regarding the role of PVT-1 in osteosarcoma are limited, and no associations with self-renewal or identification of therapeutic vulnerabilities secondary to enhanced PVT-1 expression and perturbations of downstream signaling cascades have been reported. With the use of patient-derived xenografts (PDXs) and established OS cell line models, PVT-1 was found to initiate self-renewal properties and enhance tumorigenic behaviors. Through transcriptomic and proteomic analysis, the increased expression of PVT-1 was demonstrated to enhance the expression of stem cell genes. The comprehensive identification of RNA binding proteins (ChIRP) was applied to identify the endogenous PVT-1 interactome, which revealed TRIM28 as a novel PVT-1 binding partner in 8q24.2-amplified osteosarcoma model. PVT-1/ TRIM28 was determined to inhibit TSC2 through ubiquitination resulting in subsequent proteasomal degradation. In addition, TSC2 rescue experiments resulted in reversal of PVT-1 -induced cancer stem cell capacity. To elucidate the intermediates between PVT-1 /TRIM28 and TSC2 signaling pathway, a co-immunoprecipitation assay was performed, which identified the PVT-1 /TRIM28 complex as critical for the SUMOylation of Vps34. A cell viability assay identified PVT-1 -enhanced osteosarcoma cells are more sensitive to a SUMOylation inhibitor, TAK-981, suggesting that targeting SUMOylation could be a possible therapeutic avenue used to treat 8q24.2-amplified osteosarcoma patients. In summary, relevant osteosarcoma models were applied to explore the effects of functional PVT-1 inhibition, which resulted in reduced tumorigenesis and the loss of cancer stem cell-like properties. These studies provide the first evidence of a PVT-1 /TRIM28 interaction that drives stem cell properties through the downstream ubiquitination of TSC2. The findings presented here offer a mechanism for the therapeutic targeting of PVT-1 in a high-risk molecular subgroup of osteosarcoma patients and provide further insight into how the inactivation of this mechanism suppresses tumorigenic and cancer stem-like behaviors in osteosarcoma. Methods Osteosarcoma Patient Samples: The use of patient samples and PDXs obtained from Texas Children’s Hospital was approved by the Institutional Review Boards for Baylor College of Medicine and Affiliated Institutions (H-32129 and H-32668). Migration and Invasion Assay: Migration and invasion assays were performed using HOS or TCCC-OS63 cells as previously described (19). For this study, 100,000-200,000 cells were plated in each of the Transwell chambers. Sarcosphere Assay : For the production of primary sarcospheres, single-cell suspension containing 2,000 to 5,000 HOS, CRL-7631, or TCCC-OS63 cells were seeded in a 6-well low-attachment plate (Corning: Corning, NY). The cells were cultured in DMEM F-12 supplemented with Epidermal Growth Factor, B-27, penicillin/streptomycin, and bFGF. After the primary sarcospheres developed, the spheres were dissociated into single cells and plated at a volume of 500-1500 cells/well to second generation sarcospheres. For serial dilution spheres, the cells were plated at volumes of 10,000, 1,000, 100, or 10 cells/well for secondary and tertiary formation. After 2-4 weeks the wells were imaged using a dissection microscope and the numbers and sizes of the sarcospheres were quantified using Nikon-Pro. Comprehensive identification of RNA binding proteins (ChIRP): ChIRP was conducted using TCCC-OS63 cell lines and performed experiment as reported with minor modifications (20). The modifications were, 60-mer antisense DNA probes targeting PVT-1 and lacZ are listed in Supplemental table 5. The probes cover the complete sequence of both genes (IDT). Lac Z probes was designed as a negative control. The hybridization buffer consisted of 40% formamide. The beads were directly loaded into a 10% sodium dodecyl sulfide (SDS) gel with an aliquot of each sample saved for additional confirmation studies (Bio-Rad: Hercules, CA). The gel was then stained with Coomassie-Blue stain to determine the presence of proteins and protein bands were sent to Taplin-Mass Spectrometry core for identification. Co-Immunoprecipitation (Co-IP): HOS cells from all Co-IP studies were lysed with 1% RIPA supplemented with protease and phosphatase inhibitors. For TSC2 Co-IP, the cells were transfected with HA-Ub plasmid for 48 hours before Co-IP and for TSC1 Co-IP the cells were treated with 10uM of MG-132 for 5 hours (Selleckchem, Cat#: S2619: Houston, TX). Cell lysate were pre-cleared with protein A beads for 30 minutes. The cell lysates were incubated with IgG or immunoprecipitating antibody for 24 hours at 4° C. Protein A beads were added to lysate/antibody mixture for 1 hour. Beads were washed 5 times and denatured with Laemmli/Beta-Mercaptoethanol. To detect protein to protein interaction western blot was run for each Co-IP. Samples comparison for each Co-IP prevented western loading of protein of interest pulldown, IgG pulldown, and input on one gel due to lack of well numbers. A representative image showing protein of interest pulldown, IgG pulldown, and input comes from the TSC2 Co-IP (Supplemental Fig. 6). Additional methods and materials are provided in supplemental material section . Statistics: Experiments were conducted in triplicates and confirmed performing three independent replicates unless specified. Error bars represent standard deviation (S.D.). Statistical analysis was performed using Graphpad (Version 8.1). Results High expression of PVT-1 correlates to poor prognosis in osteosarcoma. Since many cancers consist of chromosomal aneuploidy, understanding the correlation between genomic perturbations and patient survival is imperative. Comparative genomic hybridization studies performed on osteosarcoma tumors obtained from patients at Texas Children’s Hospital revealed that patients with 8q24.2 copy number gains/amplifications had a significantly lower probability of survival compared to patients presenting chromosomal balance of this amplicon (Fig. 1a). Within 8q24 resides the oncogenes MYC and PVT-1 . MYC is approximately 60 kilobases upstream of PVT-1 and due to its close proximity to PVT-1 , reports have suggested some functional interdependence between PVT-1 and MYC (10, 21). To understand the molecular relationship between PVT-1 and MYC in osteosarcoma, an analysis of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) osteosarcoma dataset was performed to compare transcript expression between patient samples with low vs. high PVT-1 and low vs. high MYC . The sampling criteria consisted of the top and bottom 25% MYC or PVT-1 expressing patients, which identified 22 patients with high MYC or PVT-1 and 22 patients with low MYC or PVT-1 . From this analysis, 4772 differentially expressed genes were identified between the low vs. high MYC groups with a fold change of at least 1.5 and adjusted p-value of less than 0.05. There were 3986 differentially expressed genes between the low vs. high PVT-1 group using the same criteria. Comparison of these two gene sets revealed that only 964 genes overlapped and the majority of the differentially expressed genes in the individual signatures were specific to the respective grouping (Fig. 1b). Additionally, Gene Set Enrichment Analysis (GSEA) determined that hyperactivation of PVT-1 can regulate pathways independent of MYC , and vice versa (Fig. 1b). Taken together these data indicates that although MYC and PVT-1 reside in close proximity on the same locus there are PVT-1 specific functions exist that are not related to MYC expression. The amplification of 8q24 was identified as a signature of poor prognosis. To gain insight into the somatic copy number alteration of PVT-1 Fluorescence In Situ Hybridization (FISH) was performed on established osteosarcoma cell lines and PDXs to understand if there is a correlation between 8q24 and PVT-1 copy number. Only 3 of 8 cell lines and 2 of 8 PDXs had chromosomal balance of PVT-1 with a majority of the analyzed samples having PVT-1 copy gain/amplification (Supplemental Table 1 and Supplemental Fig. 1a). When comparing the FISH data to previous reports describing osteosarcoma cell lines and PDXs, non-metastatic cell lines and localized PDXs were noted to present PVT-1 chromosomal balance whereas cell lines associated with higher metastatic potential and PDXs generated from patients with recurrent and therapy-resistant tumors have PVT-1 copy gain/amplification (22, 23). PVT-1 RNA expression profiling in osteosarcoma cell lines, human tumor biopsies, and PDXs revealed significantly higher levels of PVT-1 expression in a majority of the osteosarcoma samples compared to non-malignant samples (Fig. 1c). In addition, Spearman’s correlation analysis determined a significant positive association between samples with PVT-1 copy gain and samples expressing higher levels of PVT-1 (Fig. 1d) . Furthermore, when comparing patients in the osteosarcoma TARGET dataset based on top or bottom tertile of PVT-1 expression patients with higher PVT-1 levels were associated with unfavorable event-free and overall survival rates compared to patients that present lower PVT-1 expression (Fig. 1e). These PVT-1 profiling results suggests a strong relationship between PVT-1 expression and cancer progression. PVT-1 promotes osteosarcoma tumorigenesis. After demonstrating that amplification and increased expression of PVT-1 is associated with poor prognosis in osteosarcoma, further investigation was performed to understand the role of PVT-1 in promoting tumor development. The stable overexpression (o.e.) of PVT-1 was established in the balanced HOS and CRL-7631cell lines, which express low levels of PVT-1, and stably silenced PVT-1 (sh PVT-1 ) in 8q24-amplified PDX TCCC-OS63-derived cells. Efficiency of plasmid transfection was confirmed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (Supplemental Fig.1b). Subsequently, the effects of PVT-1 perturbation on osteosarcoma phenotypes were evaluated. Cells with higher levels of PVT-1 had a significant increase in the proliferation rate compared to control cells (Fig. 2a, 2b). To investigate the role of regulating metastasis behaviors PVT-1 -mediated migration and invasion assays were performed. The suppression of PVT-1 in TCCC-OS63 reduced both the cancer cells migratory and invasive capabilities, while PVT-1 overexpression in HOS cells increased both phenotypic properties (Fig. 2c, 2d, and Supplemental Fig. 1c and 1d). In summary, these studies provide evidence that PVT-1 inhibition suppresses proliferation, migration, and invasion phenotypes. PVT-1 is a driver of cancer stem-like behaviors. Because lncRNA PVT-1 expression enhances metastatic phenotypes, and lncRNAs have previously been shown to regulate stem cell properties, we investigated the role of PVT-1 in regulating cancer stem-like behaviors in osteosarcoma (24). The expression of several stem cell marker genes was examined using the established HOS PVT-1 o.e. cells. The transcriptomic and proteomic levels of the stem cell genes Oct4, Nanog, SOX2, and MYC increased in response to PVT-1 o.e. (Fig. 3a and 3b). The reduction of PVT-1 in TCCC-OS63 cells suppressed the mRNA and protein expression level of stem cell markers (Supplemental Fig. 2a). To evaluate the role of PVT-1 in regulating self-renewal capacity, a sarcosphere formation assay was performed to assess self-renewal potential (2, 4, 5, 25). The sarcosphere assay was performed using HOS PVT-1 Ctrl and PVT-1 o.e. monolayer cells to generate secondary and tertiary sarcospheres. The assay determined that enhancing PVT-1 leads to an increase in sarcosphere formation at the secondary and tertiary sarcosphere generation with no 2° sarcospheres in the HOS PVT-1 Ctrl 100 and 10 cell group (Fig. 3c and Supplemental Fig. 2b). To determine if PVT-1 expression is elevated in the cancer stem cells, the PVT-1 expression level was examined in parental HOS-adherent cells vs. parental HOS-sarcospheres. The sarcospheres had significantly higher levels of PVT-1 than the adherent cells (Fig. 3d). In addition, the sarcosphere assay was performed using TCCC-OS63 sh PVT-1 cell lines found that suppressing PVT-1 inhibited sarcosphere formation (Supplemental Fig. 2c). In summary, using multiple complementary models, we determined that PVT-1 is critical for increasing stem cell markers and cell renewal capabilities for osteosarcoma. PVT-1 drives cancer-stem like behaviors through TSC2. After demonstrating that PVT-1 participates in the molecular and phenotypic features of osteosarcoma cancer stem cells, proteomic analysis was performed to identify downstream effectors of PVT-1 . Reverse Phase Protein Array (RPPA) was performed on HOS PVT-1 Ctrl and PVT-1 o.e cells and identified more than 50 differentially expressed proteins that had a ≥1.25-fold change and a p-value less than 0.05 (Fig. 4a). GSEA analysis of these proteins revealed signaling cascades dependent upon PVT-1 (Fig. 4b). Validation of a subset of the RPPA data, focusing on the PI3K/AKT/TSC2 pathway, as this pathway has been previously reported as an inducer of cancer stem-like features (26, 27). Western blot analysis confirmed that increased expression of PVT-1 suppresses the TSC2 protein level (Fig. 4c). Interestingly, examination of the TSC2 transcript level in the HOS PVT-1 Ctrl and PVT-1 o.e. cells did not reveal a difference in TSC2 mRNA expression level (Fig. 4d). These results suggest that PVT-1 induces post-transcriptional modifications of TSC2. To understand whether PVT-1 promotes oncogenesis and self-renewal through TSC2, stable TSC2 o.e. was transfected into the HOS PVT-1 o.e. cells to rescue the expression of TSC2. Increasing TSC2 reversed the proliferation, migration, and invasion potential of PVT-1 (Fig. 4e and 4f and Supplemental Fig. 3a). The overexpression of both PVT-1 and TSC2 in osteosarcoma cells HOS and CRL-7631 caused a decrease in cancer-stem-like features, including low expression of stem cell genes at the transcript and protein level (Fig. 4g and Supplemental Fig. 4d). The role of TSC2 in regulating cancer-stem like behaviors in relation to PVT-1 was assessed by performing a serial-dilution sarcosphere assay using HOS and CRL-7631 cell lines. The results determined that PVT-1 induced sarcosphere formation was dependent upon downstream suppression of TSC2 (Fig. 4h and Supplemental Fig. 3b and 3c). These results provide evidence that PVT-1 is upstream of TSC2 and that TSC2 is a necessary intermediate for PVT-1 to induce oncogenic and cancer stem-like biological features. PVT-1 directly associates with TRIM28. After identifying TSC2 as a downstream regulator of PVT-1 , further analysis was done to assess the molecular mechanism by detecting the protein binding partners of PVT-1 . ChIRP- mass spectrometry was performed on TCCC-OS63 osteosarcoma cell line as this cell line contains high endogenous levels of PVT-1 . ChIRP analysis identified that PVT-1 interacts with TRIM28, HSP90B1, and NOP2 and these proteins were absent in the lacZ (negative control) ChIRP protein elution (Fig. 5a). The identification of NOP2 as a binding partner for PVT-1 has previously been reported, thus providing validation to the ChIRP approach (28). As TRIM28 has been characterized as a regulator of cancer stem-like cells in breast and non-small cell lung cancer, further assessment was done to examine the role of TRIM28 in regulating PVT-1 -induced behaviors (29, 30). The PVT-1 /TRIM28 interaction was validated through ChIRP-western blot (Fig. 5b) and direct PVT-1 -TRIM28 interaction was reciprocally confirmed using a TRIM28 RNA immunoprecipitation (RIP) assay in 8q24.2-amplified TCCC-OS63 cells, which found enrichment of PVT-1 in the TRIM28 pulldown versus the IgG control pulldown (Supplemental Fig. 4a). In addition, RIP of HOS PVT-1 ctrl and HOS PVT-1 o.e. cells showed HOS PVT-1 o.e. cells have enhanced PVT-1 /TRIM28 interaction compared to the HOS PVT-1 Ctrl cells (Fig. 5c). 8q24-amplification is also a marker of poor prognosis in breast cancer (31, 32). In breast cancer, PVT-1 is a mediator of disease progression and TRIM28 has been previously reported to regulate breast cancer stem-like cells (29, 30, 33, 34). Because of this concordance between breast cancer and osteosarcoma PVT-1 we examined if breast cancer cell lines with 8q24 amplification and elevated levels of PVT-1 have higher PVT-1 /TRIM28 interaction (Supplemental Fig. 4b). The 8q24 amplified breast cancer cell lines SK-BR-3, MCF7, and ZR-75-30 have enrichment of the PVT-1 /TRIM28 interaction compared to the unamplified T47D and BT-474 cell lines (Fig. 5d). From these experiments, it can be concluded that PVT-1 /TRIM28 interaction is not specific to osteosarcoma, but it may occur in to other 8q24-amplified malignancies, thus investigations into the biological significance of this complex are crucial. After the identification of the novel PVT-1 /TRIM28 complex, the biological relevance of this interaction was examined between the non-coding RNA and TRIM28. To understand the effects of PVT-1 on TRIM28 the protein expression level of TRIM28 was examined. Western blot analysis determined that TRIM28 levels are comparable independent of PVT-1 expression levels, indicating that PVT-1 has no effect on TRIM28 stability (Supplemental Fig. 4c). In addition, nuclear and cytoplasmic localization of TRIM28 was independent of PVT-1 expression (Supplemental Fig. 4d). In order to understand the functional significance of TRIM28, shTRIM28 plasmid was transfected into HOS PVT-1 o .e. cell lines (Supplemental Fig. 4e) and cell viability studies identified knockdown of TRIM28 decreases PVT-1 -induced proliferation and reduces the migratory and invasive potential of HOS PVT-1 o.e. cells (Fig. 5e and Supplemental Fig. 4f and 4g). Subsequently, we examined the importance of PVT-1 /TRIM28 axis in regulating self-renewal capacity by performing sarcosphere assays. We observed that HOS PVT-1 o.e. and shTRIM28 cells had a significant reduction in sarcosphere formation (Fig. 5f). Finally, stem cell profiling showed that PVT-1 and TRIM28 are necessary to enhance the transcriptomic and proteomic level of stem cell genes (Fig. 5g, 5h, and Supplemental Fig. 4h). In summary, PVT-1 /TRIM28 interaction significantly contributes to the PVT-1 -induced downstream phenotypes. TRIM28 interaction occurs at the 5’ end of PVT-1 . To decipher the region of PVT-1 that engages with TRIM28, the catRAPID algorithm was used to predict the binding region of RNA and target protein based upon physiochemical properties (35, 36). There were two regions of PVT-1 that were noted to have the highest propensity for TRIM28 interaction, specifically nucleotide regions 40-125 and 586-671 of PVT-1 (Supplemental Fig. 5a). Besides making the PVT-1 constructs with deletion of the predicted regions 40-125 and 586-671, three additional constructs were made that deleted the 1-978, 489-1469, or 979-1957 nucleotide regions spanning the 5’ half, 3’ half, and a middle deletion region overlapping the 5’ and 3’ regions to biochemically confirm the interacting region(s) (Fig. 6a). These five constructs along with full length PVT-1 were stably transfected into the HOS cell line and expression was confirmed by qPCR (Fig. 6b). TRIM28 RIP was used to assess differential PVT-1 enrichment for the various constructs. Analysis of the qPCR data determined that PVT-1 enrichment was negligible in the cell lines where 40-125 and 1-978 regions were deleted (Fig. 6c). PCR analysis validated the RIP data as the PVT-1 was not present in the 40-125 and 1-978 mutant cells (Supplemental Fig. 5b). To assess the biological impact caused by perturbing the direct PVT-1 /TRIM28 interaction, the oncogenic and self-renewal capacity was examined using the HOS mutant cells to full length PVT-1 o.e cells (FL). Assessment of downstream PVT-1 effectors determined that lack of PVT-1 /TRIM28 interaction impairs the enhancement of stem cell protein level caused by full length PVT-1 (Fig. 6d). Additional downstream signaling analysis determined that the lack of interaction increased TSC2 expression (Fig. 6e). Phenotypic studies determined that the lack of interaction prevented PVT-1 from promoting proliferation, migration, and invasion capacity (Fig. 6f and 6g). To assess self-renewal capacity through sarcosphere assay, it was observed that there was a lack of sarcosphere formation in the 1-978 and 40-125 mutant cells (Fig. 6h). These results collectively indicate that specific binding of PVT-1 to TRIM28 is critical for degradation of TSC2 and induction of cancer stem-like and oncogenic properties. PVT-1 /TRIM28 leads to increased TSC2 ubiquitination. After determining that PVT-1 reduces TSC2 levels, and that TSC2 is an important modulator in regulating oncogenesis and tumor-initiation maintenance, further mechanistic studies assessed whether the PVT-1 /TRIM28 axis is necessary for TSC2 reduction. The lack of TRIM28 in HOS PVT-1 o.e. cells prevent the decrease in TSC2 protein expression (Fig. 7a). As TRIM28 is known to cause TSC2 ubiquitination, a TSC2 co-IP was performed to examine the importance of PVT-1 /TRIM28 in regulating the level of TSC2 ubiquitination (37, 38). The HOS PVT-1 o.e. cells had an increase in ubiquitination of TSC2 as compared to the PVT-1 Ctrl and PVT-1 o.e. + shTRIM28 cells (Fig. 7b). Because PVT-1 requires TRIM28 for enhanced TSC2 ubiquitination, additional studies were performed to elucidate the molecular mechanism that leads to enhanced TSC2 ubiquitination. Prior reports show TRIM28 SUMOylates and activates Vps34, which leads to the recruitment of TSC1 to Vps34 and disengagement of TSC2 from the TSC1/TSC2 heterodimer (37, 38). The unbound TSC2 gets ubiquitinated and subsequently degraded (37). By utilizing Vps34 co-IP, the level of Vps34 binding to TRIM28 could be examined. PVT-1 o.e. cells showed an increase interaction of TRIM28 to Vps34, which was negligible in the PVT-1 Ctrl and PVT-1 o.e. + shTRIM28 cells (Fig. 7c). As the formation of TRIM28 and Vps34 complex causes Vps34-SUMO this modification was assessed and determined that overexpression of PVT-1 causes Vps34 SUMOylation as the molecular weight of Vps34 molecular weight increased by approximately 40 kDa due to addition of SUMO adducts (Fig. 7c) and increased Vps34/TSC1 interaction (Fig. 7c and 7d). Since the TSC1/TSC2 heterodimer decoupling is associated with TSC2 ubiquitination, the level of TSC1/TSC2 complex was examined using a TSC1 co-IP (37). To prevent TSC2 degradation, the cells were treated with MG132, a proteasome inhibitor, prior to performing the TSC1 co-IP. We observed that the PVT-1 o.e. cells lacked TSC1 binding to TSC2 (Fig. 7d). These studies suggest that PVT-1 /TRIM28 axis is critical for formation of the PVT-1 /TRIM28/Vps34-SUMO/TSC1 complex and subsequent degradation of TSC2. A representative blot from the TSC2 co-IP includes protein-of-interest pulldown, IgG pulldown, and input for the PVT-1 Ctrl and PVT-1 o.e. cells (Supplemental Fig. 6). Because PVT-1 /TRIM28 interaction leads to enhancement of Vps34 SUMOylation, an additional cell viability assay was performed to assess if PVT-1 o.e. cells would be more sensitive to a small molecule SUMOylation inhibitor, TAK-981. TAK-981 covalently binds to SUMO to form an adduct and prevents SUMO conjugation to the protein of interest (39). The in vitro drug sensitivity study determined that HOS PVT-1 o.e. cells are significantly more sensitive to TAK-981 when compared to HOS PVT-1 Ctrl cells (Fig. 7e). The data confirms increased expression of PVT-1 activates SUMOylation and targeting this post-translational modification could be a treatment for malignancies with overexpression of the PVT-1 , including 8q24.2 amplified tumors. Discussion One of the current obstacles in the development of effective osteosarcoma targeted treatment is identifying viable molecular targets (1, 40). The current study elucidated a novel mechanism underlying the interaction between PVT-1 and TRIM28, and the ability of this complex to induce cancer stem-like behaviors in osteosarcoma. Specifically, using proteomic analysis, the research identified that PVT-1 induces stem cell like behaviors by inhibiting the tumor suppressor TSC2 protein level. This work is the first to link PVT-1 to regulation of TSC2 and the research discovered that through formation of a complex with TRIM28, PVT-1 is able to ubiquitinate TSC2, while rescuing TSC2 prevents PVT-1 -induced cancer-stem like behaviors. Previous reports have identified that lncRNAs regulate biological behaviors when interacting with proteins (41, 42). The ChIRP-mass spectrometry analysis identified TRIM28, HSP90B1, and NOP2 as potential binding partners for PVT-1 . Additional studies assessed the role of TRIM28 in relationship to PVT-1 as TRIM28 has been implicated as a regulator of cancer stem-like behaviors (33,34). These additional functional studies identified that this complex is critical for the induction of tumorigenic and cancer stem-like behaviors. In addition, these studies are able to link this complex to regulation of the downstream TSC2 signaling cascade. While the studies have assessed the importance of PVT-1 /TRIM28 interaction to osteosarcoma cancer stem cell properties, there could be self-renewal capacity and/or cancer progression importance to the PVT-1 /HSP90B1 and PVT-1 /NOP2 interactions, which could not be ruled out and additional functional studies would be critical to assess the importance of these complexes. As this research has determined that enhanced PVT-1 /TRIM28 complex suppresses TSC2, mechanistically further work was performed to understand the intermediates between PVT-1 /TRIM28 and TSC2. While previous reports link TRIM28 to ubiquitination of TSC2, the role of PVT-1 in relationship to this mechanism has not been discovered (37, 38). In this study, co-IP studies identified that TRIM28 directly binds to PVT-1 causing TRIM28 interaction to Vps34 and activation of Vps34 through SUMOylation. With additional research identifying that osteosarcoma cells with Vps34 SUMOylation have increased TSC2 ubiquitination, and thus enhanced oncogenic and tumor-initiation potential. This research identified an attractive therapeutic target, the SUMOylation of Vps34. SUMOylation has been implicated in regulating cancer stem cell behaviors in different malignancies, and TAK-981, a SUMOylation inhibitor, is presently being evaluated as an alternative therapeutic intervention for both solid and hematological malignancies (ClinicalTrials.gov Identifier: NCT03648372 and ClinicalTrials.gov Identifier: NCT04074330)(43). While our research has identified that osteosarcoma cells with elevated PVT-1 are more sensitive to TAK-981, there is still an abundance of in vitro and in vivo research needed in order to understand how viable this agent is for osteosarcoma therapy. Previous reports have found that SUMOylation inhibitors can increase chemosensitivity (44, 45). Therefore, inhibition of SUMOylation in tumors with high expression of PVT-1 may be a novel therapeutic approach for patients with osteosarcoma. Our mechanistic studies have identified the downstream molecular events dictated by the PVT-1 /TRIM28 complex, however further analysis is required to understand how PVT-1 recruits TRIM28. TRIM28 is thought to recruit proteins to assist in downstream ubiquitination, as seen with TRIM28 association with TRIM24 for Speckle-type POZ protein ubiquitination (46). Therefore, a potential mechanism to how PVT-1 recruits TRIM28 could be through protein-mediated facilitation. By having insight into the formation of PVT-1 /TRIM28 axis, there may be additional avenues to inhibit PVT-1 -induced cancer stem cell behaviors. Finally, previous reports have alluded to the possibility of PVT-1 induced phenotypes being dependent upon MYC, which is a neighboring oncogene in the 8q24.2 region (10). To assess if this relationship is true in osteosarcoma, the proliferation and stem cell profile of PVT-1 o.e. +/- siMYC was examined. It was found that suppressing MYC does not prevent either PVT-1 -induced proliferation or enhancement of stem cell marker expression (Supplemental Fig. 7). This analysis was able to confirm that PVT-1 is not dependent upon MYC to promote tumorigenic and cancer-stem cell behaviors. In conclusion, the current study provides new insights linking PVT-1 and TRIM28 to activation of tumorigenesis and cancer stem-like behaviors. By understanding the PVT-1 /TRIM28 downstream signaling cascade, this research was able to target an intermediate within this pathway for potential inhibition of the cancer stem-like behaviors (Fig. 7f). Lastly, this work demonstrated that the PVT-1 /TRIM28 axis in not specific to osteosarcoma but is also present in breast cancer. By recognizing this connection, the research may be of use to cancers that present with 8q24 amplification. Declaration Financial Support: Susan V. Tsang: Cancer Prevention Institute of Texas (CPRIT) RP160283 Kimal Rajapakshe: (CPRIT) RP170005, NIH P30 shared resource grant CA125123, and NIEHS P30 Center grant 1P30ES030285 Cristian Coarfa: (CPRIT) RP170005, NIH P30 shared resource grant CA125123, and NIEHS P30 Center grant 1P30ES030285 Jason T. Yustein: The Faris D. Virani Ewing Sarcoma Center, (CPRIT) RP140022-P1 Conflict of Interest: The authors declare no potential conflicts of interest. Acknowledgement: We would like to acknowledge the Advanced Technologies Core services at the Baylor College of Medicine. This work was funded by The Faris D. Virani Ewing Sarcoma Center (to J.T. Yustein). S.V. Tsang was partially supported by Cancer Prevention Institute of Texas (CPRIT) RP160283, K. Rajapakshe and C. Coarfa were partially supported by (CPRIT) RP170005, NIH P30 shared resource grant CA125123, and NIEHS P30 Center grant 1P30ES030285; CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (CA125123 and RR024574 to the Cytometry and Cell Sorting Core at Baylor College of Medicine); and Core Facility Award (CPRIT-RP170005 to Proteomics and Metabolomics Core at Baylor College of Medicine). We thank Dr. Suzanne Fuqua and Dr. Xiang Zhang for generously providing the breast cancer cell lines. The authors would like to thank Dr. Hilsenbeck for running the generalized linear model analysis. References Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer treatment and research. 2009;152:3-13. Gibbs CP, Kukekov VG, Reith JD, Tchigrinova O, Suslov ON, Scott EW, et al. Stem-like cells in bone sarcomas: implications for tumorigenesis. Neoplasia (New York, NY). 2005;7(11):967-76. Peitzsch C, Tyutyunnykova A, Pantel K, Dubrovska A. 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1","display":"","copyAsset":false,"role":"figure","size":222530,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Tsangetal.Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/2dcc2aa4ec998d5cf5bf5b96.png"},{"id":24524200,"identity":"109067ca-5a69-4034-a217-b8128d542b5a","added_by":"auto","created_at":"2022-07-29 18:06:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1 \u003c/em\u003eenhances osteosarcoma growth and metastatic potential.\u003c/strong\u003e (a) Cell viability assessment secondary to \u003cem\u003ePVT-1\u003c/em\u003e overexpression in HOS cells. (b) Cell viability assessment secondary knockdown of \u003cem\u003ePVT-1\u003c/em\u003e in TCCC-OS63. (c) Analysis of \u003cem\u003ein vitro\u003c/em\u003e migration and invasion characterization for HOS-\u003cem\u003ePVT1\u003c/em\u003e o.e. cell lines by trans-well migration and invasion assay (d) Migration and invasion analysis for TCCC-OS63-sh\u003cem\u003ePVT-1\u003c/em\u003e cell line. Error bars represent S.D.; a Student’s t-test or one-way ANOVA was used to calculate statistical significance values for all figures. *p\u0026lt;.05; p\u0026lt;.001; *** p\u0026lt;.0001. Data represents three independent experiments.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Tsangetal.Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/2b5b772eec9d58f48ee5c42b.png"},{"id":24524199,"identity":"fcde5cba-c7b8-4eed-818e-33e5c0e6c9a6","added_by":"auto","created_at":"2022-07-29 18:06:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e induces cancer stem-like phenotypes. \u003c/strong\u003e\u0026nbsp;(a) RT-qPCR analysis for expression of stem cell genes in HOS \u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells. (b) Western blot analyzed the stem cell protein level in HOS \u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells. (c) Quantification of serial dilution secondary and tertiary sarcospheres from HOS \u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells. HOS \u003cem\u003ePVT-1\u003c/em\u003e Ctrl: 100 and 10 cell group developed no secondary sarcospheres. None Detected (N.D.). Secondary sarcospheres were generated from the primary sarcosphere and secondary sarcospheres were used for tertiary sarcosphere production.\u0026nbsp;(d) Expression level of \u003cem\u003ePVT-1\u003c/em\u003e in HOS adherent and HOS sarcospheres. * p\u0026lt;.05; ** p\u0026lt;.001; *** p\u0026lt;.0001. Data represents three independent experiments.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Tsangetal.Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/510566b58c4cf5a9668c49da.png"},{"id":24523648,"identity":"d01bb5eb-e9a3-4192-9aac-b73d07f8a666","added_by":"auto","created_at":"2022-07-29 18:01:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":167438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e drives osteosarcoma cancer stem-like behavior through TSC2.\u003c/strong\u003e (a) RPPA analysis of HOS Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells. (b) GSEA analysis of the RPPA data identified cellular pathways impacted by the enhancement of \u003cem\u003ePVT-1\u003c/em\u003e. (c) Western blot analysis of TSC2 in HOS Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells. (d) RT-qPCR of TSC2 in HOS Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells. (e-h) Rescue experiments to understand the role of TSC2 as a downstream effector of \u003cem\u003ePVT-1\u003c/em\u003e. Effects of TSC2 overexpression in HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cell lines on: (e) Cell viability. (f) Migration and invasion assay. (g) Stem cell markers via western blot. (h) Serial-Dilution Sphere formation. None Detected (N.D.). Error bars represent S.D. Statistical analysis was performed using linear regression of figure e and t-test or one-way ANOVA for the rest of the figures. * p\u0026lt;.05; **p\u0026lt;.001; *** p\u0026lt;.0001. Data represents three independent experiments.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Tsangetal.Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/a44ed19d436523a9a123d028.png"},{"id":24524203,"identity":"659d28ad-3553-404f-96ee-2e7f7417cd1e","added_by":"auto","created_at":"2022-07-29 18:06:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":164392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e interacts with TRIM28 which induces cancer stem-like behaviors.\u003c/strong\u003e (a) ChIRP-Mass Spectrometry analysis to identify proteins directly bound to \u003cem\u003ePVT-1\u003c/em\u003e. (b) ChIRP-Western Blot to validate \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction. (c) TRIM28 RIP-qPCR analysis comparing enrichment of \u003cem\u003ePVT-1\u003c/em\u003e interaction in HOS \u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells. (d) Analysis of \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction in breast cancer cell lines with 8q24 non-amplification vs. 8q24 amplification. (e-h) HOS cells with \u003c/p\u003e\u003cp\u003e\u003cem\u003e PVT-1\u003c/em\u003e\u0026nbsp;and TRIM28 expression were modified to identify which factors are critical for\u0026nbsp;\u003cem\u003ePVT-1\u003c/em\u003e-induced tumorigenic and cancer stem cell-like behaviors and TSC2 inhibition. Cell lines transfected with PVT-1 o.e. or shTRIM28 are labeled with a “+” and if the corresponding control plasmid was inserted with a “-“ (e) metastatic behaviors: migration and invasion (f) sarcosphere production (g) qPCR analysis of stem cell markers (h) Western blot to assess for protein level of stem cell markers. Error bars represent S.D. Statistical analysis was performed using one-way ANOVA. * p\u0026lt;.05; **p\u0026lt;.001; *** p\u0026lt;.0001. Data represents three independent experiments.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Tsangetal.Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/8e9623d8f80a3ef4195abc0b.png"},{"id":24525149,"identity":"012cd486-2bd0-4d89-8566-8ed3619a4914","added_by":"auto","created_at":"2022-07-29 18:11:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":274615,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Tsangetal.Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/4da59fc93af90880d8ad2bf4.png"},{"id":24523644,"identity":"07f2fb63-4e68-49f2-9cd1-c6834bc18e4c","added_by":"auto","created_at":"2022-07-29 18:01:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":231170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 axis is associated with TSC2 ubiquitination. \u003c/strong\u003eHOS cell lines transfected with PVT-1 o.e. or shTRIM28 are labeled with a “+” and if the corresponding control plasmid was inserted with a “-“ (a) Western Blot to detect TSC2 level dependent upon the perturbation of \u003cem\u003ePVT-1\u003c/em\u003e and/or TRIM28 in HOS cells. (b) HOS cells were transiently transfected with HA-Ubiquitin plasmid. 48 hours post-transfection, endogenous TSC2 was immunoprecipitated from whole cell lysate and ubiquitinated TSC2 was detected by western blot using anti-HA antibody. (c) Western blot to determine presence of Vps34 co-immunoprecipitates: SUMO1, TRIM28, and TSC1 in HOS cells. (d) Cells were treated with MG132 at 10uM for 5 hours.\u0026nbsp;Whole cell lysates of these cells were used for TSC1 co-immunoprecipitation. Immunoblots were analyzed for detection of TSC2 and Vps34. (e) CCK-8 assessment of HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cell sensitivity to SUMOylation inhibitor. (f) Proposed model of \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 signaling cascade which promotes tumorigenic and cancer-stem like phenotypes.\u0026nbsp;Error bars represent S.D. Statistical analysis was performed using one-way ANOVA. *p\u0026lt;.05; **p\u0026lt;.001; ***p\u0026lt;.0001. Data represents three independent experiments.\u003c/p\u003e","description":"","filename":"Tsangetal.Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/f6d0c0c3cbb58067b9a28283.png"},{"id":28842868,"identity":"5630a497-d18c-497f-afbc-4f6307400765","added_by":"auto","created_at":"2022-11-09 08:10:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1674828,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/dcb6aec4-d83d-4501-adfb-ecc3fbc22ce4.pdf"},{"id":24523642,"identity":"797a33d1-5820-4ce8-b3a9-7eaee77dbaec","added_by":"auto","created_at":"2022-07-29 18:01:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1982685,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials6.30.22.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1813153/v1/86f77fed597393a80bd15a35.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"LncRNA PVT-1 promotes osteosarcoma cancer stem-like properties through direct interaction with TRIM28 and TSC2 ubiquitination","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteosarcoma is the most common primary bone tumor in pediatric patients\u0026nbsp;(1). Like many malignancies, osteosarcoma is comprised of a heterogeneous population of cells, including a subpopulation of tumor-initiating cells, also known as cancer stem cells, which have the ability to self-renew and drive tumorigenesis\u0026nbsp;(2, 3). In the osteosarcoma field, no well-established cell surface markers exist to identify osteosarcoma stem cells; however, the field strongly relies on the use of characterization through stem cell marker profiling (i.e., Sox2, Oct4, Nanog, and MYC) and the ability of stem cells to form sarcospheres under specific cancer stem cell culture conditions\u0026nbsp;(2, 4, 5). \u0026nbsp;As cancer stem cells play an important role in sustaining cancer growth, therapy resistance and recurrence, there is a critical need to identify cancer stem cell drivers with the goal of developing more effective treatments for osteosarcoma patients\u0026nbsp;(2, 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA genetic anomaly of osteosarcoma is its high extent of recurrent chromosomal aneuploidy\u0026nbsp;(7, 8). Approximately 30% of osteosarcoma patients present with 8q24.2 amplification, which is associated with poor clinical prognosis\u0026nbsp;(9). Within the 8q24.2 amplicon resides \u003cem\u003ec-MYC\u003c/em\u003e (from here on referred to as \u003cem\u003eMYC\u003c/em\u003e) oncogene and \u003cem\u003ePVT-1\u003c/em\u003e (10, 11). As lncRNAs are involved in tumor proliferation and metastasis in other cancer types, the aim of this study was to characterize the role of \u003cem\u003ePVT-1\u003c/em\u003e in osteosarcoma. LncRNAs ability to control different forms of gene regulation impacts multiple tumorigenic behaviors (12,13). For instance, oncogenic lncRNA \u003cem\u003eCDKN2B-AS1\u003c/em\u003e promotes renal cell carcinoma progression by its interaction with insulin-like growth factor 2 mRNA-binding protein 3, which leads to the epigenetic activation of the Ndc80 kinetochore complex (14).\u0026nbsp;\u0026nbsp;While lncRNA, \u003cem\u003eMALAT1\u003c/em\u003e can enhance breast cancer through the regulation of alternative mRNA splicing (14). Previous reports revealed that \u003cem\u003ePVT-1\u003c/em\u003e promotes tumorigenesis and interacts with microRNAs in several cancers (10,15-18). However, studies regarding the role of \u003cem\u003ePVT-1\u003c/em\u003e in osteosarcoma are limited, and no associations with self-renewal or identification of therapeutic vulnerabilities secondary to enhanced \u003cem\u003ePVT-1\u003c/em\u003e expression and perturbations of downstream signaling cascades have been reported.\u003c/p\u003e\n\u003cp\u003eWith the use of patient-derived xenografts (PDXs) and established OS cell line models, \u003cem\u003ePVT-1\u0026nbsp;\u003c/em\u003ewas found to initiate self-renewal properties and enhance tumorigenic behaviors. Through transcriptomic and proteomic analysis, the increased expression of \u003cem\u003ePVT-1\u003c/em\u003e was demonstrated to enhance the expression of stem cell genes. The comprehensive identification of RNA binding proteins\u0026nbsp;(ChIRP) was applied to identify the endogenous \u003cem\u003ePVT-1\u003c/em\u003e interactome, which revealed TRIM28 as a novel \u003cem\u003ePVT-1\u0026nbsp;\u003c/em\u003ebinding partner in 8q24.2-amplified osteosarcoma model. \u0026nbsp;\u003cem\u003ePVT-1/\u003c/em\u003eTRIM28 was determined to inhibit TSC2 through ubiquitination resulting in subsequent proteasomal degradation. In addition, TSC2 rescue experiments resulted in reversal of \u003cem\u003ePVT-1\u003c/em\u003e-induced cancer stem cell capacity. To elucidate the intermediates between \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 and TSC2 signaling pathway, a co-immunoprecipitation assay was performed, which identified the \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 complex as critical for the SUMOylation of Vps34. A cell viability assay identified \u003cem\u003ePVT-1\u003c/em\u003e-enhanced osteosarcoma cells are more sensitive to a SUMOylation inhibitor, TAK-981, suggesting that targeting SUMOylation could be a possible therapeutic avenue used to treat 8q24.2-amplified osteosarcoma patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, relevant osteosarcoma models were applied to explore the effects of functional \u003cem\u003ePVT-1\u003c/em\u003e inhibition, which resulted in reduced tumorigenesis and the loss of cancer stem cell-like properties. These studies provide the first evidence of a \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction that drives stem cell properties through the downstream ubiquitination of TSC2. The findings presented here offer a mechanism for the therapeutic targeting of \u003cem\u003ePVT-1\u003c/em\u003e in a high-risk molecular subgroup of osteosarcoma patients and provide further insight into how the inactivation of this mechanism suppresses tumorigenic and cancer stem-like behaviors in osteosarcoma. \u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eOsteosarcoma Patient Samples:\u003c/strong\u003e The use of patient samples and PDXs obtained from Texas Children\u0026rsquo;s Hospital was approved by the Institutional Review Boards for Baylor College of Medicine and Affiliated Institutions (H-32129 and H-32668).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMigration and Invasion Assay:\u003c/strong\u003e Migration and invasion assays were performed using HOS or TCCC-OS63 cells as previously described\u0026nbsp;(19). For this study, 100,000-200,000 cells were plated in each of the Transwell chambers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSarcosphere Assay\u003c/strong\u003e: For the production of primary sarcospheres, single-cell suspension containing 2,000 to 5,000 HOS, CRL-7631, or TCCC-OS63 cells were seeded in a 6-well low-attachment plate (Corning: Corning, NY). The cells were cultured in DMEM F-12 supplemented with Epidermal Growth Factor, B-27, penicillin/streptomycin, and bFGF. After the primary sarcospheres developed, the spheres were dissociated into single cells and plated at a volume of 500-1500 cells/well to second generation sarcospheres. For serial dilution spheres, the cells were plated at volumes of 10,000, 1,000, 100, or 10 cells/well for secondary and tertiary formation. \u0026nbsp;After 2-4 weeks the wells were imaged using a dissection microscope and the numbers and sizes of the sarcospheres were quantified using Nikon-Pro. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComprehensive identification of RNA binding proteins\u003c/strong\u003e \u003cstrong\u003e(ChIRP):\u0026nbsp;\u003c/strong\u003eChIRP was conducted using \u0026nbsp;TCCC-OS63 cell lines and performed experiment as reported with minor modifications\u0026nbsp;(20). The modifications were, 60-mer antisense DNA probes targeting \u003cem\u003ePVT-1\u003c/em\u003e and lacZ are listed in Supplemental table 5. The probes cover the complete sequence of both genes (IDT). \u0026nbsp; Lac Z probes was designed as a negative control. The hybridization buffer consisted of 40% formamide. The beads were directly loaded into a 10% sodium dodecyl sulfide (SDS) gel with an aliquot of each sample saved for additional confirmation studies (Bio-Rad: Hercules, CA). The gel was then stained with Coomassie-Blue stain to determine the presence of proteins and protein bands were sent to Taplin-Mass Spectrometry core for identification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-Immunoprecipitation (Co-IP):\u0026nbsp;\u003c/strong\u003eHOS cells from all Co-IP studies were lysed with 1% RIPA supplemented with protease and phosphatase inhibitors. For TSC2 Co-IP, the cells were transfected with HA-Ub plasmid for 48 hours before Co-IP and for TSC1 Co-IP the cells were treated with 10uM of MG-132 \u0026nbsp;for 5 hours (Selleckchem, Cat#: S2619: Houston, TX). Cell lysate were pre-cleared with protein A beads for 30 minutes. The cell lysates were incubated with IgG or immunoprecipitating antibody for 24 hours at 4\u0026deg; C. Protein A beads were added to lysate/antibody mixture for 1 hour. Beads were washed 5 times and denatured with Laemmli/Beta-Mercaptoethanol. To detect protein to protein interaction western blot was run for each Co-IP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSamples comparison for each Co-IP prevented western loading of protein of interest pulldown, IgG pulldown, and input on one gel due to lack of well numbers. A representative image showing protein of interest pulldown, IgG pulldown, and input comes from the TSC2 Co-IP (Supplemental Fig. 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAdditional methods and materials are provided in supplemental material section\u003c/u\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics:\u0026nbsp;\u003c/strong\u003eExperiments were conducted in triplicates and confirmed performing three independent replicates unless specified. Error bars represent standard deviation (S.D.). Statistical analysis was performed using Graphpad (Version 8.1).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHigh expression of \u003cem\u003ePVT-1\u003c/em\u003e correlates to poor prognosis in osteosarcoma.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince many cancers consist of chromosomal aneuploidy, understanding the correlation between genomic perturbations and patient survival is imperative. Comparative genomic hybridization studies performed on osteosarcoma tumors obtained from patients at Texas Children\u0026rsquo;s Hospital revealed that patients with 8q24.2 copy number gains/amplifications had a significantly lower probability of survival compared to patients presenting chromosomal balance of this amplicon (Fig. 1a). \u0026nbsp;Within 8q24 resides the oncogenes \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003ePVT-1\u003c/em\u003e. \u003cem\u003eMYC\u003c/em\u003e is approximately 60 kilobases upstream of \u003cem\u003ePVT-1\u003c/em\u003e and due to its close proximity to \u003cem\u003ePVT-1\u003c/em\u003e, reports have suggested some functional interdependence between \u003cem\u003ePVT-1\u003c/em\u003e and \u003cem\u003eMYC\u003c/em\u003e (10, 21). To understand the molecular relationship between \u003cem\u003ePVT-1\u003c/em\u003e and \u003cem\u003eMYC\u003c/em\u003e in osteosarcoma, an analysis of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) osteosarcoma dataset was performed to compare transcript expression between patient samples with low vs. high \u003cem\u003ePVT-1\u003c/em\u003e and low vs. high \u003cem\u003eMYC\u003c/em\u003e. The sampling criteria consisted of the top and bottom 25% \u003cem\u003eMYC\u003c/em\u003e or \u003cem\u003ePVT-1\u003c/em\u003e expressing patients, which identified 22 patients with high \u003cem\u003eMYC\u003c/em\u003e or \u003cem\u003ePVT-1\u003c/em\u003e and 22 patients with low \u003cem\u003eMYC\u003c/em\u003e or \u003cem\u003ePVT-1\u003c/em\u003e. From this analysis, 4772 differentially expressed genes were identified between the low vs. high \u003cem\u003eMYC\u003c/em\u003e groups with a fold change of at least 1.5 and adjusted p-value of less than 0.05. There were 3986 differentially expressed genes between the low vs. high \u003cem\u003ePVT-1\u003c/em\u003e group using the same criteria. Comparison of these two gene sets revealed that only 964 genes overlapped and the majority of the differentially expressed genes in the individual signatures were specific to the respective grouping (Fig. 1b). \u0026nbsp;Additionally, Gene Set Enrichment Analysis (GSEA) determined that hyperactivation of \u003cem\u003ePVT-1\u003c/em\u003e can regulate pathways independent of \u003cem\u003eMYC\u003c/em\u003e, and vice versa (Fig. 1b). Taken together these data indicates that although \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003ePVT-1\u003c/em\u003e reside in close proximity on the same locus there are \u003cem\u003ePVT-1\u003c/em\u003e specific functions exist that are not related to \u003cem\u003eMYC\u003c/em\u003e expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe amplification of 8q24 was identified as a signature of poor prognosis. \u0026nbsp;To gain insight into the somatic copy number alteration of \u003cem\u003ePVT-1\u003c/em\u003e Fluorescence \u003cem\u003eIn Situ\u003c/em\u003e Hybridization (FISH) was performed on established osteosarcoma cell lines and PDXs to understand if there is a correlation between 8q24 and \u003cem\u003ePVT-1\u003c/em\u003e copy number. \u0026nbsp;Only 3 of 8 cell lines and 2 of 8 PDXs had chromosomal balance of \u003cem\u003ePVT-1\u003c/em\u003e with a majority of the analyzed samples having \u003cem\u003ePVT-1\u003c/em\u003e copy gain/amplification (Supplemental Table 1 and Supplemental Fig. 1a). When comparing the FISH data to previous reports describing osteosarcoma cell lines and PDXs, non-metastatic cell lines and localized PDXs were noted to present \u003cem\u003ePVT-1\u003c/em\u003e chromosomal balance whereas cell lines associated with higher metastatic potential and PDXs generated from patients with recurrent and therapy-resistant tumors have \u003cem\u003ePVT-1\u003c/em\u003e copy gain/amplification\u0026nbsp;(22, 23).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePVT-1\u003c/em\u003e RNA expression profiling in osteosarcoma cell lines, human tumor biopsies, and PDXs revealed significantly higher levels of \u003cem\u003ePVT-1\u003c/em\u003e expression in a majority of the osteosarcoma samples compared to non-malignant samples (Fig. 1c). In addition, Spearman\u0026rsquo;s correlation analysis determined a significant positive association between samples with \u003cem\u003ePVT-1\u003c/em\u003e copy gain and samples expressing higher levels of \u003cem\u003ePVT-1\u0026nbsp;\u003c/em\u003e(Fig. 1d)\u003cem\u003e.\u0026nbsp;\u003c/em\u003e Furthermore, when comparing patients in the osteosarcoma TARGET dataset based on top or bottom tertile of \u003cem\u003ePVT-1\u003c/em\u003e expression patients with higher \u003cem\u003ePVT-1\u003c/em\u003e levels were associated with unfavorable event-free and overall survival rates compared to patients that present lower \u003cem\u003ePVT-1\u003c/em\u003e expression (Fig. 1e). These \u003cem\u003ePVT-1\u003c/em\u003e profiling results suggests a strong relationship between \u003cem\u003ePVT-1\u003c/em\u003e expression and cancer progression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;promotes osteosarcoma tumorigenesis.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter demonstrating that amplification and increased expression of \u003cem\u003ePVT-1\u003c/em\u003e is associated with poor prognosis in osteosarcoma, further investigation was performed to understand the role of \u003cem\u003ePVT-1\u003c/em\u003e in promoting tumor development. The stable overexpression (o.e.) of \u003cem\u003ePVT-1\u0026nbsp;\u003c/em\u003ewas established in the balanced \u0026nbsp;HOS and CRL-7631cell lines, which express low levels of \u003cem\u003ePVT-1,\u003c/em\u003e and stably silenced \u003cem\u003ePVT-1\u003c/em\u003e (sh\u003cem\u003ePVT-1\u003c/em\u003e) in 8q24-amplified PDX TCCC-OS63-derived cells. Efficiency of plasmid transfection was confirmed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (Supplemental Fig.1b). Subsequently, the effects of \u003cem\u003ePVT-1\u003c/em\u003e perturbation on osteosarcoma phenotypes were evaluated. \u0026nbsp;Cells with higher levels of \u003cem\u003ePVT-1\u003c/em\u003e had a significant increase in the proliferation rate compared to control cells (Fig. \u0026nbsp;2a, 2b). To investigate the role of regulating metastasis behaviors \u003cem\u003ePVT-1\u003c/em\u003e-mediated migration and invasion assays were performed. The suppression of \u003cem\u003ePVT-1\u003c/em\u003e in TCCC-OS63 reduced both the cancer cells migratory and invasive capabilities, while \u003cem\u003ePVT-1\u003c/em\u003e overexpression in HOS cells increased both phenotypic properties (Fig. 2c, 2d, and Supplemental Fig. 1c and 1d). In summary, these studies provide evidence that \u003cem\u003ePVT-1\u003c/em\u003e inhibition suppresses proliferation, migration, and invasion phenotypes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;is a driver of cancer stem-like behaviors.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause lncRNA \u003cem\u003ePVT-1\u003c/em\u003e expression enhances metastatic phenotypes, and lncRNAs have previously been shown to regulate stem cell properties, we investigated the role of \u0026nbsp;\u003cem\u003ePVT-1\u003c/em\u003e in regulating cancer stem-like behaviors in osteosarcoma\u0026nbsp;(24). The expression of several stem cell marker genes was examined using the established HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells. \u0026nbsp;The transcriptomic and proteomic levels of the stem cell genes Oct4, Nanog, SOX2, and MYC increased in response to \u003cem\u003ePVT-1\u003c/em\u003e o.e. (Fig. 3a and 3b). The reduction of \u003cem\u003ePVT-1\u003c/em\u003e in TCCC-OS63 cells suppressed the mRNA and protein expression level of stem cell markers (Supplemental Fig. 2a). To evaluate the role of \u003cem\u003ePVT-1\u003c/em\u003e in regulating self-renewal capacity, a sarcosphere formation assay was performed to assess self-renewal potential\u0026nbsp;(2, 4, 5, 25). The sarcosphere assay was performed using HOS\u0026nbsp;\u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. monolayer cells to generate secondary and tertiary sarcospheres. The assay determined that enhancing \u003cem\u003ePVT-1\u003c/em\u003e leads to an increase in sarcosphere formation at the secondary and tertiary sarcosphere generation with no 2\u0026deg; sarcospheres in the HOS PVT-1 Ctrl 100 and 10 cell group (Fig. 3c and Supplemental Fig. 2b). To determine if \u003cem\u003ePVT-1\u003c/em\u003e expression is elevated\u0026nbsp;in the cancer stem cells, the \u003cem\u003ePVT-1\u003c/em\u003e expression level was examined in parental HOS-adherent cells vs. parental HOS-sarcospheres. The sarcospheres had significantly higher levels of \u003cem\u003ePVT-1\u003c/em\u003e than the adherent cells (Fig. 3d). In addition, the sarcosphere assay was performed using TCCC-OS63 sh\u003cem\u003ePVT-1\u003c/em\u003e cell lines found that suppressing \u003cem\u003ePVT-1\u003c/em\u003e inhibited sarcosphere formation (Supplemental Fig. 2c). In summary, using multiple complementary models, we determined that \u003cem\u003ePVT-1\u003c/em\u003e is critical for increasing stem cell markers and cell renewal capabilities for osteosarcoma. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;drives cancer-stem like behaviors through TSC2.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter demonstrating that \u003cem\u003ePVT-1\u003c/em\u003e participates in the molecular and phenotypic features of osteosarcoma cancer stem cells, proteomic analysis was performed to identify downstream effectors of \u003cem\u003ePVT-1\u003c/em\u003e. \u0026nbsp; Reverse Phase Protein Array (RPPA) was performed \u0026nbsp;on HOS\u0026nbsp;\u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e\u0026nbsp;cells and identified more than 50 differentially expressed proteins that had a \u0026ge;1.25-fold change and a p-value less than 0.05 (Fig. 4a). GSEA analysis of these proteins revealed signaling cascades dependent upon \u003cem\u003ePVT-1\u003c/em\u003e (Fig. 4b). Validation of a subset of the RPPA data, focusing on the PI3K/AKT/TSC2 pathway, as this pathway has been previously reported as an inducer of cancer stem-like features\u0026nbsp;(26, 27). Western blot analysis confirmed that increased expression of \u003cem\u003ePVT-1\u003c/em\u003e suppresses the TSC2 protein level (Fig. 4c). Interestingly, examination of the TSC2 transcript level in the HOS\u0026nbsp;\u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells did not reveal a difference in TSC2 mRNA expression level (Fig. 4d). These results suggest that \u003cem\u003ePVT-1\u003c/em\u003e induces post-transcriptional modifications of TSC2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo understand whether \u003cem\u003ePVT-1\u003c/em\u003e promotes oncogenesis and self-renewal through TSC2, \u0026nbsp;stable TSC2 o.e. was transfected into the HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells to rescue the expression of TSC2. Increasing TSC2 reversed the proliferation, migration, and invasion potential of \u003cem\u003ePVT-1\u003c/em\u003e (Fig. 4e and 4f and Supplemental Fig. 3a). The overexpression of both \u003cem\u003ePVT-1\u003c/em\u003e and TSC2 in osteosarcoma cells HOS and CRL-7631 caused a decrease in cancer-stem-like features, including low expression of stem cell genes at the transcript and protein level (Fig. 4g and Supplemental Fig. 4d). The role of TSC2 in regulating cancer-stem like behaviors in relation to \u003cem\u003ePVT-1\u003c/em\u003e was assessed by performing a serial-dilution sarcosphere assay using HOS and CRL-7631 cell lines. The results determined that \u003cem\u003ePVT-1\u003c/em\u003e induced sarcosphere formation was dependent upon downstream suppression of TSC2 (Fig. 4h and Supplemental Fig. 3b and 3c). These results\u0026nbsp;provide evidence that \u003cem\u003ePVT-1\u003c/em\u003e is upstream of TSC2 and that TSC2 is a necessary intermediate for \u003cem\u003ePVT-1\u003c/em\u003e to induce oncogenic and cancer stem-like biological features.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;directly associates with TRIM28. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter identifying TSC2 as a downstream regulator of \u003cem\u003ePVT-1\u003c/em\u003e, further analysis was done to assess the molecular mechanism by detecting the protein binding partners of \u003cem\u003ePVT-1\u003c/em\u003e. ChIRP- mass spectrometry was performed on TCCC-OS63 osteosarcoma cell line as this cell line contains high endogenous levels of \u003cem\u003ePVT-1\u003c/em\u003e. ChIRP analysis identified that \u003cem\u003ePVT-1\u003c/em\u003e interacts with TRIM28, HSP90B1, and NOP2 and these proteins were absent in the \u003cem\u003elacZ\u003c/em\u003e (negative control) ChIRP protein elution (Fig. 5a). The identification of NOP2 as a binding partner for \u003cem\u003ePVT-1\u003c/em\u003e has previously been reported, thus providing validation to the ChIRP approach\u0026nbsp;(28). \u0026nbsp;As TRIM28 has been characterized as a regulator of cancer stem-like cells in breast and non-small cell lung cancer, further assessment was done to examine the\u0026nbsp;role of TRIM28 in regulating \u003cem\u003ePVT-1\u003c/em\u003e-induced behaviors\u0026nbsp;(29, 30). The \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction was validated through ChIRP-western blot (Fig. 5b) and direct \u003cem\u003ePVT-1\u003c/em\u003e-TRIM28 interaction was reciprocally confirmed using a TRIM28 RNA immunoprecipitation (RIP) assay in 8q24.2-amplified TCCC-OS63 cells, which found enrichment of \u003cem\u003ePVT-1\u003c/em\u003e in the TRIM28 pulldown versus the IgG control pulldown (Supplemental Fig. 4a). \u0026nbsp; In addition, RIP of HOS \u003cem\u003ePVT-1\u003c/em\u003e ctrl and HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells showed HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells have enhanced \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction compared to the HOS \u003cem\u003ePVT-1\u003c/em\u003e Ctrl cells (Fig. 5c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e8q24-amplification is also a marker of poor prognosis in breast cancer\u0026nbsp;(31, 32). In breast cancer, \u003cem\u003ePVT-1\u003c/em\u003e is a mediator of disease progression and TRIM28 has been previously reported to regulate breast cancer stem-like cells\u0026nbsp;(29, 30, 33, 34). Because of this concordance between breast cancer and osteosarcoma \u003cem\u003ePVT-1\u003c/em\u003e we examined if breast cancer cell lines with 8q24 amplification and elevated levels of \u003cem\u003ePVT-1\u003c/em\u003e have higher \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction (Supplemental Fig. 4b). \u0026nbsp;The 8q24 amplified breast cancer cell lines SK-BR-3, MCF7, and ZR-75-30 have enrichment of the \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction compared to the unamplified T47D and BT-474 cell lines\u0026nbsp;(Fig. 5d). \u0026nbsp; From these experiments, it can be concluded that \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction is not specific to osteosarcoma, but it may occur in to other 8q24-amplified malignancies, thus investigations into the biological significance of this complex are crucial.\u003c/p\u003e\n\u003cp\u003eAfter the identification of the novel \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 complex, the biological relevance of this interaction was examined between the non-coding RNA and TRIM28. To understand the effects of \u003cem\u003ePVT-1\u003c/em\u003e on TRIM28 the protein expression level of TRIM28 was examined. Western blot analysis determined that TRIM28 levels are comparable independent of \u003cem\u003ePVT-1\u003c/em\u003e expression levels, indicating that \u003cem\u003ePVT-1\u003c/em\u003e has no effect on TRIM28 stability (Supplemental Fig. 4c).\u0026nbsp;In addition, nuclear and cytoplasmic localization of TRIM28 was independent of \u003cem\u003ePVT-1\u003c/em\u003e expression (Supplemental Fig. 4d). \u0026nbsp;In order to understand the functional significance of TRIM28, shTRIM28 plasmid was transfected into HOS \u003cem\u003ePVT-1 o\u003c/em\u003e.e. cell lines (Supplemental Fig. 4e) and cell viability studies identified knockdown of TRIM28 decreases \u003cem\u003ePVT-1\u003c/em\u003e-induced proliferation and reduces the migratory and invasive potential of HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells (Fig. 5e and Supplemental Fig. 4f and 4g). Subsequently, we examined the importance of \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 axis in regulating self-renewal capacity by performing sarcosphere assays. We observed that HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. and shTRIM28 cells had a significant reduction in sarcosphere formation (Fig. 5f). Finally, stem cell profiling showed that \u003cem\u003ePVT-1\u003c/em\u003e and TRIM28 are necessary to enhance the transcriptomic and proteomic level of stem cell genes (Fig. 5g, 5h, and Supplemental Fig. 4h). In summary, \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction significantly contributes to the \u003cem\u003ePVT-1\u003c/em\u003e-induced downstream phenotypes. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTRIM28 interaction occurs at the 5\u0026rsquo; end of \u003cem\u003ePVT-1\u003c/em\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo decipher the region of \u003cem\u003ePVT-1\u003c/em\u003e that engages with TRIM28, the catRAPID algorithm was used to predict the binding region of RNA and target protein based upon physiochemical properties\u0026nbsp;(35, 36). There were two regions of \u003cem\u003ePVT-1\u003c/em\u003e that were noted to have the highest propensity for TRIM28 interaction, specifically nucleotide regions 40-125 and 586-671 of \u003cem\u003ePVT-1\u003c/em\u003e (Supplemental Fig. 5a). Besides making the \u003cem\u003ePVT-1\u003c/em\u003e constructs with deletion of the predicted regions 40-125 and 586-671, three additional constructs were made that deleted the 1-978, 489-1469, or 979-1957 nucleotide regions spanning the 5\u0026rsquo; half, 3\u0026rsquo; half, and a middle deletion region overlapping the 5\u0026rsquo; and 3\u0026rsquo; regions to biochemically confirm the interacting region(s) (Fig. 6a). These five constructs along with full length \u003cem\u003ePVT-1\u003c/em\u003e were stably transfected into the HOS cell line and expression was confirmed by qPCR (Fig. 6b). TRIM28 RIP was used to assess differential \u003cem\u003ePVT-1\u003c/em\u003e enrichment for the various constructs. Analysis of the qPCR data determined that \u003cem\u003ePVT-1\u003c/em\u003e enrichment was negligible in the cell lines where 40-125 and 1-978 regions were deleted (Fig. 6c). PCR analysis validated the RIP data as the \u003cem\u003ePVT-1\u003c/em\u003e was not present in the 40-125 and 1-978 mutant cells (Supplemental Fig. 5b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the biological impact caused by perturbing the direct \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction, the oncogenic and self-renewal capacity was examined using the HOS mutant cells to full length \u003cem\u003ePVT-1\u003c/em\u003e o.e cells (FL). Assessment of downstream \u003cem\u003ePVT-1\u003c/em\u003e effectors determined that lack of \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction impairs the enhancement of stem cell protein level caused by full length \u003cem\u003ePVT-1\u003c/em\u003e (Fig. 6d). Additional downstream signaling analysis determined that the lack of interaction increased TSC2 expression (Fig. 6e). \u0026nbsp;Phenotypic studies determined that the lack of interaction prevented \u003cem\u003ePVT-1\u003c/em\u003e from promoting proliferation, migration, and invasion capacity (Fig. 6f and 6g). To assess self-renewal capacity through sarcosphere assay, it was observed that there was a lack of sarcosphere formation in the 1-978 and 40-125 mutant cells (Fig. 6h). These results collectively indicate that specific binding of \u003cem\u003ePVT-1\u003c/em\u003e to TRIM28 is critical for degradation of TSC2 and induction of cancer stem-like and oncogenic properties.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePVT-1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e/TRIM28 leads to increased TSC2 ubiquitination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter determining that \u003cem\u003ePVT-1\u003c/em\u003e reduces TSC2 levels, and that TSC2 is an important modulator in regulating oncogenesis and tumor-initiation maintenance, further mechanistic studies assessed whether the \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 axis is necessary for TSC2 reduction. The\u0026nbsp;lack of TRIM28 in HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells prevent the decrease in TSC2 protein expression (Fig. 7a). As TRIM28 is known to cause TSC2 ubiquitination, a TSC2 co-IP was performed to examine the importance of \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 in regulating the level of TSC2 ubiquitination\u0026nbsp;(37, 38). The HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells had an increase in ubiquitination of TSC2 as compared to the \u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. + shTRIM28 cells (Fig. 7b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause \u003cem\u003ePVT-1\u003c/em\u003e requires TRIM28 for enhanced TSC2 ubiquitination, additional studies were performed to elucidate the molecular mechanism that leads to enhanced TSC2 ubiquitination. Prior reports show TRIM28 SUMOylates and activates\u0026nbsp;Vps34, which leads to the recruitment of TSC1 to Vps34 and disengagement of TSC2 from the TSC1/TSC2 heterodimer\u0026nbsp;(37, 38). The unbound TSC2 gets ubiquitinated and subsequently degraded\u0026nbsp;(37). \u0026nbsp;By utilizing Vps34 co-IP, the level of Vps34 binding to TRIM28 could be examined. \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells showed an increase interaction of TRIM28 to Vps34, which was negligible in the \u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. + shTRIM28 cells (Fig. 7c). As the formation of TRIM28 and Vps34 complex causes Vps34-SUMO this modification was assessed and determined that overexpression of \u003cem\u003ePVT-1\u003c/em\u003e causes Vps34 SUMOylation as the molecular weight of Vps34 molecular weight increased by approximately 40 kDa due to addition of SUMO adducts (Fig. 7c) and increased Vps34/TSC1 interaction (Fig. 7c and 7d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince the TSC1/TSC2 heterodimer decoupling is associated with TSC2 ubiquitination, the \u0026nbsp;level of TSC1/TSC2 complex was examined using a TSC1 co-IP\u0026nbsp;(37). To prevent TSC2 degradation, the cells were treated with MG132, a proteasome inhibitor, prior to performing the TSC1 co-IP. We observed that the \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells lacked TSC1 binding to TSC2 (Fig. 7d). These studies suggest that \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 axis is critical for formation of the \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28/Vps34-SUMO/TSC1 complex and subsequent degradation of TSC2. A representative blot from the TSC2 co-IP includes protein-of-interest pulldown, IgG pulldown, and input for the \u003cem\u003ePVT-1\u003c/em\u003e Ctrl and \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells (Supplemental Fig. 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction leads to enhancement of Vps34 SUMOylation, an additional cell viability assay was performed to assess if \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells would be more sensitive to a small molecule SUMOylation inhibitor, TAK-981. TAK-981 covalently binds to SUMO to form an adduct and prevents SUMO conjugation to the protein of interest\u0026nbsp;(39). The \u003cem\u003ein vitro\u003c/em\u003e drug sensitivity study determined that HOS \u003cem\u003ePVT-1\u003c/em\u003e o.e. cells are significantly more sensitive to TAK-981 when compared to HOS \u003cem\u003ePVT-1\u003c/em\u003e Ctrl\u0026nbsp;cells (Fig. 7e). The data confirms increased expression of \u003cem\u003ePVT-1\u003c/em\u003e activates SUMOylation and targeting this post-translational modification could be a treatment for malignancies with overexpression of the \u003cem\u003ePVT-1\u0026nbsp;\u003c/em\u003e, including 8q24.2 amplified tumors.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOne of the current obstacles in the development of effective osteosarcoma targeted treatment is identifying viable molecular targets\u0026nbsp;(1, 40). The current study elucidated a novel mechanism underlying the interaction between \u003cem\u003ePVT-1\u003c/em\u003e and TRIM28, and the ability of this complex to induce cancer stem-like behaviors in osteosarcoma. \u0026nbsp;Specifically, using proteomic analysis, the research identified that \u003cem\u003ePVT-1\u003c/em\u003e induces stem cell like behaviors by inhibiting the tumor suppressor TSC2 protein level. This work is the first to link \u003cem\u003ePVT-1\u003c/em\u003e to regulation of TSC2 and the research discovered that through formation of a complex with TRIM28, \u003cem\u003ePVT-1\u003c/em\u003e is able to ubiquitinate TSC2, while rescuing TSC2 prevents \u003cem\u003ePVT-1\u003c/em\u003e-induced cancer-stem like behaviors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious reports have identified that lncRNAs regulate biological behaviors when interacting with proteins\u0026nbsp;(41, 42). The ChIRP-mass spectrometry analysis identified TRIM28, HSP90B1, and NOP2 as potential binding partners for \u003cem\u003ePVT-1\u003c/em\u003e. Additional studies assessed the role of TRIM28 in relationship to \u003cem\u003ePVT-1\u003c/em\u003e as TRIM28 has been implicated as a regulator of cancer stem-like behaviors (33,34). These additional functional studies identified that this complex is critical for the induction of tumorigenic and cancer stem-like behaviors. In addition, these studies are able to link this complex to regulation of the downstream TSC2 signaling cascade. While the studies have assessed the importance of \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 interaction to osteosarcoma cancer stem cell properties, there could be self-renewal capacity and/or cancer progression importance to the \u0026nbsp;\u003cem\u003ePVT-1\u003c/em\u003e/HSP90B1 and \u003cem\u003ePVT-1\u003c/em\u003e/NOP2 interactions, \u0026nbsp;which could not be ruled out and additional functional studies would be critical to assess the importance of these complexes. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs this research has determined that enhanced \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 complex suppresses TSC2, mechanistically further work was performed to understand the intermediates between \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 and TSC2. While previous reports link TRIM28 to ubiquitination of TSC2, the role of \u003cem\u003ePVT-1\u003c/em\u003e in relationship to this mechanism has not been discovered\u0026nbsp;(37, 38). In this study, co-IP studies identified that TRIM28 directly binds to \u003cem\u003ePVT-1\u003c/em\u003e causing TRIM28 interaction to Vps34 and activation of Vps34 through SUMOylation. With additional research identifying that osteosarcoma cells with Vps34 SUMOylation have increased TSC2 ubiquitination, and thus enhanced oncogenic and tumor-initiation potential. This research identified an attractive therapeutic target, the SUMOylation of Vps34. SUMOylation has been implicated in regulating cancer stem cell behaviors in different malignancies, and TAK-981, a SUMOylation inhibitor, is presently being evaluated as an alternative therapeutic intervention for both solid and hematological malignancies (ClinicalTrials.gov Identifier: NCT03648372 and ClinicalTrials.gov Identifier: NCT04074330)(43). While our research has identified that osteosarcoma cells with elevated \u003cem\u003ePVT-1\u003c/em\u003e are more sensitive to TAK-981, there is still an abundance of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e research needed in order to understand how viable this agent is for osteosarcoma therapy. Previous reports have found that SUMOylation inhibitors can increase chemosensitivity\u0026nbsp;(44, 45). Therefore, inhibition of SUMOylation in tumors with high expression of \u003cem\u003ePVT-1\u003c/em\u003e may be a novel therapeutic approach for patients with osteosarcoma.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur mechanistic studies have identified the downstream molecular events dictated by the \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 complex, however further analysis is required to understand how \u003cem\u003ePVT-1\u003c/em\u003e recruits TRIM28. TRIM28 is thought to recruit proteins to assist in downstream ubiquitination, as seen with TRIM28 association with TRIM24 for Speckle-type POZ protein ubiquitination\u0026nbsp;(46). Therefore, a potential mechanism to how \u003cem\u003ePVT-1\u003c/em\u003e recruits TRIM28 could be through protein-mediated facilitation. By having insight into the formation of \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 axis, there may be additional avenues to inhibit \u003cem\u003ePVT-1\u003c/em\u003e-induced cancer stem cell behaviors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, previous reports have alluded to the possibility of \u003cem\u003ePVT-1\u003c/em\u003e induced phenotypes being dependent upon MYC, which is a neighboring oncogene in the 8q24.2 region\u0026nbsp;(10). \u0026nbsp;To assess if this relationship is true in osteosarcoma, the proliferation and stem cell profile of \u003cem\u003ePVT-1\u0026nbsp;\u003c/em\u003eo.e. +/- siMYC was examined. It was found that suppressing MYC does not prevent either \u003cem\u003ePVT-1\u003c/em\u003e-induced proliferation or enhancement of stem cell marker expression (Supplemental Fig. 7). This analysis was able to confirm that \u003cem\u003ePVT-1\u003c/em\u003e is not dependent upon MYC to promote tumorigenic and cancer-stem cell behaviors. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, the current study provides new insights linking \u003cem\u003ePVT-1\u003c/em\u003e and TRIM28 to activation of tumorigenesis and cancer stem-like behaviors. By understanding the \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 downstream signaling cascade, this research was able to target an intermediate within this pathway for potential inhibition of the cancer stem-like behaviors (Fig. 7f). Lastly, this work demonstrated that the \u003cem\u003ePVT-1\u003c/em\u003e/TRIM28 axis in not specific to osteosarcoma but is also present in breast cancer. By recognizing this connection, the research may be of use to cancers that present with 8q24 amplification.\u0026nbsp;\u003c/p\u003e"},{"header":"Declaration","content":"\u003cp\u003e\u003cstrong\u003eFinancial Support:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSusan V. Tsang: Cancer Prevention Institute of Texas (CPRIT) RP160283\u003c/p\u003e\n\u003cp\u003eKimal Rajapakshe: (CPRIT) RP170005, NIH P30 shared resource grant CA125123, and NIEHS P30 Center grant 1P30ES030285\u003c/p\u003e\n\u003cp\u003eCristian Coarfa: (CPRIT) RP170005, NIH P30 shared resource grant CA125123, and NIEHS P30 Center grant 1P30ES030285\u003c/p\u003e\n\u003cp\u003eJason T. Yustein: The Faris D. Virani Ewing Sarcoma Center, (CPRIT) RP140022-P1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003eThe authors declare no potential conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the Advanced Technologies Core services at the Baylor College of Medicine. This work was funded by The Faris D. Virani Ewing Sarcoma Center (to J.T. Yustein). S.V. Tsang was partially supported by Cancer Prevention Institute of Texas (CPRIT) RP160283, K. Rajapakshe and C. Coarfa were partially supported by (CPRIT) RP170005, NIH P30 shared resource grant CA125123, and NIEHS P30 Center grant 1P30ES030285; CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (CA125123 and RR024574 to the Cytometry and Cell Sorting Core at Baylor College of Medicine); and Core Facility Award (CPRIT-RP170005 to Proteomics and Metabolomics Core at Baylor College of Medicine). We thank Dr. Suzanne Fuqua and Dr. Xiang Zhang for generously providing the breast cancer cell lines. The authors would like to thank Dr. Hilsenbeck for running the generalized linear model analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOttaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer treatment and research. 2009;152:3-13.\u003c/li\u003e\n\u003cli\u003eGibbs CP, Kukekov VG, Reith JD, Tchigrinova O, Suslov ON, Scott EW, et al. Stem-like cells in bone sarcomas: implications for tumorigenesis. 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Nature communications. 2018;9(1):5007.\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"lncRNA, PVT-1, cancer-stem cells, osteosarcoma, and TRIM28","lastPublishedDoi":"10.21203/rs.3.rs-1813153/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1813153/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Osteosarcoma, the most common pediatric bone tumor, is an aggressive heterogeneous malignancy defined by complex chromosomal aberrations. Overall survival rates remain at approximately 70%, but patients with chemoresistant or metastatic disease have extremely poor outcomes of less than 30%. A subgroup of tumors harbor amplification of chromosome 8q24.2 and increased expression of the oncogenic long noncoding RNA (lncRNA) Plasmacytoma Variant Translocation-1 (PVT-1), which is associated with an extremely poor clinical prognosis. Through in vitro assays, this study demonstrated that PVT-1 is critical for osteosarcoma tumor-initiation potential. Chromatin Hybridization by RNA Purification analysis identified Tripartite-Motif Containing Family 28 (TRIM28) as a novel PVT-1 binding partner. Mechanistically, co-immunoprecipitation studies showed the PVT-1/TRIM28 complex binds and increases SUMOylation of phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34), which leads to enhanced ubiquitination and degradation of tumor suppressor complex 2 (TSC2), thus contributing to stem cell phenotypes. Importantly, osteosarcoma cells with increased PVT-1 have enhanced sensitivity to a SUMOylation inhibitor, TAK-981. Altogether, this study elucidated a role for PVT-1 in the enhancement of cancer stem-like behaviors, including migration and invasion, in osteosarcoma, and identified the novel PVT-1/TRIM28 axis signaling cascade as a potential therapeutic target for osteosarcoma treatment.","manuscriptTitle":"LncRNA PVT-1 promotes osteosarcoma cancer stem-like properties through direct interaction with TRIM28 and TSC2 ubiquitination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-29 18:01:32","doi":"10.21203/rs.3.rs-1813153/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2022-09-05T10:29:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-08-09T12:15:33+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-07-25T14:33:35+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2022-07-25T12:58:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-01T10:10:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-06-30T20:35:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2022-06-30T20:35:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1ed99b44-77ef-40e4-a4d5-2c15ce486b62","owner":[],"postedDate":"July 29th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2022-11-09T08:10:15+00:00","versionOfRecord":{"articleIdentity":"rs-1813153","link":"https://doi.org/10.1038/s41388-022-02538-w","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2022-11-08 05:00:00","publishedOnDateReadable":"November 8th, 2022"},"versionCreatedAt":"2022-07-29 18:01:32","video":"","vorDoi":"10.1038/s41388-022-02538-w","vorDoiUrl":"https://doi.org/10.1038/s41388-022-02538-w","workflowStages":[]},"version":"v1","identity":"rs-1813153","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1813153","identity":"rs-1813153","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}