Validation of ITGB1BP1 as a novel transcriptional target of CD44-Promoted Breast Cancer Progression

Validation of ITGB1BP1 as a novel transcriptional target of CD44-Promoted Breast Cancer Progression | 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 Research Article Validation of ITGB1BP1 as a novel transcriptional target of CD44-Promoted Breast Cancer Progression Hanan Nazar, Salma M.S. Ahmad, Mizanur Rahman, Semir Vranic, Allal Ouhtit This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7083073/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background: Breast cancer (BC) is one of the most common cancers worldwide, and metastasis is its worst aspect and the first cause of death. Metastasis is a multistep process, where invasion is a recurring event. The process of BC cell invasion involves, in general, three major factors including, cell adhesion molecules (CAM), proteinases, and growth factors. CD44, a family of CAM proteins and the hyaluronic acid (HA) cell surface receptor, acts as cell differentiation, cell migration/invasion, and apoptosis regulator. To better understand the molecular mechanisms that underpin CD44-promoted BC cell invasion, our previous microarray gene expression profiling, using “TET-OFF” BC CD44-inducible cell experimental model, identified ITGB1BP1 as a novel potential transcriptional target of CD44. Methods: to test this hypothesis, both in vitro BC cell model, as well as ex-vivo tissue microarray (TMA) slides, containing adjacent sections from breast tumors of 113 BC patients were examined for the expression of both CD44 and its potential target gene expression, ITGB1BP1, by immunohistochemistry (IHC). Results: in vitro results revealed that HA-activation and induction of CD44 increased significantly the expression of its target gene, ITGB1BP1. Furthermore, IHC analysis of TMAs showed that overall 90% of the samples exhibited high CD44-immunostaining in the membrane while its target ITGB1BP1 was mainly observed in the cytoplasm and occasionally in the membrane. More interestingly, the patterns of expression of CD44 and its target ITGB1BP1 showed a parallel increase in the majority of highly invasive/metastatic tumor tissues when compared to less invasive breast tumor tissues. Consclusion: the present study provides for the first time substantial evidence supporting our hypothesis that ITGB1BP1is a potential novel transcriptional target that underpin CD44-promoted BC tumor cell progression. Breast cancer Metastasis CD44 Hyaluronan ITGB1BP1 Tissue Microarray Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Cancer is a universally fatal disease that affects millions of people worldwide [ 1 ]. It is a genetic disease that can be either inherited or acquired due to DNA damage caused by exposure to harmful environmental factors such as chemicals in tobacco and alcohol, as well as ultraviolet (UV) radiation. In 2020, 19.3 million new cancer cases were reported, and 10 million cancer-related deaths were recorded in the same year [ 2 ]. Globally, approximately 627,000 women die from BC each year, accounting for 15% of all cancer-related deaths [ 3 ]. Since BC remains the most prevalent cancer among females, data from Gulf Cooperation Council (GCC) countries align closely with international trends [ 4 ]. Risk factors for BC include hormonal exposure, menarche history, aging, physical inactivity, tobacco smoking, alcohol consumption, breast tissue characteristics, and lifestyle factors [ 5 – 6 ]. Metastasis is the most threatening aspect of cancer, and tumor cell invasion is a defining event in the metastatic process. Understanding its mechanisms is crucial for developing effective anti-metastatic therapies [ 7 ]. Cell invasion is regulated by multiple signaling pathways involving various proteins, including cell adhesion molecules (CAMs), proteases, and growth factors [ 3 , 4 ]. CAMs play a critical role in both early and late cancer development by mediating cell-cell and cell-extracellular matrix (ECM) interactions [ 5 ]. One of the key CAMs is CD44, the major hyaluronan (HA) receptor. In addition to its role in cellular adhesion, CD44 is significantly involved in intracellular signaling, regulating cell growth, proliferation, and motility [ 8 ]. While the role of CD44 in BC was initially controversial, our earlier work using a Tet-off-inducible system in a transgenic mouse model revealed that CD44 induction promotes BC metastasis to the liver [ 7 ]. To further investigate the precise molecular role of CD44 in BC cell invasion and metastasis, we applied a CHIP-microarray gene expression profiling approach to identify the transcriptional targets of CD44/HA signaling in BC. Our CD44-CHIP analysis identified over 200 novel potential downstream transcriptional target genes. Among them, three genes (Cortactin, Survivin, and TGF-β2) have been validated and published, along with their associated signaling pathways in CD44-promoted BC cell invasion [ 9 – 12 ]. In this study, we selected the large isoform of integrin subunit beta 1 binding protein 1 (ITGB1BP1)—also known as Integrin Cytoplasmic Domain-Associated Protein 1 (ICAP-1A) from the CHIP-microarray screen for further validation based on the following observations: i) ITGB1BP1 expression increased 2.5-fold upon CD44 induction in BC cells; ii) ITGB1BP1 activates KRIT-1, triggering NOTCH signaling, which regulates the PI3K/AKT pathway, promoting BC progression and survival [ 13 , 14 ]; iii) ITGB1BP1 overexpression promotes tumor cell invasion via interaction with oncogenic pathways [ 15 ]; iv) ITGB1BP1 is associated with cell adhesion, migration, and fibronectin matrix organization [ 16 ]; v) ITGB1BP1 plays a role in cell-ECM communication, allowing cells to adapt to ECM density by modifying adhesion and migration responses [ 17 ]; and vi) ITGB1BP1 is critical in angiogenesis, proliferation, differentiation, adhesion, migration, and invasion [ 16 – 18 ]. Given these observations, we hypothesize that ITGB1BP1 is a novel transcriptional target gene that mediates CD44-promoted BC cell invasion. To test this hypothesis and validate whether ITGB1BP1 protein expression correlates with CD44 levels, we employed two strategies: i) In vitro BC cell model: We examined total RNA samples and protein lysates from various BC cell lines representing different tumor stages to assess the expression of both CD44 and its potential transcriptional target; ii) Ex vivo tissue microarray analysis: We analyzed breast tumor tissue microarray slides containing adjacent sections from 113 Syrian BC patients, using immunohistochemistry (IHC) to determine the expression patterns of both CD44 and ITGB1BP1, as previously reported [ 19 ]. 2. Results 2.1.HA-activation of CD44 expression increases ITGB1BP1 RNA expression MDA-MB-231 cells were cultured in the presence or absence of HA to structurally validate ITGB1BP1 as a transcriptional target of CD44. RT-PCR was carried out to confirm an increased expression of CD44 and its target ITGB1BP1 in MDA-MB-231 cells at different time points. Transcription analysis of the results from three replicate experiments demonstrated that CD44 and its transcriptional target ITGB1BP1 mRNA levels increased simultaneously in the BC cell line MDA-MB-231 24 hours after HA treatment. As a result, no increase in ITGB1BP1 mRNA was observed when HA was not present ( Fig. 1 ) 2.2.HA-induction of CD44 expression increases ITGBPB1protein expression Analysis of ITGBPB1 protein expression in MDA-MB-231 cells after exposure to 100 µg/ml HA (MW 220 kDa) for 24 hours to ensure induction of CD44 signaling, the temporal relationship between CD44 signaling and ITGBPB1 gene regulation was also examined. According to immunoblot experiments conducted on samples extracted from MDA-MB-231 cells under identical conditions such as those used to collect mRNA samples from the microarrays, HA induces CD44 expression, as demonstrated by an immunoreactive band to the anti-human CD44 antibody at 85 kDa (Fig. 2 A and B). In MDA-MB-231 cells stimulated with HA for 24 h, immunoblotting confirmed an increase in ITGB1BP1 expression parallel to CD44 induction (Fig. 2 A and B). 2.3.Expression of CD44 and ITGB1BP1 in breast cancer cell lines To validate whether the expression of ITGB1BP1 increases upon induction of CD44 by HA, we first addressed the cell line specificity of the relationship between CD44 and the regulation of its potential target ITGB1BP1 transcription/expression using an in vitro BC cell model. In other words, to validate ITGB1BP1 as a true transcriptional target of CD44, we mimicked the BC cell progression using in vitro cell model and wondered whether the expression pattern of CD44 would follow the same pattern of expression of its transcriptional target ITGB1BP. A cell type representing each of the various stages of the BC progression (from the normal breast cell line derived from benign proliferative breast tissue and spontaneously (MCF10A), to the primary Luminal A (MCF-7) to the triple negative cell line (MDA-MB-231). In order to determine if CD44 and ITGB1BP both play equal roles in BC metastasis, we first compared the mRNA levels of CD44 and ITGB1BP1 across seven different BC cell lines ranging in aggressiveness (Table 1 : MCF10A, ZR-75-1, MCF7, SKBR3, HCC1954, MDA-MB-468, and MDA-MB-231). Figure 4. The protein expression of Both CD44 and its target ITGB1BP1 incresaeed in parallel during breast cancer cell progression. A : Western blot results showing the expression of CD44 and its transcriptional target ITGB1BP1 in various BC cell lines ranging from low to highly invasive phenotype. Beta-actions were used as an internal control. B : Semi-quantification of CD44 and its transcriptional target ITGB1BP1 relative protein expression levels in different BC cell lines; The analysis of gel bands was performed by ImageJ software and normalized to beta-actin gel bands. Mean values (n = 3) of three experiments are shown. Statistical analysis was performed using one-way analysis of variance (ANOVA). Tukey’s post-hoc test was conducted to compare treatment groups and data are statistically significant when p < 0.05 compared to the control (* p < 0.05 , ** p < 0.01 *** p < 0.001 ). 2.4.Expression of CD44 and ITGB1BP1 in breast tumor tissue microarrays Tissue microarrays (TMAs) have become an invaluable tool in cancer research to evaluate the expression and subcellular localization of proteins in cells and tissues. In order to test and validate our hypothesis that ITGB1BP1 is a transcriptional target of CD44, the expression of both CD44 and its potential transcriptional target ITGB1BP1 was examined in human breast tissue samples by immunohistochemistry using TMA slides representing 113 breast tumor tissue samples (31 patients were in grade 2 whereas 82 patients were in Grade 3) (Fig. 3 ). Using our Tet-Off CD44-inducible BC cell system in mouse model, we have previously reported that induction of CD44 in vivo caused a dramatic increase in CD44 protein expression levels that correlated with metastasis of primary breast tumors to the liver [ 5 ]. In the present investigation, we used the same approach and examined the expression patterns of both CD44 and its novel transcriptional target ITGB1BP1 in TMA slides containing adjacent sections from each of the 113 breast tumor tissues. Our results revealed that: i) the patterns of expression of CD44 and its transcriptional target ITGB1BP1 showed a parallel increase when comparing the primary breast tumors (ductal carcinoma in situ ) to the invasive tumors (Table 3 ); and ii) increased expression of CD44 and its transcriptional target ITGB1BP1 correlated with tumor malignancy and invasiveness (Table 3 ). On the other hand, CD44 immunostaining was strongly membrane-associated and was present in both basal and luminal cells of most samples (Fig. 5 ), but immunostaining showed a gradient of decreasing intensity from basal to luminal cell type (More aggressive to less aggressive BC). ITGB1BP1 immunostaining was both membrane-associated and cytoplasmic and followed a pattern similar to CD44, being strongly expressed in basal cells, but was low or absent in benign tumor cells (Fig. 5 ) . Table 2 The percentage of CD44 and ITGB1BP1 immuno-reactivity in all breast tissues investigated. Breast Tumor type # of cases CD44-immunopositive (% ) ITGB1BP1-immunopositive ( % ) Grade 2 31 20/31 (64.5%) 22/31 (71%) Grade 3 82 77/82 (93.3%) 80/82 (97.6%) Metastatic 38 38/38 (100%) 38/38 (100%) Overall Tumors 113 97/113 (85.8%) 101/113 (90.2%) 3. Discussion Based on our initial work and findings from the literature, CD44’s role in cancer metastasis has been controversial [ 22 – 24 ]. To address this discrepancy and investigate the function of the standard form CD44s in breast cancer (BC) invasion and metastasis, while further elucidating its downstream signaling, we developed a tetracycline (Tet)-Off-regulated expression system for CD44s, both in vitro and in vivo [ 5 , 10 ]. Using a microarray technique (12K CHIP from Affymetrix), we identified more than 200 potential CD44s target genes. Among these, we have already validated three novel transcriptional targets—Cortactin, Survivin, and TGF-β2—along with their molecular signaling pathways that underpin CD44/HA-promoted BC cell invasion [ 9 – 12 ]. In the present study, ITGB1BP1 was selected as an additional novel transcriptional target gene based on the following observations: i) It showed a 2.5-fold upregulation upon CD44 induction; ii) ITGB1BP1 plays a key role in cell adhesion, migration, and invasion [ 12 – 18 ]; and iii) ITGB1BP1 interacts with the PI3K/AKT pathway, promoting breast tumor progression and survival [ 13 – 14 ]. Our results showed that both CD44 and its transcriptional target ITGB1BP1 increased simultaneously at the mRNA and protein levels in the BC cell line MDA-MB-231 after 24 hours of activation by its major ligand, HA. Furthermore, both CD44 and ITGB1BP1 exhibited parallel expression patterns at the mRNA and protein levels in a BC cell model that mimics BC progression from normal/benign to malignant/metastatic stages. More interestingly, TMA slides containing breast tumor tissues from 113 BC patients, examined by IHC analysis, revealed a parallel increase in CD44 and ITGB1BP1 expression from less invasive to highly invasive breast tumors. Approximately 85% of the samples showed CD44 localization on both the membrane and cytoplasm of BC tissues, consistent with the Human Protein Atlas, which previously reported CD44 expression in BC tissues. Previous studies validated CD44 membrane localization due to its activity as a receptor for multiple ligands—including HA—to facilitate BC progression and metastasis [ 25 – 27 ]. Moreover, recent studies on melanoma confirmed cytoplasmic CD44 localization, attributed to WNT pathway activation, which enhances direct CD44–Cortactin interactions and upregulates CD44 and c-Myc, indicating a positive feedback loop that promotes tumor proliferation, migration, and invasion [ 28 ]. Additionally, ITGB1BP1 was localized in the cytoplasm of BC tissues, consistent with studies on cerebral cavernous malformation, which demonstrated ITGB1BP1 expression in both the cytoplasm and nucleus [ 17 ]. Notably, ITGB1BP1 enhances cell proliferation and survival by binding Nm23-H2, leading to nuclear translocation and induction of cyclin D1 and c-Myc gene expression [ 29 ]. Our study further demonstrated that CD44 activation via HA binding led to the induction of ITGB1BP1, with their expression patterns paralleling one another in both our in vitro BC progression model and breast tumor tissues (from less invasive to highly metastatic stages). These findings suggest that CD44-induced ITGB1BP1 expression promotes BC cell invasion and progression. Several studies have already confirmed the crucial role of HA-CD44 signaling in BC proliferation and invasion [ 25 , 27 , 29 , 30 ]. Specifically, CD44 induced cell invasion by activating the RhoA GTPase/ROCK-1 signaling pathway [ 30 ]. ITGB1BP1 and Nm23-H2 interact via RhoA-GTPase, further supporting CD44-driven tumor proliferation and invasion [ 29 ]. Additionally: i) CD44 upregulates c-Myc expression [ 31 ]; ii) CD44 activates ERK, leading to cyclin D1 upregulation [ 32 ]; iii) ERK phosphorylation promotes BC cell proliferation and migration [ 33 ]; iv) ROCK-1 and ITGB1BP1 form a complex at the plasma membrane, where RhoA co-localizes with β1 integrin [ 29 ]; and v) RhoA GTPase/ROCK pathway activation by ITGB1BP1 enhances cell migration [ 29 ]. Interestingly, CD44-HA interaction also activates RhoA GTPase, which recruits IP3 receptors to intracellular calcium storage organelles, leading to calcium release [ 33 ]. Collectively, these findings—combined with our results—support the hypothesis that ITGB1BP1 is a novel transcriptional target of CD44, driving cell proliferation, adhesion, migration, and invasion, as we have recently reported [ 18 ]. Furthermore, KRIT-1 binds to ITGB1BP1 through its PTB domain, competing with β1 integrin for binding [ 34 , 35 ]. Additionally, ITGB1BP1 and KRIT-1 promote Notch signaling, activating PI3K/AKT and enhancing tumor cell survival and invasion [ 14 ]. A major component of CD44's role in tumor survival and motility is its regulation of the PI3K/AKT pathway [ 18 – 29 , 32 , 33 , 36 ]. In fact, we previously demonstrated that CD44 promotes BC invasion via the PI3K/AKT pathway [ 9 – 12 ]. 4. Materials and Methods 4.1.Cell culture Six human BC cell lines (MDA-MB-231, MDA-MB-468, HCC-1954, SkBr3, ZR751, and MCF-7), as well as the immortalized human breast cell line MCF10A, supplied by our collaborator Dr. Al-Moustafa, were used in the present study as described in Table 1 . The cell lines MDA-MB-231 and MDA-MB-468 were cultured in 90% Dulbecco's Modified Eagle Medium (DMEM), while the HCC-1954, SkBr3, MCF-7 and ZR751 cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640). All culture media were supplemented with 1% L-Glutamate, 50 ml Fetal Bovine Serum Heat Inactivated Collected in South America (FBS) (Thermo Scientific, USA), and 1% Penicillin Streptomycin antibiotic (Pen Strep) (Gibco, USA). According to our protocol, MCF10A breast cells were cultured in DMEM and 2% glutamine. All the cells were then incubated in a 37°C humidified incubator with 5% CO 2 . Cells were incubated in the presence or absence of 100 µ g/ml HA, and protein lysates and RNA were collected at both 12 and 24 hours after HA treatment. Table 1 The different cell lines used for the in vitro models. Name Cancer Subtype Type of Tumor BRCA1 Mutation HER2 ER PR MDA-MB-231 Triple-Negative A Adenocarcinoma Wildtype - - - MDA-MB-468 Triple-Negative A Adenocarcinoma Wildtype - - - HCC1954 HER2 Positive Ductal Carcinoma Wildtype + - - SKBR3 HER2 Positive Adenocarcinoma Wildtype + - - MCF7 Luminal A Invasive Ductal Carcinoma Wildtype - + + ZR751 Luminal A Invasive Ductal Carcinoma Wildtype - + +/- MCF10A Basal Epithelial Cell Line Fibrocystic Disease Wildtype - - - 4.2.RNA Extraction and cDNA synthesis Gene transcription analysis was performed following HA treatment of MDA-MB-231 cells, and RNA samples were collected at 12 and 24 hours time points. Briefly, total cellular RNA was obtained using the TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA concentration and purity were determined by NanoDrop ND 1,000 Spectrophotometer (Nanodrop Technologies Inc., Delaware, USA). Ribosomal RNA band integrity was carried using 1% agarose gel electrophoresis. Synthesis of cDNA was performed only on samples whose 260/280 ratio values ranged from 1.8 to 2.0. Reverse transcription was performed with 2 µg of RNA, using random primers and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, UK), according to the manufacturer's instructions. Mock reverse transcription reaction without reverse transcriptase was utilized as a negative control. The RT-PCR reaction was carried as we have previously reported [ 10 ], and RT-PCR products were run on 2% agarose gel containing 5 mg/ml ethidium bromide. Annealing temperatures for the CD44, ITGB1BP1, and GAPDH genes, along with the oligonucleotide primers used, are shown in Table 2 . Table 3 Temperature and specific oligonucleotide primers used for Reverse Transcription-PCR. Genes Temperature (◦C) Oligonucleotide Primers CD44 56 5'-TTT GCA TTG CAG TCA ACA GTC-3' (Forward) 5'-GTT ACA CCC CAA TCT TCA TGT CCA C-3' (Reverse) ITGB1BP1 50 5'-TTT GCA TTG CAG TCA ACA GTC-3' (Forward) 5'-GTT ACA CCC CAA TCT TCA TGT CCA C-3' (Reverse) GAPDH 52 5'-GAAGGCCATGCCAGTGAGCT-3' (Forward) 5'-CCGGGAAACTGTGGCGTGAT-3' (Reverse) 4.3.Real-time qRT‐PCR Real-time qRT-PCR was carried out as we have previously reported [ 20 ]. Briefly, 1 µg of total RNA extracted using GeneJET RNA Purification Kit (Thermo Fisher Scientific) was reversed transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-qPCR assay was performed to determine the expression of both CD44 and its target gene ITGB1BP1 using TaqMan® Fast Advanced Master Mix (Applied Biosystems). For each reaction, 1ul of diluted cDNA (1:4) was added to 10 µl of TaqMan Master Mix, 1 µl of BRIP1 Gene Expression TaqMan® Assays FAM‐MGB Probe (4351372, Applied Biosystems), and 1µl of GAPDH Gene Expression TaqMan® Assays VIC‐MGB Probe (4448489, Applied Biosystems) used as an endogenous control, and 7µl of nuclease‐free water. The reaction was performed using the QuantStudio™ 6 Flex Real‐Time PCR System (Applied Biosystems, Inc) as previously reported [ 20 ]: 10 minutes at 95°C (stage 1); 20 seconds at 95°C and 20 seconds at 60°C for 40 cycles (stage 2). The results were analysed using the QuantStudio™ 6 software. 4.4.Western Blotting Whole cell lysates were collected from MDA-MB-231 cells following HA treatment 12 and 24 post-treatment, using RIPA buffer supplemented with protease inhibitors (Pierce, USA). According to the manufacturer's detailed protocol, PierceTM BCA Protein Assay Kit (Thermo ScientificTM) was used to quantify proteins. Denatured cell lysates were analyzed by SDS-PAGE, using 20 µg from each protein sample. Membranes were probed with primary antibodies overnight at 4°C, using the Rabbit polyclonal antiCD44 antibody (ab157107, Abcam), and Rabbit polyclonal anti-ITGB1BP1 antibody (PA5-97883, Thermo Fisher Scientific). The membranes were then probed with HRP-conjugated anti-rabbit secondary antibody (A0545, Sigma) for CD44 and ITGB1BP1, and anti-mouse secondary antibody (PAB0096, Abnova) to detect the β‐Actin control protein. The signal was revealed with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and was developed using Chemiluminescent GeneGnome (Syngene). Densitometric quantitative analysis was performed using Image software (NIH Image Software). 4.5.Patients and breast tumors In this study, 113 tumor samples were collected from female Syrian BC patients with a range of (26–66) years as described in our previous publication [ 19 ]. The tumor samples were fixed in Formalin (buffered neutral aqueous 10% solution) and paraffin-embedded as we have previously reported [ 19 ]. Paraffin-embedded tumor materials were obtained from the Department of Pathology, Faculty of Medicine, University of Aleppo. The use of these specimens and their corresponding data in research was approved by the Ethics Committee of the Faculty of Medicine of Aleppo University (Syria) as we have previously reported [ 19 ]. 4.6. Tissue microarray The tissue microarray (TMA) slides were obtained as previously described [ 21 ]. Tissue cylinders with a diameter of 0.6 mm were punched from representative tumors areas of a ‘donor’ tissue block using a semiautomatic robotic precision instrument. Two adjacent sections of the TMA blocks were transferred to adhesive coated slides (Instrumedic Inc., Hackensack, NJ, USA). In this study, each TMA slide contained adjacent sections from 113 Syrian breast tumor tissues. The slides were then used for IHC as described below. 4.7. Immunohistochemistry Immunohistochemical analysis, examining the expression of both CD44 and its transcriptional target ITGB1BP1, was carried out as we have previously described [ 5 , 19 ]. Briefly, the primary specific antibodies used in the IHC analysis are Rabbit polyclonal antiCD44 antibody (ab157107, Abcam), and the Rabbit polyclonal anti-ITGB1BP1 antibody (PA5-97883, Thermo Fisher Scientific). Briefly, TMA sections were de-paraffinized, rehydrated, and blocked using 3% hydrogen peroxide in methanol to express the endogenous peroxidase activity at room temperature for ten minutes. Antigen retrieval was accomplished by boiling the slides for 10 minutes in 10 mM sodium citrate solution (pH 6.0). The slides were cooled and pre-incubated in Optimax™ washing buffer, then incubated overnight at 4°C with primary CD44 and ITGB1BP1 antibodies. Sections were then washed thoroughly, and Invitrogen DAB substrate was used (Thermofisher, USA) and the appropriate secondary HRP-conjugated antibody was applied for 1h at room temperature (Ultra-sensitive ABC Rabbit IgG Kit CAT = 32054). The slides were then counterstained with hematoxylin and eosin and mounted for analysis of immunostaining. 4.8.Statistical analysis Protein quantification was assessed statistically using Student's two-tailed test. We compared the statistical significance of the two groups using an ANOVA test and a Student's t -test. In other experiments, Microsoft Excel 2013 and GraphPad Prism 8 were used to perform a non-parametric Student's t -test. The data were presented as means SD for three independent experiments unless otherwise specified. It was regarded as statistically significant if the P value was less than 0.05. 5. Conclusions In summary, using a BC in vitro model and TMA containing breast tumor tissues ranging from less invasive to highly invasive/metastatic, our results revealed that CD44 expression significantly increased from benign/primary to invasive breast tumor tissues, paralleling the expression pattern of ITGB1BP1. Notably, HA-induced CD44 activation significantly upregulated ITGB1BP1 expression at both RNA and protein levels. Furthermore, our recent literature review, combined with this study’s findings, supports our hypothesis that ITGB1BP1 is a potential novel transcriptional target of CD44 signaling in BC progression. Ongoing experiments integrating functional, molecular, pharmacological, and bioinformatics approaches aim to validate ITGB1BP1 as a key mediator of CD44-promoted BC cell invasion and to identify the precise molecular mechanisms linking HA-CD44 activation to ITGB1BP1 transcription. Declarations Author Contributions: Conceptualization, AO; methodology: SA and HN; software: AO, SA and HN; validation: AO, SA and HN; formal analysis: AO, SA and HN; investigation: AO, SA, HN and SV; resources: AO; data curation, AO, SA and HN; writing—original draft preparation: AO, SA and HN; writing—review and editing, AO, SA, HN, and MR; supervision, AO; project administration: AO; funding acquisition, AO. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Qatar University (Internal grant number (QUST-1-CAS2019-22)), and the Qatar National Research Fund: UREP24-117-1-027 and UREP29-186-3-059. Hanan Nazar was awarded a PhD scholarship from the Kuwaiti Government. Special thanks to Ms. Qubaa Ahmed Elzubair for her help in cutting the slides for TMAs. The American University of Iraq-Baghdad supported the publication fee of this paper. 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In Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression , Seminars in cancer biology, 2008; Elsevier: 2008; pp 251–259. Faurobert E, Albiges-Rizo C. Recent insights into cerebral cavernous malformations: a complex jigsaw puzzle under construction. FEBS J. 2010;277(5):1084–96. Zheng Y, Qiu J, Hu J, Wang G. Concepts and hypothesis: integrin cytoplasmic domain-associated protein-1 (ICAP-1) as a potential player in cerebral cavernous malformation. J Neurol. 2013;260:10–9. Degani S, Balzac F, Brancaccio M, Guazzone S, Retta SF, Silengo L, Eva A, Tarone G. The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading. J Cell Biol. 2002;156(2):377–87. Additional Declarations No competing interests reported. Supplementary Files OriginalBlotsOuhtitetal2025.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 08 Jan, 2026 Editor assigned by journal 09 Jul, 2025 Submission checks completed at journal 09 Jul, 2025 First submitted to journal 09 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7083073","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":571796176,"identity":"255ec74c-4e40-45cd-84b3-ee2c05be2099","order_by":0,"name":"Hanan Nazar","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Hanan","middleName":"","lastName":"Nazar","suffix":""},{"id":571796177,"identity":"7166557a-bc20-4338-9db2-623fb65c348d","order_by":1,"name":"Salma M.S. 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09:23:05","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121637,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7083073/v1/f0ad7c1143e47003eddbadb3.html"},{"id":100367639,"identity":"b50fdbfc-3f17-42e4-83dd-556c61969ece","added_by":"auto","created_at":"2026-01-16 07:57:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-induction of CD44 increased ITGB1BP1 mRNA expression in MDA-MB-231 cells.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e: RNA gene expression levels of CD44 and its target ITGB1PB1 in MDA-231 BC cell line 24hrs post-HA treatment. -HA: control (non-treated), +HA: HA treated. \u003cstrong\u003eB\u003c/strong\u003e: Semi-quantitative analysis of CD44 and its target gene ITGB1BP1 was carried out using student’s two-tailed \u003cem\u003et\u003c/em\u003e-test, and data are statistically significant when p\u0026lt;0.05 compared to the control (**\u003cem\u003ep\u0026lt; 0.01\u003c/em\u003e ***\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e). When the CD44 expression was induced by its ligand HA, the mRNA expression of its transcriptional target ITGB1BP1 was up-regulated in the treated 24hr MDA-MB-231 cells, when compared to its corresponding control.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7083073/v1/26d4a3c9cf878a6feb5f1fd0.png"},{"id":100125553,"identity":"d829a67f-8487-4eb0-a871-1fd86d8a8d4b","added_by":"auto","created_at":"2026-01-13 09:23:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-induction of CD44 protein increased ITGB1BP1 protein expression in MDA-MB-231 cells.\u003c/strong\u003e \u003cstrong\u003eA: \u003c/strong\u003eWestern blot results showing the expression of CD44 and its transcriptional target ITGB1BP1 in the MDA-MB-231 BC cell line. Beta-Actin was used as an internal control. \u003cstrong\u003eB:\u003c/strong\u003e Semi-quantification of CD44 and its transcriptional target ITGB1BP1 protein expression levels in the MDA-MB-231 BC cell line was performed by ImageJ software analysis of the gel bands and normalized to Beta-Actin expression levels. Mean values (n=3) of three experiments are shown. Statistical analysis was performed using one-way analysis of variance (ANOVA). The student’s two-tailed \u003cem\u003et\u003c/em\u003e-test was conducted to compare treatment groups and data are statistically significant when p\u0026lt;0.05 (*\u003cem\u003ep\u0026lt; 0.05\u003c/em\u003e, **\u003cem\u003ep\u0026lt; 0.01\u003c/em\u003e ***\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7083073/v1/55fa0cd72d0ee9ec34d1319e.png"},{"id":100125554,"identity":"75c86b8c-7189-4321-b34a-0264d3f6044f","added_by":"auto","created_at":"2026-01-13 09:23:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":138505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mRNA expression levels of both CD44 and its target ITGB1BP1 in the breast cancer cell model. \u003c/strong\u003eSemi-quantification of the relative mRNA levels of both CD44 and its transcriptional target ITGB1BP1 in different BC cell lines mimicking a typical BC cell progression model from less to highly invasive BC cells. Quantification was performed by ImageJ software analysis of gel bands, and normalized to β-actin control bands. Mean values (n=3) of three experiments are shown. Statistical analysis was performed using one-way analysis of variance (ANOVA). Tukey’s post-hoc test was conducted to compare treatment groups and data are statistically significant when p\u0026lt;0.05 compared to the control (*\u003cem\u003ep\u0026lt; 0.05\u003c/em\u003e, **\u003cem\u003ep\u0026lt; 0.01\u003c/em\u003e ***\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7083073/v1/99a3047a7bc1ddfa3d051571.png"},{"id":100125566,"identity":"d602c38c-642f-4b24-a822-dc9afe7f983a","added_by":"auto","created_at":"2026-01-13 09:23:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":232944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe protein expression of Both CD44 and its target ITGB1BP1 incresaeed in parallel during breast cancer cell progression. A:\u003c/strong\u003e Western blot results showing the expression of CD44 and its transcriptional target ITGB1BP1 in various BC cell lines ranging from low to highly invasive phenotype. Beta-actions were used as an internal control. \u003cstrong\u003eB:\u003c/strong\u003eSemi-quantification of CD44 and its transcriptional target ITGB1BP1 relative protein expression levels in different BC cell lines; The analysis of gel bands was performed by ImageJ software and normalized to beta-actin gel bands. Mean values (n=3) of three experiments are shown. Statistical analysis was performed using one-way analysis of variance (ANOVA). Tukey’s post-hoc test was conducted to compare treatment groups and data are statistically significant when p\u0026lt;0.05 compared to the control (*\u003cem\u003ep\u0026lt; 0.05\u003c/em\u003e, **\u003cem\u003ep\u0026lt; 0.01\u003c/em\u003e ***\u003cem\u003ep\u0026lt;0.001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7083073/v1/09a9a68b92f860268aaaa8af.png"},{"id":100366063,"identity":"f59bdfd8-fb3d-495e-a012-1315686ef109","added_by":"auto","created_at":"2026-01-16 07:55:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":945238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of ITGB1BP1 as a novel target of CD44 by immunohistochemistry using breast tumor tissue microarrays.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Grade-2 tumor (negative for CD44) \u003cem\u003eversus\u003c/em\u003e \u003cstrong\u003eB)\u003c/strong\u003e Grade-3 tumor (positive for CD44). \u003cstrong\u003eC)\u003c/strong\u003e Grade-2 tumor (negative for ITGB1BP1) \u003cem\u003eversus\u003c/em\u003e \u003cstrong\u003eD)\u003c/strong\u003e Grade-3 tumor (positive for ITGB1BP1). The expression patterns of CD44 were accompanied by a parallel expression patterns of its transcriptional target ITGB1BP1.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7083073/v1/268c7d93ba52c2cc1953af14.png"},{"id":100421569,"identity":"dc438792-a306-490c-b82b-f186a1f24cab","added_by":"auto","created_at":"2026-01-16 13:34:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2816296,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7083073/v1/1cd9ce30-3a44-42cf-acd4-8d55419cce54.pdf"},{"id":100366074,"identity":"b1c28230-bacd-452b-9d4c-49a8b608ce9f","added_by":"auto","created_at":"2026-01-16 07:55:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":560855,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalBlotsOuhtitetal2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7083073/v1/c6b87435effd3de1c529542d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Validation of ITGB1BP1 as a novel transcriptional target of CD44-Promoted Breast Cancer Progression","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCancer is a universally fatal disease that affects millions of people worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is a genetic disease that can be either inherited or acquired due to DNA damage caused by exposure to harmful environmental factors such as chemicals in tobacco and alcohol, as well as ultraviolet (UV) radiation. In 2020, 19.3\u0026nbsp;million new cancer cases were reported, and 10\u0026nbsp;million cancer-related deaths were recorded in the same year [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGlobally, approximately 627,000 women die from BC each year, accounting for 15% of all cancer-related deaths [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Since BC remains the most prevalent cancer among females, data from Gulf Cooperation Council (GCC) countries align closely with international trends [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRisk factors for BC include hormonal exposure, menarche history, aging, physical inactivity, tobacco smoking, alcohol consumption, breast tissue characteristics, and lifestyle factors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMetastasis is the most threatening aspect of cancer, and tumor cell invasion is a defining event in the metastatic process. Understanding its mechanisms is crucial for developing effective anti-metastatic therapies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Cell invasion is regulated by multiple signaling pathways involving various proteins, including cell adhesion molecules (CAMs), proteases, and growth factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. CAMs play a critical role in both early and late cancer development by mediating cell-cell and cell-extracellular matrix (ECM) interactions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the key CAMs is CD44, the major hyaluronan (HA) receptor. In addition to its role in cellular adhesion, CD44 is significantly involved in intracellular signaling, regulating cell growth, proliferation, and motility [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While the role of CD44 in BC was initially controversial, our earlier work using a Tet-off-inducible system in a transgenic mouse model revealed that CD44 induction promotes BC metastasis to the liver [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further investigate the precise molecular role of CD44 in BC cell invasion and metastasis, we applied a CHIP-microarray gene expression profiling approach to identify the transcriptional targets of CD44/HA signaling in BC. Our CD44-CHIP analysis identified over 200 novel potential downstream transcriptional target genes. Among them, three genes (Cortactin, Survivin, and TGF-β2) have been validated and published, along with their associated signaling pathways in CD44-promoted BC cell invasion [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we selected the large isoform of integrin subunit beta 1 binding protein 1 (ITGB1BP1)\u0026mdash;also known as Integrin Cytoplasmic Domain-Associated Protein 1 (ICAP-1A) from the CHIP-microarray screen for further validation based on the following observations: i) ITGB1BP1 expression increased 2.5-fold upon CD44 induction in BC cells; ii) ITGB1BP1 activates KRIT-1, triggering NOTCH signaling, which regulates the PI3K/AKT pathway, promoting BC progression and survival [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; iii) ITGB1BP1 overexpression promotes tumor cell invasion via interaction with oncogenic pathways [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]; iv) ITGB1BP1 is associated with cell adhesion, migration, and fibronectin matrix organization [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]; v) ITGB1BP1 plays a role in cell-ECM communication, allowing cells to adapt to ECM density by modifying adhesion and migration responses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]; and vi) ITGB1BP1 is critical in angiogenesis, proliferation, differentiation, adhesion, migration, and invasion [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven these observations, we hypothesize that ITGB1BP1 is a novel transcriptional target gene that mediates CD44-promoted BC cell invasion.\u003c/p\u003e \u003cp\u003eTo test this hypothesis and validate whether ITGB1BP1 protein expression correlates with CD44 levels, we employed two strategies: i) \u003cem\u003eIn vitro\u003c/em\u003e BC cell model: We examined total RNA samples and protein lysates from various BC cell lines representing different tumor stages to assess the expression of both CD44 and its potential transcriptional target; ii) Ex vivo tissue microarray analysis: We analyzed breast tumor tissue microarray slides containing adjacent sections from 113 Syrian BC patients, using immunohistochemistry (IHC) to determine the expression patterns of both CD44 and ITGB1BP1, as previously reported [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.1.HA-activation of CD44 expression increases ITGB1BP1 RNA expression\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMDA-MB-231 cells were cultured in the presence or absence of HA to structurally validate ITGB1BP1 as a transcriptional target of CD44. RT-PCR was carried out to confirm an increased expression of CD44 and its target ITGB1BP1 in MDA-MB-231 cells at different time points. Transcription analysis of the results from three replicate experiments demonstrated that CD44 and its transcriptional target ITGB1BP1 mRNA levels increased simultaneously in the BC cell line MDA-MB-231 24 hours after HA treatment. As a result, no increase in ITGB1BP1 mRNA was observed when HA was not present \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2.HA-induction of CD44 expression increases ITGBPB1protein expression\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAnalysis of ITGBPB1 protein expression in MDA-MB-231 cells after exposure to 100 \u0026micro;g/ml HA (MW 220 kDa) for 24 hours to ensure induction of CD44 signaling, the temporal relationship between CD44 signaling and ITGBPB1 gene regulation was also examined. According to immunoblot experiments conducted on samples extracted from MDA-MB-231 cells under identical conditions such as those used to collect mRNA samples from the microarrays, HA induces CD44 expression, as demonstrated by an immunoreactive band to the anti-human CD44 antibody at 85 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). In MDA-MB-231 cells stimulated with HA for 24 h, immunoblotting confirmed an increase in ITGB1BP1 expression parallel to CD44 induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3.Expression of CD44 and ITGB1BP1 in breast cancer cell lines\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo validate whether the expression of ITGB1BP1 increases upon induction of CD44 by HA, we first addressed the cell line specificity of the relationship between CD44 and the regulation of its potential target ITGB1BP1 transcription/expression using an \u003cem\u003ein vitro\u003c/em\u003e BC cell model. In other words, to validate ITGB1BP1 as a true transcriptional target of CD44, we mimicked the BC cell progression using \u003cem\u003ein vitro\u003c/em\u003e cell model and wondered whether the expression pattern of CD44 would follow the same pattern of expression of its transcriptional target ITGB1BP. A cell type representing each of the various stages of the BC progression (from the normal breast cell line derived from benign proliferative breast tissue and spontaneously (MCF10A), to the primary Luminal A (MCF-7) to the triple negative cell line (MDA-MB-231). In order to determine if CD44 and ITGB1BP both play equal roles in BC metastasis, we first compared the mRNA levels of CD44 and ITGB1BP1 across seven different BC cell lines ranging in aggressiveness (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e: MCF10A, ZR-75-1, MCF7, SKBR3, HCC1954, MDA-MB-468, and MDA-MB-231).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eFigure 4. The protein expression of Both CD44 and its target ITGB1BP1 incresaeed in parallel during breast cancer cell progression. A\u003c/b\u003e: Western blot results showing the expression of CD44 and its transcriptional target ITGB1BP1 in various BC cell lines ranging from low to highly invasive phenotype. Beta-actions were used as an internal control. \u003cb\u003eB\u003c/b\u003e: Semi-quantification of CD44 and its transcriptional target ITGB1BP1 relative protein expression levels in different BC cell lines; The analysis of gel bands was performed by ImageJ software and normalized to beta-actin gel bands. Mean values (n\u0026thinsp;=\u0026thinsp;3) of three experiments are shown. Statistical analysis was performed using one-way analysis of variance (ANOVA). Tukey\u0026rsquo;s post-hoc test was conducted to compare treatment groups and data are statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to the control (*\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, **\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e ***\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4.Expression of CD44 and ITGB1BP1 in breast tumor tissue microarrays\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTissue microarrays (TMAs) have become an invaluable tool in cancer research to evaluate the expression and subcellular localization of proteins in cells and tissues. In order to test and validate our hypothesis that ITGB1BP1 is a transcriptional target of CD44, the expression of both CD44 and its potential transcriptional target ITGB1BP1 was examined in human breast tissue samples by immunohistochemistry using TMA slides representing 113 breast tumor tissue samples (31 patients were in grade 2 whereas 82 patients were in Grade 3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Using our Tet-Off CD44-inducible BC cell system in mouse model, we have previously reported that induction of CD44 \u003cem\u003ein vivo\u003c/em\u003e caused a dramatic increase in CD44 protein expression levels that correlated with metastasis of primary breast tumors to the liver [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the present investigation, we used the same approach and examined the expression patterns of both CD44 and its novel transcriptional target ITGB1BP1 in TMA slides containing adjacent sections from each of the 113 breast tumor tissues. Our results revealed that: i) the patterns of expression of CD44 and its transcriptional target ITGB1BP1 showed a parallel increase when comparing the primary breast tumors (ductal carcinoma \u003cem\u003ein situ\u003c/em\u003e) to the invasive tumors (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); and ii) increased expression of CD44 and its transcriptional target ITGB1BP1 correlated with tumor malignancy and invasiveness (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On the other hand, CD44 immunostaining was strongly membrane-associated and was present in both basal and luminal cells of most samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e), but immunostaining showed a gradient of decreasing intensity from basal to luminal cell type (More aggressive to less aggressive BC). ITGB1BP1 immunostaining was both membrane-associated and cytoplasmic and followed a pattern similar to CD44, being strongly expressed in basal cells, but was low or absent in benign tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe percentage of CD44 and ITGB1BP1 immuno-reactivity in all breast tissues investigated.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBreast Tumor type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e# of cases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCD44-immunopositive (% )\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eITGB1BP1-immunopositive ( % )\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrade 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20/31 (64.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22/31 (71%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrade 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77/82 (93.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80/82 (97.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMetastatic\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38/38 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38/38 (100%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOverall Tumors\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97/113 (85.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101/113 (90.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBased on our initial work and findings from the literature, CD44\u0026rsquo;s role in cancer metastasis has been controversial [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To address this discrepancy and investigate the function of the standard form CD44s in breast cancer (BC) invasion and metastasis, while further elucidating its downstream signaling, we developed a tetracycline (Tet)-Off-regulated expression system for CD44s, both in vitro and in vivo [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Using a microarray technique (12K CHIP from Affymetrix), we identified more than 200 potential CD44s target genes. Among these, we have already validated three novel transcriptional targets\u0026mdash;Cortactin, Survivin, and TGF-β2\u0026mdash;along with their molecular signaling pathways that underpin CD44/HA-promoted BC cell invasion [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, ITGB1BP1 was selected as an additional novel transcriptional target gene based on the following observations: i) It showed a 2.5-fold upregulation upon CD44 induction; ii) ITGB1BP1 plays a key role in cell adhesion, migration, and invasion [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; and iii) ITGB1BP1 interacts with the PI3K/AKT pathway, promoting breast tumor progression and survival [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results showed that both CD44 and its transcriptional target ITGB1BP1 increased simultaneously at the mRNA and protein levels in the BC cell line MDA-MB-231 after 24 hours of activation by its major ligand, HA. Furthermore, both CD44 and ITGB1BP1 exhibited parallel expression patterns at the mRNA and protein levels in a BC cell model that mimics BC progression from normal/benign to malignant/metastatic stages.\u003c/p\u003e \u003cp\u003eMore interestingly, TMA slides containing breast tumor tissues from 113 BC patients, examined by IHC analysis, revealed a parallel increase in CD44 and ITGB1BP1 expression from less invasive to highly invasive breast tumors. Approximately 85% of the samples showed CD44 localization on both the membrane and cytoplasm of BC tissues, consistent with the Human Protein Atlas, which previously reported CD44 expression in BC tissues.\u003c/p\u003e \u003cp\u003ePrevious studies validated CD44 membrane localization due to its activity as a receptor for multiple ligands\u0026mdash;including HA\u0026mdash;to facilitate BC progression and metastasis [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, recent studies on melanoma confirmed cytoplasmic CD44 localization, attributed to WNT pathway activation, which enhances direct CD44\u0026ndash;Cortactin interactions and upregulates CD44 and c-Myc, indicating a positive feedback loop that promotes tumor proliferation, migration, and invasion [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, ITGB1BP1 was localized in the cytoplasm of BC tissues, consistent with studies on cerebral cavernous malformation, which demonstrated ITGB1BP1 expression in both the cytoplasm and nucleus [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Notably, ITGB1BP1 enhances cell proliferation and survival by binding Nm23-H2, leading to nuclear translocation and induction of cyclin D1 and c-Myc gene expression [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study further demonstrated that CD44 activation via HA binding led to the induction of ITGB1BP1, with their expression patterns paralleling one another in both our in vitro BC progression model and breast tumor tissues (from less invasive to highly metastatic stages). These findings suggest that CD44-induced ITGB1BP1 expression promotes BC cell invasion and progression.\u003c/p\u003e \u003cp\u003eSeveral studies have already confirmed the crucial role of HA-CD44 signaling in BC proliferation and invasion [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Specifically, CD44 induced cell invasion by activating the RhoA GTPase/ROCK-1 signaling pathway [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. ITGB1BP1 and Nm23-H2 interact via RhoA-GTPase, further supporting CD44-driven tumor proliferation and invasion [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally: i) CD44 upregulates c-Myc expression [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]; ii) CD44 activates ERK, leading to cyclin D1 upregulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; iii) ERK phosphorylation promotes BC cell proliferation and migration [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; iv) ROCK-1 and ITGB1BP1 form a complex at the plasma membrane, where RhoA co-localizes with β1 integrin [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]; and v) RhoA GTPase/ROCK pathway activation by ITGB1BP1 enhances cell migration [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, CD44-HA interaction also activates RhoA GTPase, which recruits IP3 receptors to intracellular calcium storage organelles, leading to calcium release [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Collectively, these findings\u0026mdash;combined with our results\u0026mdash;support the hypothesis that ITGB1BP1 is a novel transcriptional target of CD44, driving cell proliferation, adhesion, migration, and invasion, as we have recently reported [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, KRIT-1 binds to ITGB1BP1 through its PTB domain, competing with β1 integrin for binding [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Additionally, ITGB1BP1 and KRIT-1 promote Notch signaling, activating PI3K/AKT and enhancing tumor cell survival and invasion [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A major component of CD44's role in tumor survival and motility is its regulation of the PI3K/AKT pathway [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In fact, we previously demonstrated that CD44 promotes BC invasion via the PI3K/AKT pathway [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.1.Cell culture\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSix human BC cell lines (MDA-MB-231, MDA-MB-468, HCC-1954, SkBr3, ZR751, and MCF-7), as well as the immortalized human breast cell line MCF10A, supplied by our collaborator Dr. Al-Moustafa, were used in the present study as described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The cell lines MDA-MB-231 and MDA-MB-468 were cultured in 90% Dulbecco's Modified Eagle Medium (DMEM), while the HCC-1954, SkBr3, MCF-7 and ZR751 cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640). All culture media were supplemented with 1% L-Glutamate, 50 ml Fetal Bovine Serum Heat Inactivated Collected in South America (FBS) (Thermo Scientific, USA), and 1% Penicillin Streptomycin antibiotic (Pen Strep) (Gibco, USA). According to our protocol, MCF10A breast cells were cultured in DMEM and 2% glutamine. All the cells were then incubated in a 37\u0026deg;C humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were incubated in the presence or absence of 100 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml HA, and protein lysates and RNA were collected at both 12 and 24 hours after HA treatment.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe different cell lines used for the \u003cem\u003ein vitro\u003c/em\u003e models.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCancer Subtype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType of Tumor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBRCA1\u003c/p\u003e \u003cp\u003eMutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHER2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMDA-MB-231\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTriple-Negative A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMDA-MB-468\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTriple-Negative A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHCC1954\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHER2 Positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuctal Carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSKBR3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHER2 Positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMCF7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLuminal A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInvasive Ductal Carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eZR751\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLuminal A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInvasive Ductal Carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+/-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMCF10A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBasal Epithelial Cell Line\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFibrocystic Disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.2.RNA Extraction and cDNA synthesis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGene transcription analysis was performed following HA treatment of MDA-MB-231 cells, and RNA samples were collected at 12 and 24 hours time points. Briefly, total cellular RNA was obtained using the TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA concentration and purity were determined by NanoDrop ND 1,000 Spectrophotometer (Nanodrop Technologies Inc., Delaware, USA). Ribosomal RNA band integrity was carried using 1% agarose gel electrophoresis. Synthesis of cDNA was performed only on samples whose 260/280 ratio values ranged from 1.8 to 2.0. Reverse transcription was performed with 2 \u0026micro;g of RNA, using random primers and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, UK), according to the manufacturer's instructions. Mock reverse transcription reaction without reverse transcriptase was utilized as a negative control. The RT-PCR reaction was carried as we have previously reported [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and RT-PCR products were run on 2% agarose gel containing 5 mg/ml ethidium bromide. Annealing temperatures for the CD44, ITGB1BP1, and GAPDH genes, along with the oligonucleotide primers used, are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTemperature and specific oligonucleotide primers used for Reverse Transcription-PCR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTemperature (◦C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOligonucleotide Primers\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCD44\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TTT GCA TTG CAG TCA ACA GTC-3' (Forward)\u003c/p\u003e \u003cp\u003e5'-GTT ACA CCC CAA TCT TCA TGT CCA C-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eITGB1BP1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TTT GCA TTG CAG TCA ACA GTC-3' (Forward)\u003c/p\u003e \u003cp\u003e5'-GTT ACA CCC CAA TCT TCA TGT CCA C-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGAPDH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GAAGGCCATGCCAGTGAGCT-3' (Forward)\u003c/p\u003e \u003cp\u003e5'-CCGGGAAACTGTGGCGTGAT-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.3.Real-time qRT‐PCR\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eReal-time qRT-PCR was carried out as we have previously reported [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, 1 \u0026micro;g of total RNA extracted using GeneJET RNA Purification Kit (Thermo Fisher Scientific) was reversed transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-qPCR assay was performed to determine the expression of both CD44 and its target gene ITGB1BP1 using TaqMan\u0026reg; Fast Advanced Master Mix (Applied Biosystems). For each reaction, 1ul of diluted cDNA (1:4) was added to 10 \u0026micro;l of TaqMan Master Mix, 1 \u0026micro;l of BRIP1 Gene Expression TaqMan\u0026reg; Assays FAM‐MGB Probe (4351372, Applied Biosystems), and 1\u0026micro;l of GAPDH Gene Expression TaqMan\u0026reg; Assays VIC‐MGB Probe (4448489, Applied Biosystems) used as an endogenous control, and 7\u0026micro;l of nuclease‐free water. The reaction was performed using the QuantStudio\u0026trade; 6 Flex Real‐Time PCR System (Applied Biosystems, Inc) as previously reported [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]: 10 minutes at 95\u0026deg;C (stage 1); 20 seconds at 95\u0026deg;C and 20 seconds at 60\u0026deg;C for 40 cycles (stage 2). The results were analysed using the QuantStudio\u0026trade; 6 software.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.4.Western Blotting\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWhole cell lysates were collected from MDA-MB-231 cells following HA treatment 12 and 24 post-treatment, using RIPA buffer supplemented with protease inhibitors (Pierce, USA). According to the manufacturer's detailed protocol, PierceTM BCA Protein Assay Kit (Thermo ScientificTM) was used to quantify proteins. Denatured cell lysates were analyzed by SDS-PAGE, using 20 \u0026micro;g from each protein sample. Membranes were probed with primary antibodies overnight at 4\u0026deg;C, using the Rabbit polyclonal antiCD44 antibody (ab157107, Abcam), and Rabbit polyclonal anti-ITGB1BP1 antibody (PA5-97883, Thermo Fisher Scientific). The membranes were then probed with HRP-conjugated anti-rabbit secondary antibody (A0545, Sigma) for CD44 and ITGB1BP1, and anti-mouse secondary antibody (PAB0096, Abnova) to detect the β‐Actin control protein. The signal was revealed with SuperSignal\u0026trade; West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and was developed using Chemiluminescent GeneGnome (Syngene). Densitometric quantitative analysis was performed using Image software (NIH Image Software).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.5.Patients and breast tumors\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this study, 113 tumor samples were collected from female Syrian BC patients with a range of (26\u0026ndash;66) years as described in our previous publication [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The tumor samples were fixed in Formalin (buffered neutral aqueous 10% solution) and paraffin-embedded as we have previously reported [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Paraffin-embedded tumor materials were obtained from the Department of Pathology, Faculty of Medicine, University of Aleppo. The use of these specimens and their corresponding data in research was approved by the Ethics Committee of the Faculty of Medicine of Aleppo University (Syria) as we have previously reported [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Tissue microarray\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe tissue microarray (TMA) slides were obtained as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Tissue cylinders with a diameter of 0.6 mm were punched from representative tumors areas of a \u0026lsquo;donor\u0026rsquo; tissue block using a semiautomatic robotic precision instrument. Two adjacent sections of the TMA blocks were transferred to adhesive coated slides (Instrumedic Inc., Hackensack, NJ, USA). In this study, each TMA slide contained adjacent sections from 113 Syrian breast tumor tissues. The slides were then used for IHC as described below.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Immunohistochemistry\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eImmunohistochemical analysis, examining the expression of both CD44 and its transcriptional target ITGB1BP1, was carried out as we have previously described [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, the primary specific antibodies used in the IHC analysis are Rabbit polyclonal antiCD44 antibody (ab157107, Abcam), and the Rabbit polyclonal anti-ITGB1BP1 antibody (PA5-97883, Thermo Fisher Scientific). Briefly, TMA sections were de-paraffinized, rehydrated, and blocked using 3% hydrogen peroxide in methanol to express the endogenous peroxidase activity at room temperature for ten minutes. Antigen retrieval was accomplished by boiling the slides for 10 minutes in 10 mM sodium citrate solution (pH 6.0). The slides were cooled and pre-incubated in Optimax\u0026trade; washing buffer, then incubated overnight at 4\u0026deg;C with primary CD44 and ITGB1BP1 antibodies. Sections were then washed thoroughly, and Invitrogen DAB substrate was used (Thermofisher, USA) and the appropriate secondary HRP-conjugated antibody was applied for 1h at room temperature (Ultra-sensitive ABC Rabbit IgG Kit CAT\u0026thinsp;=\u0026thinsp;32054). The slides were then counterstained with hematoxylin and eosin and mounted for analysis of immunostaining.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.8.Statistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProtein quantification was assessed statistically using Student's two-tailed test. We compared the statistical significance of the two groups using an ANOVA test and a Student's \u003cem\u003et\u003c/em\u003e-test. In other experiments, Microsoft Excel 2013 and GraphPad Prism 8 were used to perform a non-parametric Student's \u003cem\u003et\u003c/em\u003e-test. The data were presented as means SD for three independent experiments unless otherwise specified. It was regarded as statistically significant if the P value was less than 0.05.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn summary, using a BC \u003cem\u003ein vitro\u003c/em\u003e model and TMA containing breast tumor tissues ranging from less invasive to highly invasive/metastatic, our results revealed that CD44 expression significantly increased from benign/primary to invasive breast tumor tissues, paralleling the expression pattern of ITGB1BP1. Notably, HA-induced CD44 activation significantly upregulated ITGB1BP1 expression at both RNA and protein levels. Furthermore, our recent literature review, combined with this study\u0026rsquo;s findings, supports our hypothesis that ITGB1BP1 is a potential novel transcriptional target of CD44 signaling in BC progression. Ongoing experiments integrating functional, molecular, pharmacological, and bioinformatics approaches aim to validate ITGB1BP1 as a key mediator of CD44-promoted BC cell invasion and to identify the precise molecular mechanisms linking HA-CD44 activation to ITGB1BP1 transcription.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, AO; methodology: SA and HN; software: AO, SA and HN; validation: AO, SA and HN; formal analysis: AO, SA and HN; investigation: AO, SA, HN and SV; resources: AO; data curation, AO, SA and HN; writing\u0026mdash;original draft preparation: AO, SA and HN; writing\u0026mdash;review and editing, AO, SA, HN, and MR; supervision, AO; project administration: AO; funding acquisition, AO. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by Qatar University (Internal grant number (QUST-1-CAS2019-22)), and the Qatar National Research Fund: UREP24-117-1-027 and UREP29-186-3-059. Hanan Nazar was awarded a PhD scholarship from the Kuwaiti Government.\u0026nbsp;Special thanks to Ms. Qubaa Ahmed Elzubair for her help in cutting the slides for TMAs. The American University of Iraq-Baghdad supported the publication fee of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e The use of these specimens and their corresponding data in research was approved by the Ethics Committee of the Faculty of Medicine of Aleppo University (Syria).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest. The founders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript, or in the decision to publish the results.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCheek DM, Naxerova K. Mapping the long road to cancer. 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Novel CD44-downstream signaling pathways mediating breast tumor invasion. Int J Biol Sci. 2018;14(13):1782.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUebelhoer M, Boon LM, Vikkula M. Vascular anomalies: from genetics toward models for therapeutic trials. Cold Spring Harbor Perspect Med. 2012;2(8):a009688.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo S, Liu M, Gonzalez-Perez RR. Role of Notch and its oncogenic signaling crosstalk in breast cancer. Biochim et Biophys Acta (BBA)-Reviews Cancer. 2011;1815(2):197\u0026ndash;213.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStroeken PJ, Alvarez B, Rheenen Jv, Wijnands YM, Geerts D, Jalink K, Roos E. Integrin cytoplasmic domain-associated protein‐1 (ICAP‐1) interacts with the ROCK‐I kinase at the plasma membrane. J Cell Physiol. 2006;208(3):620\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouin A-P, Kyurmurkov A, R\u0026eacute;gent-Kloeckner M, Ribba A-S, Faurobert E, Fournier H-N, Bourrin-Reynard I, Manet-Dup\u0026eacute; S, Oddou C, Balland M. ICAP-1 monoubiquitylation coordinates matrix density and rigidity sensing for cell migration through ROCK2\u0026ndash;MRCKα balance. J Cell Sci. 2017;130(3):626\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrunner M, Millon-Fr\u0026eacute;millon A, Chevalier G, Nakchbandi IA, Mosher D, Block MR, Albig\u0026egrave;s-Rizo C, Bouvard D. Osteoblast mineralization requires β1 integrin/ICAP-1\u0026ndash;dependent fibronectin deposition. J Cell Biol. 2013;201(4):643.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad SM, Nazar H, Rahman MM, Rusyniak RS, Ouhtit A. ITGB1BP1, a Novel Transcriptional Target of CD44-Downstream Signaling Promoting Cancer Cell Invasion. 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CD44 splice isoform switching determines breast cancer stem cell state. Genes Dev. 2019;33(3\u0026ndash;4):166\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorak CE, Lee JH, MARSHALL JC, Shreeve SM, Steeg PS. The role of metastasis suppressor genes in metastatic dormancy. Apmis. 2008;116(7\u0026ndash;8):586\u0026ndash;601.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGross N, Balmas K, Brognara CB. Absence of functional CD44 hyaluronan receptor on human NMYC-amplified neuroblastoma cells. Cancer Res. 1997;57(7):1387\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa R, Feng Y, Lin S, Chen J, Lin H, Liang X, Zheng H, Cai X. Mechanisms involved in breast cancer liver metastasis. J translational Med. 2015;13:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoreth D, Schuette M, Zinke J, Mohme M, Piffko A, Schneegans S, Stadler J, Janning M, Loges S, Joosse SA. CD74 and CD44 expression on CTCs in cancer patients with brain metastasis. Int J Mol Sci. 2021;22(13):6993.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaor D, Nedvetzki S, Golan I, Melnik L, Faitelson Y. CD44 in cancer. Crit Rev Clin Lab Sci. 2002;39(6):527\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei C-Y, Zhu M-X, Yang Y-W, Zhang P-F, Yang X, Peng R, Gao C, Lu J-C, Wang L, Deng X-Y. Downregulation of RNF128 activates Wnt/β-catenin signaling to induce cellular EMT and stemness via CD44 and CTTN ubiquitination in melanoma. J Hematol Oncol. 2019;12:1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFournier H-N, Dup\u0026eacute;-Manet S, Bouvard D, Luton F, Degani S, Block MR, Retta SF, Albiges-Rizo C. Nuclear translocation of integrin cytoplasmic domain-associated protein 1 stimulates cellular proliferation. 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J Biol Chem. 2008;283(46):31823\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBourguignon LY. In \u003cem\u003eHyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression\u003c/em\u003e, Seminars in cancer biology, 2008; Elsevier: 2008; pp 251\u0026ndash;259.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaurobert E, Albiges-Rizo C. Recent insights into cerebral cavernous malformations: a complex jigsaw puzzle under construction. FEBS J. 2010;277(5):1084\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng Y, Qiu J, Hu J, Wang G. Concepts and hypothesis: integrin cytoplasmic domain-associated protein-1 (ICAP-1) as a potential player in cerebral cavernous malformation. J Neurol. 2013;260:10\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDegani S, Balzac F, Brancaccio M, Guazzone S, Retta SF, Silengo L, Eva A, Tarone G. The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading. J Cell Biol. 2002;156(2):377\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, Metastasis, CD44, Hyaluronan, ITGB1BP1, Tissue Microarray","lastPublishedDoi":"10.21203/rs.3.rs-7083073/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7083073/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Breast cancer (BC) is one of the most common cancers worldwide, and metastasis is its worst aspect and the first cause of death. Metastasis is a multistep process, where invasion is a recurring event. The process of BC cell invasion involves, in general, three major factors including, cell adhesion molecules (CAM), proteinases, and growth factors. CD44, a family of CAM proteins and the hyaluronic acid (HA) cell surface receptor, acts as cell differentiation, cell migration/invasion, and apoptosis regulator. To better understand the molecular mechanisms that underpin CD44-promoted BC cell invasion, our previous microarray gene expression profiling, using “TET-OFF” BC CD44-inducible cell experimental model, identified ITGB1BP1 as a novel potential transcriptional target of CD44.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethods: to test this hypothesis, both \u003cem\u003ein vitro\u003c/em\u003e BC cell model, as well as \u003cem\u003eex-vivo\u003c/em\u003etissue microarray (TMA) slides, containing adjacent sections from breast tumors of 113 BC patients were examined for the expression of both CD44 and its potential target gene expression, ITGB1BP1, by immunohistochemistry (IHC).\u003c/p\u003e\n\u003cp\u003eResults: \u003cem\u003ein vitro\u003c/em\u003e results revealed that HA-activation and induction of CD44 increased significantly the expression of its target gene, ITGB1BP1. Furthermore, IHC analysis of TMAs showed that overall 90% of the samples exhibited high CD44-immunostaining in the membrane while its target ITGB1BP1 was mainly observed in the cytoplasm and occasionally in the membrane. More interestingly, the patterns of expression of CD44 and its target ITGB1BP1 showed a parallel increase in the majority of highly invasive/metastatic tumor tissues when compared to less invasive breast tumor tissues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsclusion: the present study provides for the first time substantial evidence supporting our hypothesis that ITGB1BP1is a potential novel transcriptional target that underpin CD44-promoted BC tumor cell progression.\u003c/p\u003e","manuscriptTitle":"Validation of ITGB1BP1 as a novel transcriptional target of CD44-Promoted Breast Cancer Progression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 09:23:00","doi":"10.21203/rs.3.rs-7083073/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-01-08T11:30:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T11:09:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-09T11:08:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2025-07-09T10:47:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46f8a57f-43d2-442d-a2b4-c095e290c19b","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-13T09:23:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 09:23:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7083073","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7083073","identity":"rs-7083073","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}