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An Interplay between miRNA and SOX4 in the regulation of Epithelial mesenchymal transition and cancer progression
Running Title: miRNA and SOX4
MicroRNA (miRNA) are abundant class of small, non-coding RNA which is emerging as a potential therapeutic target for various cancers. In cancers of epithelial origin and in some others, growing evidences have revealed a close association between miRNAs and SOX4. Sex-determining region Y-related high-mobility group box 4 (SOX4) is an important member of SOX family and a crucial master regulator of epithelial mesenchymal transition (EMT) that has been implicated in tumor growth and progression. Most miRNAs modulate SOX4 by directly binding to SOX4 3’-UTR, thereby regulating cancer cell proliferation, invasion and EMT. Therefore, understanding miRNA/SOX4 interactions may provide useful for manipulating them for development of potential targeted therapeutic approaches. In this review, we highlight the interaction of miRNAs with SOX4 and discuss the possibility of combined miRNA-SOX4 based treatment that may serve as a targeted therapy for several cancers.
Key words: SOX4, microRNA, EMT, cancer
Metastasis is the primary cause of mortality and morbidity in various types of cancers. It is a complex process, during which cancer cells disseminate from the primary tumor through the circulation, and establish their growth at distant organs. When compared to benign tumors, metastatic tumors are difficult to remove by surgical resection and/or by irradiation, and are resistant to treatment with chemotherapeutic drugs, hence often have poor prognosis. Besides, chemotherapeutics are developed in such a way to target proliferation of cancer cells rather than dissemination. Therefore, there is an utmost need to develop potential therapeutics targeting the metastasis of cancer cells.
Epithelial–mesenchymal transition- A critical player in metastasis
Epithelial–mesenchymal transition (EMT) is a significant regulatory process in embryogenesis as well as in cancer progression, metastasis and recurrence  wherein differentiated epithelial cells change to dedifferentiated and migratory mesenchymal cells  . Cancer cells colonize, acquire stemness and drug resistance through EMT [4-7]. EMT is characterized by loss of epithelial marker E‐cadherin expression  and increase in mesenchymal marker Vimentin expression. This crucial process is regulated by several oncogenic events and signaling pathways such as transforming growth factor- (TGF-) , epidermal growth factor (EGF), Wnt-Notch signaling  and by several transcription factors such as Snail, Twist, Zeb1/2, SOX4 and FOXO family .
SOX4 – A master regulator of EMT and its role in cancer progression
Sex-determining region Y-related high-mobility group box 4 (SOX4) is one of the master transcription factor that regulates EMT and contributes to cell survival and metastasis [12, 13] by TGF- induced upregulation of mesenchymal EMT markers such as Vimentin and N- Cadherin [14-16] and loss of epithelial markers such as E-Cadherin by transcription factor repressors-Snail, Zeb, and Twist  or via an epigenetic regulator-polycomb-group protein EZH2, [15, 18].
SOX4 is a 47-kDa protein member of the Sex-determining region Y-related high-mobility group box (Sox) transcription factor family  and the gene is located on chromosome 6p22.3 which encodes a protein of 474 amino acids with three distinct domains: an HMG box-DNA-binding region, a glycine-rich region which is a novel functional region for promoting apoptotic cell death, and a serine-rich region that acts as a transactivation domain [20, 21]. SOX4 is a necessary transcription factor in many embryonic development processes such as the thymocyte differentiation, B cell development , T-cell development  pancreatic cell differentiation  osteoblast differentiation  in development of nervous system, and embryonic cardiovascular system [22, 26] and survival of multipotent neural and mesenchymal stem cells  thereby functioning as a putative stem cell marker crucial for cell fate determination . Many genes that are involved in embryogenesis have also been implicated in tumorigenesis. Among 20 members of Sox family, SOX4 has been recognized as a “cancer signature gene”  based on its oncogenic role in multiple human cancers; majority having epithelial origin such as breast, ovarian, lung cancer, hepatocellular carcinoma, and some others sarcomas . In normal adults, expression of SOX4 is restricted to certain cell types, including hematopoietic stem cells, mammary stem cells and hair follicle stem cells [30-32]. Of note, several lines of evidence suggest that SOX4 overexpression could promote tumor progression by inhibiting apoptosis, cell proliferation and cell survival, expansion of cancer stem cells (CSCs) and initiation of EMT  through regulating transcription of genes involved in cancer signaling pathway such as Wnt/-catenin pathway, Notch1 pathway, and p53 pathway [33, 34] or by interaction with several miRNAs components of miRNA processing machinery such as Dicer, Argonaute 1 and RNA Helicase A [28, 35]. In contrast, SOX4 can also serve as tumor suppressor which is evident from the fact that increased expression of SOX4 has been found to correlate with slower cancer progression, hence prolonged patient survival in bladder cancer, colon carcinoma, HCC, melanoma and gallbladder cancer [36-38] [21, 39] by inhibition of tumor initiation through DNA-damage signaling or direct transcriptional regulation of Dicer  and/or activation of apoptosis . This differential expression of SOX4 in various tumor types may relate to either amplification of SOX4 chromosome loci such as in bladder and lung cancer [41, 42], or activity of signaling pathways such as TGF- and Wnt, hormone regulation such as in breast cancer  and ovarian cancer, post-transcriptional modifications  or may occur through miRNA-mediated regulation of SOX4 mRNA stability and protein translation.
MicroRNAs directly target SOX4 to regulate cancer progression
MicroRNAs (miRNAs) are small (18–25 nucleotides in length) class of endogenous non-coding RNAs that regulate gene expression by binding to 3’-untranslated region (3’-UTR) of their target messenger RNAs (mRNAs) thereby either degrading or suppressing their translation . miRNAs are critical regulators of cell proliferation, differentiation, metabolism and apoptosis in normal cells . Deregulation of miRNAs such as downregulation or aberrant amplification/deletion of miRNA mapped to genomic loci have been implicated in tumorigenesis, making them either tumor suppressors or oncogenes respectively [46, 47]. Most miRNA have been observed to be downregulated in many human cancers . Recent studies have demonstrated that miRNAs can modulate SOX4; moreover, SOX4 also regulates the expression of several miRNAs. Most of the miRNAs are reported to be downregulated in many cancers that causes an upregulation of SOX4. In this review, we provide a comprehensive overview of interactions between miRNAs and SOX4 and their roles in cancer while focusing on how we can utilize this knowledge in novel cancer therapies. Functions of these miRNAs in cancer progression and metastasis are discussed below.
MiR-338-3p is located on chromosome 17q25.3 on 7th intron of apoptosis-associated tyrosine kinase (AATK) gene. Recent studies have identified that miR338-3p is downregulated in gastric cancers , ovarian cancer , breast cancer  , colorectal cancers  and highly metastatic non- small cell lung carcinoma cells (NSCLC) and metastatic tissue specimens , renal cell carcinoma (RCC) cell lines and tissues  and breast cancer . Recently Sun et al reported that downregulated mir338-3p enhanced proliferation and suppressed apoptosis in NSCLC by targeting RAB14 . Overexpression experiments caused suppression of cancer cell migration and invasion and EMT through directly targeting SOX4 on 3’UTR thereby decreasing its mRNA and protein expression both in vitro and in vivo . Of note, upregulation of miR338-3p also reported to be inhibited cell proliferation, migration and invasion of RCC  and breast cancer  and caused downregulation of SOX4, thereby modulating SOX4 mRNA and protein levels. This suggests that mi338-3p and SOX4 expression are inversely correlated in NSCLC, RCC and breast cancer. Li et al found that histone H3 lys27 (H3K27me3) methylation but not DNA methylation unlike in gastric cancer  is activated in the promoter region of mir338-3p which is responsible for downregulation of miR-338-3p suggesting an epigenetic regulatory mechanism of this miRNA in cancer progression .
MiR-25 is located on chromosome 7 (7q22.1) in the 13th intron of the MCM7 gene, and is a member of the miR-106b-25 cluster (consisting of miR-106b, miR-25, miR-93, miR-363-3p, and miR-367) . MiR- 25 functions as an oncogene in NSCLC  breast cancer  ovarian cancer  cervical cancer  and gastric cancer whereas it acts as tumor suppressor in colon cancer , glioblastoma multiforme . Recent studies by Chen et al revealed that miR-25 acts as tumor suppressor which is evident from the fact that miR-25 is downregulated in osteosarcoma and is associated with advanced tumor–node–metastasis stage and lymph node metastasis . However, introduction of miR-25 by overexpression experiments, showed a suppression in cell proliferation, migration, and invasion of osteosarcoma cells in vitro and in vivo. This study also showed a direct association of miR-25 with SOX4 using bioinformatics analysis and luciferase reporter assay. Furthermore, this tumor suppression effects of miR-25 is partially reversed by SOX4 following interaction thus suggesting an inverse correlation of miR-25 with SOX4 expression in osteosarcoma tissues .
miR-138 is known to be frequently downregulated in colorectal, esophageal, bladder cancers and NSCLC through PD-L1 downregulation, repression of EZH2 or targeting YAP1 [68-70]. Recent studies showed that downregulated miR-138 is associated with increased expression of SOX4 and up-regulation of miR-138 inhibited proliferation, invasion, metastasis and EMT of NSCLC cells  and highly invasive ovarian cancer cells both in vitro and in vivo . Additionally, authors used bioinformatics analysis such as TargetScan and luciferase reporter assay to predict that SOX4 is a direct potential target of miR-138 which binds to 3’UTR of SOX4 mRNA. Hence, based on this study, miR138 negatively regulates SOX4. Furthermore, earlier studies reveal that SOX4 can suppress the effects of p53  as p53 being a downstream target of Sox4 . Li et al also demonstrated that in NSCLC cells, decreased SOX4 expression could increase the level of miR-138 via up-regulation of p53. Hence, introduction of miR-138 inhibited growth, invasion and EMT of NSCLC cells through SOX4/p53 feedback loop . Similar results were shown by other studies in ovarian cancer providing a potential use of miR138 for suppression of non-small cell lung carcinoma as well as ovarian cancer by targeting SOX4 via p53 [70, 71].
Reports have identified miR-129-5p deregulation in some cancers such as esophageal cancers and chondrosarcoma , which resulted in upregulation of SOX4. A recent study by Zhang et al demonstrated that overexpressed miR-129-5p caused suppression of cell proliferation, migration and promoted apoptosis both in vitro and in vivo xenograft models by targeting SOX4 and causing its downregulation . Aberrant Wnt/B catenin signaling has been observed in many cancers including chorndrosarcoma  and is associated with SOX4 in melanoma cells . Moreover miR-129-5p also inhibited over-activation of Wnt signaling components such as b catenin, Cyclin D1and c-Myc by targeting SOX4 bringing about regulation of proliferation and migration in chondrosarcoma cells .
miR-129-2 is located on chromosome 11 at locus 11p11.2 . MiR129-2 is often reported to be downregulated in bladder cancers, colorectal carcinoma , b-catenin/TCF mediated hepatocarcinoma  gastric cancer , endometrial cancer  and esophageal cancer cells and adjacent non-tumor tissues from patients who underwent primary surgical resection for esophageal cancer (8) and correlates with advanced TNM stage in esophageal cancers. This is further associated with increased expression of SOX4 and SOX-4 mediated epigenetic silencing of miR129-2. SOX4 is found to be inversely correlated with epigenetic repression of miR0129-2, however, restoration of miR-129-2 brought a downregulation of its direct target-SOX4 expression along with a reduction in migration, proliferation and cell arrest of the cancer cells   .
miR-449a is a member of miR-34/miR-449 family consisting of miR-34a, miR-34b, miR-34c, miR- 449a, miR-449b, and miR-449c . Notably, one of its family member miR-34a is the first currently tested miRNA in a clinical trial for patients with primary liver cancer. MiR449 is known as a tumor suppressor in prostate , gastric, bladder , and lung cancer  and HCC . Sandbothe et al in their recent study demonstrated an epigenetic regulation of miR-449a induced by TSA-mediated HDAC inhibition in HCC and normal liver cell lines  suggesting a regulation by histone acetylation. Using target gene prediction databases, the authors identified putative targets for miR449 family members and SOX4 to be among one of the target which is overexpressed in HCC tissues and have poor prognosis. Moreover, the authors have also shown that overexpression of miR-449 family members was inhibited by TGF-b- mediated SOX4 overexpression which further inhibits cell migration .
MicroRNA-381 (miR-381) is reported to be a tumor suppressor which is downregulated in breast cancer , chondrosarcoma , osteo-sarcoma  and ovarian cancer  through targeting Cx43, VEGF-C, LRRC4 and YY1. In a recent novel study by Zhang et al, identified miR-381 to be downregulated in gastric cancer . However, overexpression of miR-381 inhibited migration and invasion and EMT in gastric cancer cells through direct targeting and downregulating SOX4. Mechanistic studies by Zhang at al revealed that a long non-coding RNA taurine 1 (TUG1) negatively regulates miR-381 expression in gastric carcinoma .
Earlier, MiR-204 is known to suppress tumor initiation in esophageal cancer, lung cancer and gastric cancer [90-92]. Few known targets of miR204 are USP47, SOX4, RAB22A, SIRT1 and Snai1 [90, 93, 94]. In a very recent study by Yuan et al reported that miR-204 suppressed gastric cancer cell proliferation and metastasis and negatively correlate with its target SOX4  . Further, a comprehensive analysis with clinicopathological parameters of gastric cancer patients revealed that miR-204 and SOX4 are associated with lymph node metastasis and tumor stages I-IV . Another study by Yin et al identified downregulation of miR204 in T-cell acute lymphoblastic leukaemia (T-ALL) and overexpression by mimics inhibited the cell proliferation, migration and invasion ability and SOX4 expression thereby negatively modulating SOX4 expression by binding to its 3’UTR .
A recent study identified that frequent downregulation of microRNA miR-320 occurs in primary colon cell carcinoma (CRC) tissues and cell lines. Lentiviral-mediated overexpression of miR-320 is reported to inhibit HCT116 CRC growth and migration in vitro and inhibit tumor formation in SCID mice. Furthermore, there exists an inverse correlation between the expression of miR-320 and its targets such as SOX4, FOXM1, and FOXQ1 .
Like other miRNAs discussed here, miR-363-3p and miR140 are tumor suppressors which is downregulated in colorectal cancer tissue specimens with lymph node metastasis and gastric cancer tissues and cell lines thus promoting cell migration and invasion and EMT induction both in vitro and in vivo via its direct target SOX4 . Hu et al  further demonstrated that exogenous miR-363-3p reversed the EMT and phenotypic metastasis in CRC cells. Zhou et al revealed that miR140 overexpression in HGC-27 cells inhibited SOX4 expression resulting in decreased cell viability, colony formation, and resulted in G0/G1 cell arrest .
A recent study by Zhang et al has shown that miR-187 is downregulated in colorectal cancer (CRC) cell lines and tissues and SOX4 is upregulated which is correlated with poor disease prognosis. On the contrary, exogenous miR-187 can inactivate TGF-B mediated Smad pathway, thus preventing EMT and suppressing cell proliferation and migration in vitro and inhibited CRC growth and progression in vivo. Furthermore, authors also found SOX4-upstream effector of Smad as a direct target of that miR-187 .
Several studies revealed miR-191 to be an estrogen and hypoxia responsive miRNA that promotes ER positive breast cancer cell proliferation and migration. Apart from its role in breast cancer, miR- 191-5p has been identified in other cancers such as lung cancer, colon cancer, gastric cancer as an oncogenic miRNA . A recent study by Sharma et al demonstrated that p53 downregulates miR-191-5p in breast cancer, whereas SOX4-a direct target of miR191increases p53 protein levels. In fact, overexpression of miR191 caused a down-regulation of SOX4 and p53 levels .
MiR-212 and miR-132 are located in chromosome 17 and is derived from the miR-212/132 cluster . Earlier studies reported miR-212 and miR-132 are tumor suppressor and are downregulated in ovarian cancer by targeting E2F5 and HBEGF  and NSCLC by targeting SOX4 [97. SOX4 is a downstream effector of miR-212 and miR-132 modulating EMT of ovarian cancer cells. A recent study by Lin et al identified that SOX4/EZH2 complex can silence miR-212/132 expression by binding to its promoter region and promoting H3K27me3, whereas miR-212 and miR-132 can directly bind to the 3′UTR of SOX4 and suppress its expression and thus forms a MiR-132/212-SOX4/EZH2-H3K27me3 feedback loop in ovarian cancer cells . Enhancer of zeste homolog 2 (EZH2) is a subunit of the Polycomb repressor complex 2 (PRC2), that promotes DNA methylation . Additionally, EZH2 can catalyze histone 3 (H3) (H3K27me3) trimethylation, an epigenetic modification that silences gene transcription . Therefore, the downregulation of miR212 and miR132 in ovarian cancer cells is due to epigenetic upregulation of EZH2 and SOX4. The authors further showed and interaction between EZH2, SOX4 and HDAC3 that form a co-repressor complex to silence miR212 and miR132 expression in ovarian cancer cells . Exogenous introduction of miR‐132 significantly inhibit proliferation and EMT and induce cell cycle arrest and apoptosis in ovarian cells  and osteosarcoma cells .
miR-211 plays a dual role as tumor suppressor in epithelial ovarian cancer, hepatocellular carcinoma and breast cancer    as well as an oncogene in colon cancer, oral carcinoma, head and neck squamous cell carcinoma, often associated with poor prognosis of patients . In one of the studies in gastric cancer, Wang et al, demonstrated that miR-211 is down-regulated and its over-expression inhibited gastric cancer cell proliferation and invasion in vitro by targeting and downregulating SOX4. This highlights the role of miR21 as tumor suppressor by interacting with SOX4 .
microRNA-30a (miR-30a) plays an important role in development of endometrial carcinoma , hepatocellular carcinoma , and chronic myelogenous leukemia . One of the recent study has shown that miR30a is downregulated in chondrosarcoma patients , prostate cancer and pancreatic cancer cells and is negatively correlated with SOX4 expression. MiR-30a directly targets SOX4 and its overexpression downregulates SOX4 expression thereby suppressing proliferation, migratory, and invasive capacity of SW1353 chondrosarcoma cells in vitro and TGF-B mediated EMT in prostate cancer cells .
microRNA-133a (miR-133a) is a muscle specific miRNA where the gene is located on chromosome 18 and chromosome 20 as two copies . Several studies have shown that low levels of this miRNA are expressed in several cancers including ovarian cancer , colorectal cancer , bladder cancer , breast cancer , prostate cancer and esophageal cancer . Li et al have demonstrated that miR-133a is downregulated in esophageal squamous cell carcinoma (ESCC) cell lines and clinical ESCC tissue samples and is inversely correlated with tumor progression in ESCCs suggesting that miR-133a may function as a novel tumor suppressor . Furthermore, Sox4 is reported to be a direct target of miR133a with the 3’UTR thereby regulating EMT markers as well. Notably, there is a reciprocal effect of miR133a over tumor progression upon targeting SOX4 similar to other miRNAs discussed above. For instance, following overexpression of miR133a, Sox4 is decreased thereby decreasing migration and invasion and EMT in ESCC which clearly suggest that miR-133a could act as a potential tumor suppressor in ESCC through targeting Sox4 and the EMT process .
CONCLUDING REMARKS AND FUTURE DIRECTIONS
Metastasis is a complex phenomenon and despite of various advancements in therapeutics, recurrence due to metastasis remains a severe issue for cancer patients. EMT is associated with cancer recurrence and metastasis and is often displayed by cancer stem cells (CSCs). CSCs are self-renewing and heterogenous population of cells that usually remain in resting phase thus are chemoresistant. Hence, there is an urgent need of prognosis detecting EMT and thereby regulating metastatic growth and mortality. Challenges faced in curbing metastasis and developing prognostic tools is mainly due to the clinical and molecular hetrerogeniety. Not all cells undergo EMT simultaneously and the cells which have undergone EMT not usually metastasize simultaneously. In fact, extracellular and intracellular signaling, environmental changes and epigenetic factors affect the EMT and metastatic process. Drug resistance is another serious issue that accompanies EMT and metastasis. For instance, in breast and pancreatic cancer higher resistance to drugs such as oxaliplatin and paclitaxel [4, 125] is reported to be seen in cells expressing EMT markers. Therefore, a more robust prognostication is required to overcome this challenge.
In recent years, miRNA based therapy is emerging rapidly. As a multicenter phase I study, miR-34a is currently in first clinical trial for miRNA replacement therapy since 2013 to be used in patients with unresectable primary liver cancer or other selected solid or hematologic malignancies (www.clinicaltrials.gov/ct2/show/NCT01829971). MicroRNAs act as both oncogene and tumor suppressor by regulating or are involved in the regulation of multiple signaling pathways such as Hedgehog pathway, Notch pathway, TGF β Pathway and Wnt/ β Catenin pathway. SOX4 acts as a downstream effector of some microRNAs, modulating EMT and is often reported to be overexpressed in many aggressive cancers. MiRNAs modulate the function of SOX4-a master regulator of EMT by binding to 3’UTRs and initiate their degradation at translational level. Moreover, there exists an inverse correlation between various miRNAs and SOX4 in different types of cancers. For instance, miRNA’s are downregulated in various aggressive cancers predominantly of epithelial origin and some of non-epithelial ones correlated with an upregulation of SOX4 expression levels. Some studies reported that epigenetic repression of microRNAs can lead to overexpression of SOX4 and exogenous expression of microRNAs can downregulate SOX4 expression and potentially inhibit metastasis in vitro and in vivo by inhibiting cancer cell proliferation, migration, invasion and induce apoptosis. Notably, miRNA-SOX4 overexpression and downregulation may serve as a prognostic marker for cancer patients. Further research is required to understand the molecular mechanism and the epigenetic mechanism involved in the interaction of various miRNAs with SOX4 in order to develop novel anti-cancer therapeutic approaches that involve modulation of miRNA-SOX4 interaction by epigenetic drugs. In summary, upregulation/restoration of tumor suppressor miRNA’s along with downregulation of SOX4 with an epigenetic approach or signaling pathway targeted approach could possibly evolve as a promising option to improve patient survival and decrease mortality.