Oncogene (2013) 32, 3397–3409 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

REVIEW The role of SRY-related HMG box 4 (SOX4) in tumorigenesis and metastasis: friend or foe?

SJ Vervoort1,2, R van Boxtel1 and PJ Coffer1,2

Development and progression of cancer are mediated by alterations in transcriptional networks, resulting in a disturbed balance between the activity of oncogenes and tumor suppressor . Transcription factors have the capacity to regulate global transcriptional profiles, and are consequently often found to be deregulated in their expression and function during tumorigenesis. Sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4) is a member of the group C subfamily of the SOX transcription factors and has a critical role during embryogenesis, where its expression is widespread and controls the development of numerous tissues. SOX4 expression is elevated in a wide variety of tumors, including leukemia, , lung cancer and breast cancer, suggesting a fundamental role in the development of these malignancies. In many cancers, deregulated expression of this developmental factor has been correlated with increased cancer cell proliferation, cell survival, inhibition of apoptosis and tumor progression through the induction of an epithelial-to-mesenchymal transition and metastasis. However, in a limited subset of tumors, SOX4 has also been reported to act as a tumor suppressor. These opposing roles suggest that the outcome of SOX4 activation depends on the cellular context and the tumor origin. Indeed, SOX4 expression, transcriptional activity and target specificity can be controlled by signaling pathways, including the transforming growth factor-b and the WNT pathway, as well as at the post-translational level through regulation of stability and interaction with specific cofactors, such as TCF, syntenin-1 and p53. Here, we provide an overview of our current knowledge concerning the role of SOX4 in tumor development and progression.

Oncogene (2013) 32, 3397–3409; doi:10.1038/onc.2012.506; published online 17 December 2012 Keywords: SOX4; cancer; metastasis

INTRODUCTION including prostate, colorectal and breast cancer (Table 1).7 The discovery of the sex-determining region Y (SRY) gene more Correspondingly, SOX4 has been recognized as one of the 64 than two decades ago revealed the existence of a family of ‘cancer signature’ genes, suggesting a fundamental role in tumor 7 evolutionary conserved SRY-related high-mobility-group (HMG)- development. Nevertheless, many aspects of SOX4 function box (SOX) transcription factors, characterized by their high degree remain undefined or imperfectly understood. Here, we will discuss of with the SRY HMG DNA-binding domain.1 the molecular mechanisms regulating SOX4 functionality and To date, 20 members of this family have been identified in discuss our current understanding concerning the role of this vertebrate species, which in mammals are subdivided into eight transcription factor in development, differentiation and cancer. groups (A–H), of which most are represented by a single gene in invertebrates.1 SOX family members are critical for many developmental processes regulating both the maintenance of SOX4 DOMAIN STRUCTURE AND FUNCTION stem cells and controlling terminal differentiation of a wide variety SOX4 belongs to the highly conserved group C SOX (SOXC) of cell types.2 Consequently, deregulation and mutation of SOX transcription factors, which consists of two additional members: genes have been implicated in a variety of hereditary human SOX11 and SOX12.1 SOXC members are single genes, diseases and several SOX members have been demonstrated to grouped on the basis of the high degree of sequence similarity in be involved in tumorigenesis, such as SOX9 in breast cancer and the HMG domain located at the N-terminal half of the protein, and SOX18 in prostate cancer.2,3 are further distinguished from other SOX transcription factors by At the time of its discovery more than 15 years ago by Clevers their conserved C-terminal transactivation domain (TAD)8 and co-workers,4 SOX4 was recognized as the first ‘classical’ (Figure 1). Outside these two conserved domains, the homology transcription factor within the SOX family composed of separate between SOXC members is relatively low. DNA-binding and transactivation domains, functioning as a The SOX4 HMG domain (amino acids (aa) 59–138) is responsible transcriptional activator in lymphocytes. Subsequently, SOX4 was for DNA binding and DNA bending.4,9 It binds with high affinity to demonstrated to be critical for cardiac outflow tract development the minor groove of the heptamer DNA motif AACAAAG, with the and the differentiation of pro-B and T cells.5,6 More recent studies optimal 10 bp sequence containing 50 AG and 30 G nucleotides have identified SOX4 mRNA as one of the most frequently directly adjacent to the core sequence.4,10,11 The elucidation of the upregulated transcripts in a wide variety of human cancers, solution structure of the SOX4 HMG domain identified an

1Department of Cell Biology, University Medical Centre Utrecht, Utrecht, The Netherlands and 2Division of Pediatrics, Wilhelmina Children’s Hospital, Utrecht, The Netherlands. Correspondence: Professor PJ Coffer, Department of Cell Biology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3584CX, The Netherlands. E-mail: [email protected]. Received 17 July 2012; revised 18 September 2012; accepted 18 September 2012; published online 17 December 2012 Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3398 Table 1. Overview of tumors reported with elevated SOX4 expression

Cancer Role of SOX4 Reference

HCC SOX4 is overexpressed in intrahepatic metastasis tissue compared with HCC tissue and contributes to 62,107 HCC progression by promoting metastasis Medulloblastoma Significant increased expression compared with normal cerebellum. Trend toward prolonged patient 99,130–132 survival with elevated SOX4 expression Prostate cancer SOX4 increased expression compared with normal prostate tissue. Increased levels of SOX4 59,86,133–135 expression correlate with more aggressive tumors and poor prognosis Lung cancer Gene amplification of SOX4 at 6p in lung cancer cell lines and primary tumors. Ectopic 87,135–137 SOX4 expression increases transforming ability in NIH-3T3 cells. SOX4 has been suggested as a lung tumor vaccine antigen Colorectal cancer High levels of SOX4 in microsatellite stable stage II cancers correlate with recurrence, linked to 78,79,84 metastatic capacity of primary tumor ACC Increased expression of SOX4 relative to normal tissue. Knockdown of SOX4 results in reduced 58,60 viability and increased apoptosis. SOX4 contributes to malignant phenotype Bladder cancer Elevated SOX4 expression in bladder cancer compared with normal tissue. In high-grade urothelial 100,110,111,120,138 carcinomas, the genomic segment 6p22 is amplified, resulting in increased expression of SOX4 Endometrial cancer SOX4 expression is significantly higher in tumor tissues compared with normal tissue; SOX4 is 89 targeted by miRNA129-2, the expression of which is lost in endometrial cancer; hypermethylation of this miRNA is linked to poor overall survival SOX4 is upregulated in glioblastoma compared with the normal brain with especially high expression 73,74 multiforme in the malignant glioma-initiating cells (cancer stem cells). Reduced expression of SOX4 abrogates tumorigenic activity Gallbladder carcinoma Elevated SOX4 overexpression in primary gall bladder carcinoma compared with normal epithelium 102 of the gall bladder. SOX4 expression correlates with low-grade and better overall disease-free survival Gastric cancer SOX4 is upregulated in gastric cancer compared with benign gastric tissue. SOX4 expression is 90 inversely correlated with epigenetic silencing of miRNA129-2, which is associated with poor outcome Breast cancer SOX4 expression is elevated in breast cancer compared with normal mammary tissue and positively 63,70,114 correlates with tumor grade. SOX4 expression induces EMT and promotes metastasis in vitro and in xenograft models in vivo Myeloma SOX4 is upregulated in myeloma cell lines and primary tumors. SOX4 is critical for the survival of 57 CD56 þ myeloma cells Pineoblastoma High expression levels of SOX4 compared with normal tissue 139 Splenic marginal zone The SOX4 gene is a common retroviral integration site, and is associated with disease progression 43 lymphomas Myeloid leukemia SOX4 is an oncogene in myeloid leukemia and is a common site for retroviral integration. SOX4 41,42,44,45,48,52 expression is increased in AML patient samples containing the AML-ETO1 translocation Uterine leiomyoma SOX4 overexpression compared with normal myometrium 140 Pancreatic cancer SOX4 upregulation relative to normal tissue 141 Pheochromocytoma SOX4 upregulated relative to normal tissue 142 Abbreviations: ACC, adenoid cystic carcinoma; EMT, epithelial-to-mesenchymal transition; HCC, hepatocellular carcinoma; SOX4, sex-determining region Y- related high-mobility-group box transcription factor 4.

PP PPP A AAA A P P P

YYS SS KKKKK S S S 123/126 136/143/148 192/196/197/202/217 276 354 424 SOX4 HMG-Box GRR SRR TAD/DD 1 59 135 152 227 333 397 441 474 HMGS395X TAD/DD Interaction partners ubc9, p53, syntenin-1, GATA3, TBX3 Brn1, p53, syntenin-1, GATA3

ND: TCF4, β−catenin, p300, plakoglobin, Oct-4 Figure 1. Domain structure, post-translational modifications and cofactors of SOX4. Green boxes indicate domains identified in SOX4. HMG domain: DNA-binding domain; GRR: glycine-rich region; SRR: serine-rich region; TAD/DD: transactivation domain/degradation domain; ND: not determined. Post-translational modifications present on SOX4 identified by mass spectrometry recorded on phoshosite.org. Red circles indicate and yellow indicates acetylation. Y: tyrosine; K: lysine; S: serine. S395X indicates the mutation found in lung cancer, resulting in a truncated protein.

L-shaped structure consisting of three a-helices connected by crystal structure of the SOX4 HMG domain in complex with DNA loop structures, which are stabilized by a highly conserved revealed slight structural differences in the DNA-binding interface hydrophobic core.12 Binding of the HMG domain to the minor of SOX4 compared with the previously identified structures of the groove in the DNA helix induces a sharp bend, altering local HMG domains in SOX2 and SOX17.16 This structural difference chromatin architecture and enabling the formation of functional allows the SOX4 HMG domain to accommodate both a primary transcriptional enhancer complexes.9,12–15 The recently solved AACAAAG motif and a secondary AATTGTT motif.16 The reported

Oncogene (2013) 3397 – 3409 & 2013 Macmillan Publishers Limited Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3399 variation in binding motifs is in accordance with previous present in mesenchymal and neural progenitor cells, where they observations demonstrating a marked variability in the act redundantly to ensure survival of these multipotent cells.8,25 sequences bound by SOX4 in the promoters of its target genes, Consistent with their role in these cells, SOXC triple knockout mice such as Tubulin-beta 3 (Tubb3) (TATTGTC, CATTGTG), Tead2 die of severely impaired organogenesis at midgestation (E8).25 (CTTTGTC, CTTTGTT) and the AACAATA motif in the CD2 SOX4-deficient animals also suffer from defects in pancreatic islet enhancer.8,17,18 This variation in enhancer element sequence formation, which results in impaired insulin secretion and glucose expands the repertoire of target genes regulated by SOX4. tolerance and models increased risk of type 2 diabetes and obesity Similar to other SOX transcription factors, the SOXC HMG in humans.26–29 In addition, loss of SOX4 during hematopoiesis domain has been demonstrated to interact with a variety of other blocks development of B cells at the pro-B-cell stage and severely transcription factors and thereby cooperatively regulate gene affects the development of T lymphocytes, its activity playing an expression.19 For example, SOX4 associates with BRN2 and TBX3, important role in mediating the transforming growth factor-b which results in synergistic activation of the Nestin gene in (TGF-b)-induced block in T-helper type 2 cell differentiation.5,6,30 neurons and the Gja1 gene in the heart, respectively.19 The During hematopoiesis, SOX4 also controls differentiation of transcription complexes formed upon binding distinct myeloid lineages regulated by the interleukin (IL)-5 receptor transcription factors represent an additional level of target gene signaling complex, through direct interaction with the adaptor specificity and transcriptional activation in a given cell system. protein syntenin-1.22,31 The C-terminal 33 residues in the SOX4 protein constitute Finally, mice that are heterozygous for the SOX4 knockout allele the TAD. Deletion of this domain completely abrogates the show diminished bone formation and osteoblast development, transactivation capacity of SOXC members.8 In SOX11, the TAD which is possibly due to SOX4-mediated effects on parathyroid is predicted to adopt a stable uninterrupted a-helical structure, and parathyroid-related hormone receptor signaling in osteoblast which is shorter and interrupted in SOX4 and SOX12.8 cells.32,33 Interestingly, in a genome-wide association study in Correspondingly, the SOX11 TAD possesses the strongest humans the SOX4 has been associated with bone mineral transactivating capacity followed by SOX4 and SOX12, density and fracture risk in postmenopausal women and respectively.8 The mechanism through which the TAD of SOXC reduced expression of SOX4 correlated with susceptibility to transcription factors controls transcriptional activation remains to osteoporosis.34,35 be investigated. However, it is likely that the SOX4 TAD recruits co- After birth, expression of SOX4 becomes restricted as cells activators, as the homologous domain in SOX2 has been become terminally differentiated. In adult tissue, SOX4 expression demonstrated to recruit co-activators such as the histone acetyl appears to be more limited to specific cell types, including transferase p300.20 pancreatic islet cells, gonads, female reproductive tissues, thymus, Interestingly, the TAD of SOX4 also controls proteasomal hematopoietic stem cells, mammary stem cells and intestinal crypt degradation and thereby regulates protein stability and expres- stem cells.4,36–40 This restricted expression in adult tissues sion.21 In a variety of cancer cell lines, SOX4 was observed to be indicates that SOX4 activity has to be constrained to a limited rapidly degraded by the , which was independent of subset of cells and potentially reflects the activity of various polyubiquitination, but rather depended on the presence of the signaling pathways, controlling SOX4 expression and stability. The TAD. Proteasomal degradation could be prevented by association important developmental role of SOX4 in the control of cell fate of the SOX4 TAD to its interaction partner syntenin-1, resulting in and differentiation suggests that, similar to other tumor-related reciprocal stabilization in the nucleus and increasing the developmental transcription factors, aberrant regulation of SOX4 transcriptional output of SOX4.21,22 and associated developmental processes has a significant impact Two additional functional domains have been identified in on tumorigenesis. SOX4: a glycine-rich region (aa 152–227), which was demonstrated to be involved in proapoptotic events, and a serine-rich region (aa 333–397), previously thought to be part of the TAD.23 SOX4-MEDIATED REGULATION OF TUMORIGENESIS AND In addition to the functions detailed here, SOX4 domains can be TUMOR PROGRESSION the acceptor site for post-translational modifications (PTMs), In recent years, large-scale studies have been modulating the functionality of these regions, and consequently applied to identify novel cancer-related genes, which could be SOX4 activity during development and tumorigenesis (Figure 1). used as prognostic markers or therapeutic targets. Such studies The role of context-dependent regulation of SOX4 will be have identified elevated SOX4 expression in nearly all major discussed below. human cancers, including breast, lung, brain, prostate, colorectal, bladder, pancreatic and ovarian cancer, indicating a central role in the development of multiple tumors7 (Table 1). SOX4 REGULATES EMBRYONIC DEVELOPMENT AND Some of the most compelling evidence pointing towards a pro- DIFFERENTIATION oncogenic role for SOX4 originates from genome-wide retroviral To understand the role of SOX4 in malignancies, it is important to tagging studies in cancers of hematopoietic origin. Retroviral first highlight its role in normal vertebrate development and insertional mutagenesis can result in activation of proto-onco- differentiation, as aberrant recapitulation of developmental genes and more rarely inactivation of tumor suppressor genes, processes in adult cells can underlie tumorigenesis. SOX4 and tumor-promoting insertions are identified by their selective expression is widespread during embryonic development, over- outgrowth. Such studies have identified SOX4 as the most lapping with other SOXC members in the central and peripheral common retroviral integration site in mouse myeloid leukemia, nervous system, and mesenchyme of developing organs.8,24 In B/T-cell lymphomas and splenic marginal zone lymphomas addition, SOX4 is expressed in the developing thymus, spleen, and (a B-cell lymphoma subset), indicating that, in addition to normal hair follicles.8 As suggested by its expression pattern, SOX4 has an hematopoietic development, SOX4 can induce malignant hema- important role in a plethora of developmental processes. Indeed, topoietic development.41–43 Indeed, forced expression of only SOX4-deficient animals suffer from many developmental defects SOX4 in mouse bone marrow cells is sufficient for cellular (Table 2), resulting in lethality at embryonic day 14 (E14) as a result transformation by blocking myeloid differentiation and inducing of circulatory failure caused by malformation of the cardiac the formation of myeloid leukemia in the majority of transplanted outflow tract.5 The late embryonic lethality can be attributed to animals.44,45 Retroviral insertions in the SOX4 gene can also act the significant degree of redundancy between SOXC transcription synergistically with pre-existing or retrovirus-induced genetic factors.25 High expression levels of all three SOXC genes are lesions to promote development of myeloid leukemia, such as

& 2013 Macmillan Publishers Limited Oncogene (2013) 3397 – 3409 Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3400 Table 2. Phenotype of SOXC knockout model organisms

Alleles Phenotype Reference

Sox4 À / À Die in utero at E14.5 due to defects in cardiac outflow tract development 5 Sox4 À / À Block in B-cell development at pro-B-cell stage and decreased development of 6 hematopoietic cells T cells Sox4b À / À Defect in pancreatic a cell differentiation in zebrafish (D. rerio)29 Sox4 À / À Impaired pancreatic islet cell development 28 Sem-2 À / À Absence of vulval and uterine muscles, resulting in egg laying defect in C. elegans. Sem-2/SOXC promotes 143 development of proliferative sex myeloblast cells over differentiated body wall muscle cells Sox14 À / À Lethal in D. melanogaster, die at prepupae stage. Numerous morphological and internal defects 144 Sox4 þ / À Impaired osteoblast development and lower bone mass due to diminished bone formation 33 Sox11 À / À Die at birth from defects in cardiac development, additional abnormalities observed in the lung, stomach, 145 pancreas and eyelids Sox11 À / À In sensory neurons, SOX11 deletion results in impaired survival of sensory neuron and a block in axonal 146 outgrowth Sox12 À / À No defects observed because of non-reciprocal redundancy with SOX4 and SOX11 147 Sox4 À / À , Sox11 À / À Increased cell death in neural cell types in the central nervous system during development, resulting in 148 hypoplasia of the spinal cord Sox4 À / À , Sox11 À / À , SOXC triple knockouts die in utero at E8.5 because of impaired organogenesis caused by impaired survival 25 Sox12 À / À of mesenchymal and neuronal progenitor cells Abbreviations: E14.5, embryonic day 14.5; SOX4, sex-determining region Y-related high-mobility-group box transcription factor 4; SOXC, group C SOX.

loss of Cdkn2a, Sfpi1 and p15Ink4b, proviral integration in the EVI1 to investigate transcriptional networks in isolated human oncogene, MADS box transcription enhancer factor 2C (Mef2C) and hematopoietic cells, SOX4 and the known HSC-related CREB, and viral integration of the large T antigen.41,44,46–48 In a transcription factor PBX1 were identified as key regulators of an search for cooperating cancer genes, leukemias derived from HSC and progenitor gene expression profile.56 This suggests a SOX4-transduced bone marrow-transplanted mice were analyzed fundamental role for SOX4 in HSC development and maintenance, for insertional mutations induced by the SOX4 carrying retrovirus, which could be mirrored by similar functions in leukemic stem revealing integrations in various cancer-related genes, including cells. Nevertheless, the mechanisms whereby SOX4 functions in multiple integrations in the SfpiI (PU.1 in humans) gene and the human HSCs or causality in the development of human myeloid Mef2C gene.45,49 The presence of multiple independent leukemia remain unresolved. In addition, in multiple myeloma, a integrations at the loci of these important myeloid and distinct hematological malignancy of the B-cell lineage, SOX4 lymphoid differentiation factors is indicative for a cooperative expression has been linked to the expression of the neural cell role with the introduced SOX4 oncogene. Indeed, co-transduction adhesion molecule (NCAM/CD56).57 SOX4 directly regulates of bone marrow cells with Mef2C was shown to accelerate expression of this neuronal marker, the expression of which significantly the SOX4-mediated development of myeloid occurs in the majority of multiple myelomas and is associated leukemia.45 Similarly, haploinsufficiency of the SfpiI gene with tumor development, lytic bone lesions and poor disease encoding the transcription factor PU.1 acted synergistically with prognosis, suggesting that SOX4 contributes to the development SOX4 overexpression to promote murine myeloid leukemia.50 In de of CD56 þ multiple myelomas.57 novo tumors, SOX4 might actively repress SfpiI expression by Taken together, it appears that deregulation of SOX4 during directly binding an upstream regulatory element in the SfpiI gene, hematopoiesis has the capacity to give rise to distinct hemato- which is corroborated by an inverse correlation between SOX4 and poietic malignancies. However, it remains to be investigated how PU.1 expression in acute myeloid leukemia (AML) patient genetic alteration or aberrant regulation of SOX4 contributes to samples.50 In AML, SOX4 also acts in a cooperative manner with the development of hematopoietic tumors in humans. the proto-oncogenic transcription factor CREB to promote myeloid Elevated expression of SOX4 has also been demonstrated in a transformation by increasing cell survival and self-renewal.51 wide variety of solid tumors (Table 1). However, in contrast to SOX4 was shown to directly regulate the expression of CREB hematological malignancies, disparate functions for SOX4 have by binding to an upstream CREB promoter element and induc- been reported in the development and progression of solid ing transcription. Accordingly, SOX4 and CREB protein expres- tumors. In a manner reminiscent of c-MYC, SOX4 may act as either sion were observed to be elevated and positively correlated a tumor suppressor gene or oncogene, depending on the type of in pediatric AML patient samples.51 Increased expression of tumor, the cellular context and the stage of tumor development. SOX4 in human AML also occurs in patient samples contain- ing the common MOZ-TIF2, AML1-ETO and NUP98-HOXA9 translocations.52–54 These findings indicate that SOX4 acts as a proto-oncogene in both myeloid leukemia and lymphomas by PRO-ONCOGENIC ROLES OF SOX4 IN SOLID MALIGNANCIES acting in concert with a variety of distinct genetic lesions. There is compelling evidence to suggest that SOX4 acts as a proto- Many oncogenic transcription factors in myeloid leukemias oncogene in a wide variety of solid tumors. Increased expression influence hematopoietic stem cell (HSCs) development and the of SOX4 has been correlated with tumor development or tumor induction or maintenance of leukemic stem cells.55,56 Also, SOX4 progression in colorectal cancer, breast cancer, prostate cancer, has been recognized as a potential regulator of HSC activity in hepatocellular carcinoma (HCC), glioma, gastric cancer, adenoid both mouse and human hematopoiesis.38 In a gain-of-function cystic carcinoma (ACC), lung cancer and endometrial cancer screen in mouse HSCs, SOX4 was identified as one of the 18 (Table 1). Depending on the tumor type, SOX4 expression may factors conferring increased HSC activity, demonstrated by impact on a variety of distinct processes, including inhibition of increased repopulating capacity of the transduced HSCs.38 apoptosis, increased cell invasion and metastasis, and induction Moreover, in a recent study using gene expression profiling and maintenance of cancer-initiating cells.

Oncogene (2013) 3397 – 3409 & 2013 Macmillan Publishers Limited Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3401 Contribution of SOX4 to tumor initiation by inhibition of apoptosis demonstrated to be required for TGF-b-mediated induction of and stimulation of cell proliferation and cell survival EMT. Moreover, ectopic expression of SOX4 in mammary epithelial Similar to its prosurvival role in mesenchymal and neural cells was sufficient to induce EMT and confer metastatic properties progenitor cell development, several studies have provided in vitro such as increased migration and enhanced stem cell evidence that SOX4 promotes tumorigenesis primarily through properties. In mouse xenograft experiments, SOX4 potentiated RAS- inhibition of apoptosis and stimulation of cell survival. SOX4 induced tumorigenesis in MCF10A cells and has also been shown to expression is distinctly higher in primary ACC and prostate tumors reduce the metastatic potential of highly metastatic derivatives of compared with the corresponding healthy tissue.58–60 RNA MDA-MB-231 cells.63,70 Moreover, immunohistochemical analysis of interference-mediated downregulation of SOX4 in ACC-, SOX4 expression in patient breast cancer samples revealed a prostate- and breast cancer-derived cell lines induced apoptotic positive correlation with tumor-grade and triple-negative breast cell death in vitro, suggesting that SOX4 contributes to cell cancers.69 These findings demonstrate an important role for SOX4 survival.59,60 The prosurvival function of SOX4 could be related to in the progression and metastasis of high-grade breast cancers. its transcriptional regulation of the transcription factor TEAD2, A role for SOX4 in the maintenance of cancer-initiating cells has which is part of the HIPPO signaling pathway and confers SOX4- been described in glioma, a tumor type that arises from glial cells mediated survival signals during organogenesis. Interestingly, the in the brain or the spine. SOX4 expression is responsive to TGF-b in prosurvival role of the HIPPO pathway has also been implicated in a variety of cell types, including highly aggressive glioma-initiating the development and metastasis of a large variety of tumors, cells. In glioma-initiating cells, SOX4 expression is directly induced suggesting that SOX4-mediated modulation of this pathway may, by SMAD2/3 downstream of TGF-b signaling.71–74 Subsequently, in part, underlie its prosurvival function.61 SOX4 induces the expression of the stem cell marker SOX2 In addition to this prosurvival function, stable expression of through cooperative binding with OCT-4 to the SOX2 promoter, SOX4 in non-transformed prostate cells was sufficient to confer which was shown to be critical for TGF-b1-mediated maintenance oncogenic properties as illustrated by the increased colony- of stemness in glioma-initiating cells.74,75 As SOX4 is found to be forming capacity in soft agar and anchorage-independent growth, highly expressed in stem cells and early progenitors from a variety suggesting that SOX4 exerts additional pro-oncogenic effects.59 of tissues, it is possible that the stemness-promoting activity of SOX4 observed in glioma-initiating cells contributes to tumor progression in diverse malignancies. Promoting tumor progression through expansion of cancer stem Taken together, these data indicate that tumor-specific cells, initiation of an epithelial-to-mesenchymal transition and increased expression of the developmental transcription factor tumor metastasis SOX4 contributes to tumor development and progression. In addition to its role in the initial stage of tumor development, Although this idea is heavily supported by the evidence reviewed SOX4 can contribute to tumor progression by supporting here, the proposed mechanisms through which SOX4 exerts this expansion of highly aggressive tumor-initiating cells and by pro-oncogenic function are diverse and appear to vary greatly promoting tumor cell metastasis. In HCC and breast cancer, SOX4 depending on the tumor type. mRNA expression has been demonstrated to be particularly elevated in metastases, which was associated with disease progression, recurrence and poor-metastasis-free survival.62,63 SOX4 and the WNT/b-catenin signaling pathway SOX4 has been demonstrated to function as an oncogene in more The canonical , which signals through b- advanced stages of HCC by promoting intrahepatic metastasis.62 catenin and TCF transcription factors, is critical for correct Depletion of SOX4 in invasive HCC cell lines greatly suppresses embryonic development and the maintenance of adult stem cells. in vivo tumor growth, invasion into smooth muscle layers and Unsurprisingly, aberrant regulation of this pathway has been formation of metastatic tumor foci. Moreover, SOX4 knockdown implicated in the pathogenesis of a wide variety of malignancies.76 in HCC cell lines was associated with distinct morphological SOX4 expression is upregulated by WNT signaling in both benign changes in vitro, including reduced vimentin expression and and malignant intestinal tissues.37,77,78 In the intestine, SOX4 is altered actin organization. This phenotype resembles a reversion predominantly expressed in the part of the crypt containing the of the metastasis-associated mesenchymal phenotype to an intestinal stem cell compartment.37 Although SOX4 has not been epithelial state, a process that is termed the mesenchymal-to- functionally implicated in the development or maintenance of epithelial transition (MET). This suggests that SOX4 is involved in these cells, SOX4 has been demonstrated to potentiate canonical the promotion of an epithelial-to-mesenchymal transition (EMT) WNT signaling through a variety of mechanisms. First, SOX4 during metastatic conversion of HCC cells.64 Using a bioinformatics directly interacts with TCF and b-catenin, which stabilizes the b- approach combined with chromatin immunoprecipitation and catenin/TCF complex. This results in increased WNT target gene SOX4 knockdown, 31 potential SOX4 target genes were identified activation and subsequently proliferation of colon carcinoma in HCC.62 Two of these genes, neuropilin-1 (NRP-1)andsemaphorin cells.79,80 Second, it has been proposed that SOX4 can stabilize 3C (SEMA3C), have previously been linked to tumor development b-catenin by transcriptional induction of casein-kinase 2, which is and are part of a signaling pathway that regulates axonal known to directly phosphorylate b-catenin, thereby protecting it guidance. Notably, this process is often aberrantly used by tumors from degradation.80,81 Third, SOX4 has been proposed to promote to promote metastasis.65–68 Subsequently, SEMA3C and NRP1 were WNT/b-catenin signaling in endometrial carcinomas through direct demonstrated to be potential effectors of the SOX4-induced transcriptional activation of TCF4 expression, resulting in WNT metastatic phenotype, as knockdown of these genes in HCC cells target gene activation.82 Finally, SOX4 has been described to reduced cell migration and invasion. This suggests that the interact, in a WNT3a-dependent manner, with the E-cadherin and metastatic potential of HCC cells is at least, in part, modulated the desmosome-associated junction protein plakoglobin.83 WNT3a- through SOX4-mediated regulation of metastatic effectors. induced nuclear shuttling of junction protein plakoglobin increases Recently, SOX4 has been demonstrated to induce EMT in the interaction with SOX4 and competes with the binding of SOX4 human mammary epithelial cells. Ectopic expression of SOX4 in to b-catenin, resulting in nuclear export of SOX4 and reduction in these cells induced a mesenchymal phenotype, which was the activity of WNT signaling pathway83 (Figure 2). Consistent with a associated with increased stem cell properties, cellular migration role as an ‘enhancer’ of WNT signaling, increased SOX4 expression in and invasion in vitro.69 SOX4 expression itself was shown to be colorectal cancer correlates with poor outcome.84 Taken together, regulated by TGF-b, a potent inducer of EMT in a variety of these findings indicate an important role for SOX4 in the WNT cancers. Importantly, in mammary epithelial cells, SOX4 was signaling pathway and WNT-driven colorectal tumorigenesis. In

& 2013 Macmillan Publishers Limited Oncogene (2013) 3397 – 3409 Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3402 Wnt3a TGF-β1 IL-5 TGF-βR1 IL-5R Frizzled E-Cadherin

Prostaglandins, DNA-Damaging Smad2/3 β-Cat (B) agents Syntenin-1 + and Parathyroid hormone P ++(E) (A) TA (G) JUP D

P Sox4 +

p300/CBP Smad2/3 P TA p53 D Smad4 PR

Sox4 P + SOX4 mRNA Smad2/3 -

Smad4 SOX4 promoter TA D miRNA-335 Syntenin-1 miRNA-129 (C) Sox4 (F) miRNA-93 miRNA-129-2 Sox4 TAD TAD (D) Syntenin-1 Ubc9 + SOX4 target genes (e.g. Tubb3, Tead2) Sox4 Sox4 p53 TF AACAAAG AACAAAG Proteasome Target gene promoter (I) TA GATA3 D (H) TAD β-Cat + p53, TCF Sox4 and other TF target genes Rapid degradation Sox4 TCF TF p53

Distinct TF target gene

Figure 2. Schematic overview of the pathways controlling SOX4 transcriptional activity through transcriptional and post-translational regulation. (a) Regulation of SOX4 mRNA transcript levels occurs through the activation of the canonical TGF-b pathway via direct binding of SMAD2/3 in complex with SMAD4 to the SOX4 promoter region. (b) SOX4 expression is also controlled through distinct pathways in hormone- sensitive tissues through prostaglandins and parathyroid hormone. (c) SOX4 mRNA levels can be regulated post-transcriptionally by miRNA- 335, miRNA-129 and miRNA-93. (d) In addition, SOX4 expression is regulated on a post-translational level. SOX4 is rapidly degraded by the proteasome, resulting in a short half-life of approximately 1 h. Proteasome-mediated degradation can be halted via its interaction with the receptor adaptor protein syntenin-1, which results in stabilization in the nucleus. (e) SOX4 stability may also be controlled upon DNA damage via the ATM/ATR pathway. Upon DNA damage, SOX4 interacts with p53 and p300/CBP, preventing p53 degradation and increasing stability, resulting in elevated expression of DNA-damage target genes. (f) SOX4 transcriptional activity is regulated by Ubc9 in a sumoylation- independent manner and the junction protein plakoglobin (JUP) (g), which has been proposed to regulate SOX4 localization. Finally, SOX4 expression impacts on the activity of various transcription factors and signaling pathways. (h) For example, SOX4 expression promotes WNT signaling by stabilizing b-catenin, resulting in the activation of TCF target genes. SOX4 also interacts with a number of transcription factors, resulting in specific target gene selection and synergistic activation. (i) In contrast, SOX4 has been demonstrated to interact with GATA3, resulting in inhibition of DNA binding and prevent target gene activation by occupying the GATA3 target gene interleukin-5 promoter.

addition to colorectal cancer, aberrant WNT signaling has been genes. A number of studies have investigated SOX4-mediated implicated in the development of a large number of distinct human regulation of transcriptional targets in a variety of human cancers. cancers, such as melanoma, breast cancer and leukemia.85 However, Analysis of potential SOX4 target genes in prostate cancer, ACC, it remains to be determined whether SOX4 contributes to WNT- HCC and lung cancer revealed that SOX4 could be involved in the driven tumorigenic effects in these cancers. regulation of many key cellular processes, including apoptosis, control, microRNA (miRNA) processing, differentiation and growth factor signaling.59,60,62,86 SOX4-mediated transcriptional regulation In lung tumors and cell lines, SOX4 expression is elevated As SOX4 is a transcription factor, its function in cancer most because of genetic amplification of the SOX4 locus.87 Based likely relies on its direct regulation of transcriptional target on this tumor-specific expression of SOX4 in lung tumors

Oncogene (2013) 3397 – 3409 & 2013 Macmillan Publishers Limited Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3403 and the presence of naturally occurring SOX4 antibodies progression in a variety of cancers, including bladder cancer, colon in small-cell lung cancer patients, SOX4 has been considered carcinoma, HCC, melanoma and gallbladder cancer.99–102 This as a possible antigen for lung tumor vaccination.80 SOX4 suggests that SOX4 in these malignancies can act as a tumor knockdown in lung cancer cells carrying a SOX4 amplification suppressor. In bladder cancer, colon carcinoma and HCC, the was demonstrated to result in differential expression of numerous tumor-suppressive function of SOX4 has been attributed to its genes involved in neuronal development, such as TEAD2, the ability to induce apoptosis, which may occur downstream of PCDHB gene cluster, MYB and RBP1.88 The SOX4 gene signature in various stimuli, including prostaglandins and DNA damage.100,103–106 lung cancer was particularly prominent in small-cell lung cancer, which is characterized by its neuroendocrine nature and elevated expression of SOX2, SOX4 and SOX11.88 Investigation of direct transcriptional targets of SOX4 in the Inhibition of tumor initiation through DNA-damage signaling and prostate cancer cell line LNCaP, using chromatin immunoprecipi- activation of apoptosis tation-based genome-wide promoter binding analysis and micro- SOX4 has been identified as an effector of the DNA-damage array gene expression profiling, revealed SOX4-mediated pathway, which is required for DNA-damage-induced activation of regulation of genes involved in WNT signaling, inflammation p53.103 Upon treatment of the colon carcinoma cell line HCT116 and apoptosis59,86 A large number of genes were characterized as with DNA-damage-inducing agents and subsequent activation of direct targets based on SOX4 promoter binding combined with DNA-damage signaling through ATM/ATR kinases, SOX4 protein altered gene mRNA transcript levels upon modulation of SOX4 expression and stability were observed to be increased in an ATM/ expression by either knockdown or overexpression. The set of ATR-dependent but p53-independent manner.103,104 This DNA- proposed direct transcriptional targets consists of important damage-induced increase in SOX4 expression was not associated cancer-related genes, including EGFR, DICER, Tenascin C, ADAM10 with increased mRNA expression, suggesting a yet unknown post- and Frizzled-5, regulating miRNA processing, embryonic translational mechanism.103,104 In the presence of DNA damage, development, growth factor signaling and tumor metastasis.86 SOX4 also directly interacts with p53, resulting in increased Microarray analysis of SOX4 knockdown in ACC cells revealed expression of the latter through recruitment of p300/CBP to the differential expression of genes involved in cell cycle and apoptosis SOX4–p53 complex. Subsequent acetylation of p53 disrupts pathways, including BIRC5, BNIP3 and Cyclin-B1, suggesting that in binding to its ubiquitin ligase MDM2, thereby decreasing p53 both prostate cancer and ACC increased tumor-specific SOX4 polyubiquitination, inhibiting nuclear export and increasing p53 expression activates a variety of pro-oncogenic pathways through stability.103 In support of these findings, SOX4 was demonstrated the induction of SOX4 target genes.60 However, the functional to induce apoptosis and cell cycle arrest in vitro, and impair tumor contribution of individual target genes to the SOX4-mediated growth in vivo in a p53-dependent manner in HCT116 cells.103 tumorigenic responses remains to be determined. Similar to ACC Similar to colon carcinoma, in primary HCC, SOX4 acts as a and prostate cancer, SOX4 knockdown in gastric and endometrial tumor suppressor by interacting with the DNA-damage pathway. cancer cell lines decreases proliferation and increases apoptosis.89,90 Etoposide-mediated activation of the DNA-damage pathway in Taken together, these data suggest that SOX4 expression in a HCC cell lines has also been demonstrated to result in SOX4 variety of diverse tumors promotes cell survival and transformation, stabilization and interaction with p53.107 However, in contrast to through increased proliferation and inhibition of apoptosis. previous findings, SOX4 overexpression decreased p53-mediated Although several studies have attempted to identify SOX4 transcriptional activation of the proapoptotic BAX gene, and transcriptional targets in human tumors, comparative analysis reduced p53-induced apoptosis upon DNA damage.107 of SOX4 transcriptional targets between cancers types reveals a Nonetheless, increased SOX4 expression was observed to relatively minor degree of overlap (Figure 3a). Although the correlate with diminished risk of recurrence and improved limited overlap could be the result of limitations in the different overall survival in patients, suggesting a tumor-suppressive experimental setups, it is more likely that it rather reflects tumor- function.107 These findings are in contrast with previous reports specific and context-dependent mechanisms in target gene stating a metastasis-promoting function of SOX4 in advanced selection. In agreement with this is the finding that the core stages of HCC.62 This apparent contrast could illustrate stage- SOX4-regulated genes, such as the tyrosine kinase signaling- specific effects of SOX4 in the context of HCC. While SOX4 may act associated EGFR and VEGF, are present in multiple studies as a tumor suppressor in the initial stages of the disease by (Figure 3b). Notably, EGFR and VEGF are well known for their promoting apoptosis, in more advanced stages of tumorigenesis, pivotal role in the development of cancer by regulating cellular it may acquire metastasis-promoting capacity due to mutational transformation and angiogenesis, respectively.91,92 In addition, inactivation of the DNA-damage pathway and activation of other SOX4 targets within this group, such as SLCO5A1, CCNG2, prometastatic genes. SOX4 has recently been suggested to have GPR56, GRN, MGAT4B, SKP2, TRIB3 and CD24, have been a role in the DNA-repair pathway upstream of p53 in demonstrated to have diverse roles in the tumorigenesis of a medulloblastomas. In medulloblastoma cell lines, SOX4 variety of cancers (Figure 3b).92–98 The context-dependent expression was rapidly induced upon g-irradiation, a process activation of target genes is reminiscent of other transcriptional that could be prevented by ectopic expression of the secreted effectors involved in signaling pathways such as the TGF-b protein acidic and rich in cysteine protein (SPARC), a glycoprotein pathway, in which the SMAD transcription factors elicit distinct that is involved in radiotherapy sensitization.108 Ectopic SPARC responses in a cell-type- and tumor-stage-dependent manner. The expression as well as small interfering RNA-mediated SOX4 consequences for TGF-b-induced tumor cell fate outcome are that depletion resulted in decreased DNA repair and increased in early-stage tumor development, the pathway acts in a tumor- radiotherapy sensitivity which was associated with reduced suppressive manner, but promotes metastasis in more advanced p53 phosphorylation and increased DNA-damage, suggesting a tumors. The wide variety of target genes in different cell systems potential role for SOX4 in the p53-mediated DNA-repair pathway. might suggest that this process is mirrored by SOX4. SOX4 mRNA expression has also been shown to be rapidly induced by treatment of HCT116 cells with the chemotherapeutic agents 5-fluorouracil and oxaliplatin, and its expression remained TUMOR SUPPRESSOR FUNCTIONS OF SOX4 elevated in therapy-resistant cells compared with non-resistant In direct contrast to the tumor- and metastasis-promoting role of parental lines.109 These findings suggest that SOX4 has dual roles SOX4, several studies have shown that increased SOX4 expression in cancer therapy treatment, promoting either resistance or correlated with prolonged patient survival and slower disease sensitization depending on the cellular context and tumor type.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3397 – 3409 Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3404

noma

124 genes SOX4 knockdown 2 273 genes stic carci Lung cancer 1 ancer 1 2 SOX4 knockdown cancer ncer cy Prostate cancer 4 49 genes g ca noid

Prostate Prostate c Lun Ade SOX4 knockdown 3 CCNG2 Adenoid cystic carcinoma RNF150 SOX4 overexpression PCDHB5 SLCO5A1 SOX4 knockdown MAN1C1 345 genes GPR56 SOX4 overexpression GRN Prostate cancer MGAT4B SKP2 VEGF TRIB3 CD24 EGFR

UP DOWN

SOX4 knockdown Overlap Genes UP Genes DOWN Adenoid cystic carcinoma 273 genes SOX4 knockdown A Prostate cancer 124 genes SOX4 knockdown 49 genes 1 Lung cancer 9 (B) B 41 (C) 345 genes C 19 (A) SOX4 overexpression Prostate cancer

44 (D)

2616 genes Prostate cancer D SOX4 ChIP on chip

B+C

Figure 3. Gene expression analysis of SOX4 target genes in prostate cancer, ACC and lung cancer. Gene expression data sets taken from selected publications investigating SOX4 target genes in prostate, ACC and lung cancer in which SOX4 promotes tumorigenesis. SOX4 target genes were defined as significantly regulated genes that were differentially expressed 4 2-fold. (a) Overlap in SOX4 target genes between published data sets. (b) SOX4 target genes13 represented in more than one expression data set with the indicated directionality of regulation. (b) Overlap between SOX4 target genes, determined by gene expression analysis, and SOX4-bound genes, as determined by chromatin immunoprecipitation (ChIP), on chip in prostate cancer.

In bladder cancer, SOX4 has been suggested to function as a line decreases cell viability and induces apoptosis.100 The tumor- tumor suppressor in a manner independent of the DNA-damage suppressive role of SOX4 in these cells appears to be related to pathway. In a number of bladder carcinoma cell lines, the genomic activation of proapoptotic and antiproliferative SOX4 target region 6p22.3, which harbors the SOX4 gene, is amplified, genes.100 Microarray analysis of HU609 cells, which overexpress resulting in gene duplication. Although this copy number variation SOX4, revealed differential expression of 130 potential SOX4 cannot be directly related to increased SOX4 expression, SOX4 target genes, involved in angiogenesis, cell cycle arrest and mRNA and protein levels are significantly elevated in bladder apoptosis, such as interleukin-8, NEDD9 and PIK3R3.100 A small carcinoma samples when compared with normal tissue, which subset of 27 genes also correlated with SOX4 expression in correlates with prolonged patient survival.100,110,111 Similar to HCC, primary bladder tumors, suggesting that the majority might not increased expression of SOX4 in the HU609 bladder carcinoma cell represent direct target genes or that additional factors are

Oncogene (2013) 3397 – 3409 & 2013 Macmillan Publishers Limited Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3405 involved in their modulation. These findings indicate that the progesterone control the growth and differentiation of the tumor-suppressive function of SOX4 can at least, in part, be mammary gland. However, both these hormones also explained by proapoptotic effects through interaction with the profoundly influence the development of breast cancer.118,119 It p53 DNA-damage pathway or through the induction of is thus possible that, in the context of hormone-sensitive cancers proapoptotic and antiproliferative SOX4 target genes. such as breast cancer and ovarian cancer, the tumorigenic effects exerted by SOX4 depend on the expression and activation of the Inhibition of tumor suppression by repressing tumor metastasis and progesterone receptors, providing an example of context-specific regulation, even between cancer subtypes. A recent study investigating the effects of SOX4 in human Tumor-type- and stage-specific expression of SOX4 can also occur cutaneous melanoma revealed that the tumor-suppressive activities through miRNA-mediated regulation of mRNA stability and protein of SOX4 might also stem from its ability to regulate tumor cell translation. In bladder cancer, genomic profiling of miRNAs revealed migration and invasion.101,112 Ectopic SOX4 expression decreased that expression of the SOX4 targeting miRNA-129 positively melanoma cell migration, invasion and stress fiber formation correlated with disease progression. The expression of miRNA-129 through direct transcriptional repression of the prometastatic factor inversely correlated with SOX4 expression levels and was associated nuclear factor-kB p50. Markedly reduced expression of SOX4 was with poor outcome, suggesting that in the context of bladder cancer observed in metastatic melanoma compared with primary tumors, miRNA-mediated repression of SOX4 expression promotes tumor which inversely correlated with nuclear factor-kBp50expression 120 101 progression. In contrast, loss of SOX4 targeting miRNAs in gastric and was related to diminished patient survival. In addition, SOX4- cancer, endometrial cancer and breast cancer has been induced suppression of melanoma metastasis can be mediated by demonstrated to promote tumor progression.63,89,90 In both direct transcriptional regulation of DICER, a critical component of endometrial and gastric cancer, SOX4 mRNA expression can be the miRNA processing pathway.112 In patient samples, DICER repressed by miRNA-129-2, the expression of which is frequently lost expression positively correlated with SOX4 expression and because of hypermethylation-mediated silencing, resulting in reduced expression of DICER was linked to disease progression elevated SOX4 expression.89,90 In endometrial cancer, loss of and decreased patient survival.112 Moreover, in vitro knockdown of miRNA-129-2 was demonstrated to promote cell proliferation and DICER increased melanoma invasion in matrigel and its correlated with poor overall survival in patients,89 whereas in gastric overexpression was sufficient to revert the proinvasive phenotype cancer, depletion of SOX4 through ectopic expression of miRNA- of SOX4 knockdown melanoma cells. Interestingly, knockdown of 129-2 or small interfering RNA-mediated knockdown was shown to SOX4 and the resulting decreased DICER expression greatly altered induce apoptosis.90 It thus appears that miRNA-mediated regulation the melanoma miRNA profile, suggesting that changes in miRNA of SOX4 expression in gastric and endometrial cancer has an expression underlie the metastasis-suppressive role of SOX4 in important role during tumorigenesis. melanoma.112 In a similar manner, SOX4 mRNA expression has been shown to Taken together, these studies suggest that, depending on the be directly regulated through miRNA-335 in the breast cancer cell type of cancer and cellular context, SOX4 may function as a tumor line MDA-MB-231.63 The expression of miRNA-335 was lost in suppressor, exerting its effects through the induction of apoptosis MDA-MB-231 derivative breast cancer cell lines, which readily or inhibition of tumor metastasis. metastasize to either lung or bone.63 Ectopic expression of miRNA- 335 or SOX4 knockdown in these highly metastatic MDA-MB-231 CELLULAR CONTEXT MODULATES SOX4 EXPRESSION AND cells altered breast cancer cell morphology and decreased ACTIVITY migration and invasion in vitro and significantly reduced metastatic foci in the lungs in vivo, suggesting that, in part, Context-dependent and tumor-specific effects of SOX4 can miRNA-335 exerts its antimetastatic effects through regulation of originate from underlying differences in the control of its mRNA SOX4 expression.63 In the breast cancer cell line SUM159, SOX4 has expression and differential regulation of its protein stability and been demonstrated to be the target of miRNA-93, which is activity (Figure 2). frequently overexpressed in a variety of human cancers and affects stem cell differentiation and proliferation in both normal Regulation of SOX4 mRNA expression mammary tissue and breast cancer.121 Ectopic expression of SOX4 mRNA expression can vary greatly in a tumor-type-specific miRNA-93 in a xenograft mouse model of breast cancer abrogated manner through amplification of the SOX4 locus and the tumorigenesis and metastasis. Moreover, in SUM159 cells miRNA- concerted activity of signaling pathways and microRNAs. The 93 was observed to promote MET and maintain an epithelial state SOX4 6p22 locus has been observed to be amplified in bladder by decreasing TGF-b signaling and the expression of stem cell- and lung cancer, resulting in increased SOX4 expression in the related genes such as AKT3, EZH1 and SOX4 in vitro121 Thus, latter.87,111,113 Nevertheless, it remains to be determined whether miRNA-335- and miRNA-93-mediated modulation of SOX4 amplification of the SOX4 locus occurs in a larger variety of human expression appears to influence breast cancer progression by cancers and to which extent this contributes to the elevated affecting the generation of cancer stem cells, EMT and tumor expression of SOX4 in observed in human cancers. metastasis. These findings indicate that elevated SOX4 expression Differential expression of SOX4 could also be related to the might, in part, result from hypermethylation-mediated silencing of activity of a variety of signaling pathways, such as the previously specific miRNAs, adding another level of complexity to the mentioned TGF-b and WNT signaling pathways (Figure 2). In regulation of SOX4 mRNA expression. addition to TGF-b and WNT signaling, SOX4 expression is under Taken together, these findings indicate that context-dependent the control of estrogen and progesterone in female reproductive and tumor-specific mechanisms regulate SOX4 expression on a tissues, as well as in normal and malignant breast tissue.114 SOX4 number of levels, suggesting that tight regulation of this transcription expression is rapidly induced upon activation of the progesterone factor is important during development and that aberrant regulation receptor, resulting in enhanced transcriptional activity.114,115 may impact on various aspects of tumorigenesis. Interestingly, SOX4 activity in hormone-sensitive tissues has the potential to induce a positive feedback loop by regulating expression of the gonadotropin-releasing hormone (GnRH), which Regulation of SOX4 protein expression and activity subsequently induces the release of estrogen and proge- In addition to regulation of SOX4 on the mRNA level, modulation sterone through follicle-stimulating hormone and luteinizing of SOX4 transcriptional activity and expression can occur through hormone.116,117 During normal development, estrogen and PTMs and protein–protein interactions. Despite the crucial role of

& 2013 Macmillan Publishers Limited Oncogene (2013) 3397 – 3409 Role of SOX4 in tumorigenesis and metastasis SJ Vervoort et al 3406 post-translational regulation for the proper activation of most occurring mutations in p53, resulting in contrasting effects of transcription factors, the knowledge about the regulation of SOX4 SOX4 activation on cell survival depending on the tumor type. on this level remains limited. The SOX4 protein has a very short half-life, which potentially allows for the tight control of SOX4 protein levels.21 The rapid turnover also indicates the FUTURE PERSPECTIVES requirement to evaluate SOX4 protein levels as post-translational From the time of its discovery, SOX4 has been demonstrated to be regulation of protein expression could result in a large discrepancy critical for numerous aspects of embryogenesis and various facets between mRNA and protein expression. The rapid protein of tumorigenesis and tumor progression. In the development of turnover of SOX4 is reminiscent of important transcriptional human cancers, SOX4 has disparate roles acting as either a tumor regulators such as p53 and MYC of which the protein expression suppressor or an oncogene and affects key processes related to is tightly controlled on the post-translational level. For several tumor biology, including apoptosis, cell proliferation, migration, members of the SOX family of transcription factors, PTMs, includ- metastasis, EMT and cancer stemness. The opposite roles of SOX4 ing phosphorylation, acetylation and sumoylation, have been in these processes could be related to, yet unexplored, regulation described to regulate protein expression, activity and localization. of expression levels and protein activity of this transcription factor However, there is currently no direct evidence showing that the through transcriptional, post-transcriptional and post-translational transcriptional activity of SOX4 is regulated in this manner. events. This is influenced by cofactors, gene mutations, miRNAs Nevertheless, it is likely that similar to other SOX family members, and various signaling pathways, the cellular context and tumor- SOX4 activity is regulated through distinct PTMs.122 Multiple SOX4 type of which thus determines whether SOX4 acts as a friend or a PTMs identified by mass spectrometry have been reported, foe. Nevertheless, although this dual role of SOX4 in tumorigenesis although the function of these modifications remains to be and progression has recently been appreciated, it warrants further determined (www.phosphosite.org) (Figure 1).123 investigation into its function and regulation. Transcriptional activity and target gene specificity of SOX4 is Future insight into the regulation of SOX4 and its downstream also controlled through cooperative interactions with distinct target genes in the context of cancer development and progression transcription factors and cofactors. Syntenin-1 has been identified could prove useful to design pharmacological compounds that as an important regulator of SOX4 protein stability and transcrip- modulate the activity of this important transcription factor. tional activity.21 Syntenin-1 interacts with a large variety of cell However, despite the prospective therapeutic benefits of such surface , including interleukin-5 receptor, syndecans, compounds, it is imperative to carefully assess the cellular context ephrin and frizzled receptors, and its expression contributes to of the tumor and role of SOX4 herein to prevent adverse effects metastasis in breast cancer, gastric cancer and melanoma.124,125 brought on by inhibition of its tumor-suppressive function. Thus, syntenin-1-mediated regulation of SOX4 stability might contribute to tumorigenesis and metastasis. In addition, SOX4 has been shown to associate with the E2 ubiquitin-ligase Ubc9, which CONFLICT OF INTEREST inhibits the transcriptional activation of SOX4 downstream of the The authors declare no conflict of interest. progesterone pathway independent of its SUMO-1-conjugating activity.115 SOX4 also interacts with a number of transcription factors, ACKNOWLEDGEMENTS including TCF and OCT-4, to synergistically activate target genes in Ruben van Boxtel is supported by a ‘Center for Translational Molecular Medicine’ colorectal cancer and glioma, respectively.75,79 In colon cancer, (CTMM) grant. SOX4 has been shown to cooperate with the oncogenic transcription factor ETS-1 to promote transcription of the lymphocyte-specific protein tyrosine kinase, a gene which has REFERENCES been correlated with high recurrence of liver metastases.126,127 1 Bowles J, Schepers G, Koopman P. 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