Role Of SOX2 Research Articles

Abstract 3348: The role of SOX2 in promoting resistance to AR-targeted therapies in prostate cancer

Abstract Prostate cancer is the most common type of cancer in men, with approximately 181,000 new cases diagnosed in 2016. Due to the central role of the androgen receptor (AR) in prostate development and more importantly prostate cancer cell survival and proliferation, strategies targeting AR have been the mainstay therapy for over 70 years. However, despite potent inhibition of AR pathway activation, many patients develop castration-resistant prostate cancer (CRPC). Second-line therapies, such as enzalutamide, has increased overall survival in CRPC, but resistance to these therapies inevitably emerges, suggesting that other pathways apart from AR signalling are contributing to the failure of treatments. We have previously demonstrated that SOX2 [SRY (sex determining region Y)-box 2] is an AR-repressed gene that is expressed in a large percentage of high Gleason grade prostate tumours, as well as in most metastases. Additionally, expression of SOX2 within a castration-sensitive cell line is sufficient to enable castration-resistant tumor formation in vivo, and enzalutamide resistance in vitro. In prostate cancer, SOX2 is not found with its normal stem cell co-factors, NANOG and OCT4, leading to the hypothesis that SOX2 is interacting with a novel co-factor in prostate cancer to regulate expression of genes promoting castration-resistance. A chromatin-immunoprecipitation experiment in a castration-resistant prostate cancer cell line was performed to determine if SOX2 binds the same genes and activates similar pathways in prostate cancer as in embryonic stem cells. Approximately half of the SOX2 bound genes in the CRPC cell line were shared with the embryonic stem cell line, and these genes are involved in signalling pathways and maintenance of stem cell pluripotency; the genes bound uniquely in the CRPC cell line are present in pathways involved with metabolic processes. To identify potential binding partners of SOX2, computational analysis of the sites bound by SOX2 in the promoter regions of target genes determined the FOXA1 motif is within close proximity to the SOX2 motif, and the physical interaction of these proteins was confirmed through co-immunoprecipitation. Further understanding of SOX2 target genes and the pathways that SOX2 activates in the presence of enzalutamide will be crucial to identify mechanisms of resistance and enable the development of novel therapies for castration-resistant prostate cancer. Citation Format: Larischa de Wet, Anthony Williams, Marc Gillard, Steve Kregel, Tzintzuni Garcia, Erin McAuley, Ryan Brown, Donald Vander Griend. The role of SOX2 in promoting resistance to AR-targeted therapies in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3348.

Abstract 404: Amplification of SOX4 promotes PI3K/Akt signaling in human breast cancer

Abstract Basal-like or triple-negative breast cancer, if considered its own disease, would rank as the 5th leading cause of cancer deaths for women in the USA each year with ~10,000 deaths annually. Given the lack of drug-able targets expressed by TNBC tumors, few therapeutic options exist beyond currently utilized cytotoxic therapies, and the overall prognosis for these patients remains poor. The oncogenic PI3K/Akt signaling axis contributes to the dysregulation of many dominant features in breast cancer including cell proliferation, survival, metabolism, motility, and genomic instability. While multiple studies have demonstrated that basal-like or triple-negative breast tumors have uniformly high PI3K/Akt activity, genomic alterations that mediate dysregulation of this pathway in this subset of highly aggressive breast tumors remain to be determined. In this study, we present an integrated genomic analysis based on the use of a PI3K gene expression signature as a framework to analyze orthogonal genomic data in human breast tumors from TCGA (The Cancer Genome Atlas) including RNA expression, DNA copy number alterations, and protein expression. These analyses identified copy number alterations associated with PI3K activity in human breast tumors (n=1,031), including known and potentially novel drivers of PI3K signaling. Data from a genome-wide RNA interference (RNAi) screen were used to discriminate between essential and non-essential genes within identified amplicons. Integrating DNA copy number and RNAi analyses identified amplified SOX4 as a novel and essential regulator of PI3K/Akt signaling. Importantly, by analyzing protein expression data (n=733), we determined that SOX4 amplified tumors have increased activation of pAkt substrates. Moreover, depleting SOX4 levels in HCC38 and HCC1143, basal-like breast cancer cell lines with high PI3K activity and high SOX4 expression, demonstrated significant abrogation of p-Akt levels. This validated our in silico findings and confirmed the role of SOX4 in regulating PI3K pathway activity. To identify the potential mechanism(s) by which SOX4 mediates PI3K pathway activity, we performed genome-wide analyses of these cell lines following SOX4 inhibition. Transcriptomic profiling by RNA-sequencing, protein profiling by Reverse Phase Protein Array (RPPA) and Kinome profiling through multiplexed kinase inhibitor beads and mass spectrometry (MIB-MS) based approach identified TGFBR2, as a potential regulator of SOX4-mediated PI3K/Akt signaling. Further studies will elucidate the mechanism by which SOX4 modulates TGFBR2 expression and drives the oncogenic PI3K/Akt signaling pathway in basal-like breast cancer. Citation Format: Gaurav Mehta, Michael Gatza, Steve Angus, Gary Johnson. Amplification of SOX4 promotes PI3K/Akt signaling in human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 404.

Abstract 4221: Association between SOX9 expression in Mexican patients with early colon cancer stage

Abstract Introduction. Colon cancer is the fourth leading cause of mortality worldwide. Between 30-40% of patients are diagnosed at early stage (I or II), sadly 20% will relapse. It is necessary to have a biomarker that identifies patients at high risk. SOX 9 is a transcription factor required for differentiation of different cells and is important for cell proliferation, senescence and lineage commitment. The exact role of SOX9 in carcinogenesis and cancer progression is controversial because could have oncogenic and tumor-suppressing functions. Reports show that strong levels of SOX 9 in colon cancer stage II are linked to low risk of relapse. Objective. The aim of this study was to investigate if the protein expression of SOX9 was associated with the stage, clinical characteristics and relapse of Mexican colon cancer patients. Methods. We obtained 97 samples embedded in paraffin. A pathologist blinded to the clinical information determined the immunoreactivity of SOX9 into 2 groups: low and high expression (by evaluating the percentage of positive nucleus and the tissue staining intensity). All statistical analyses were conducted with SPSS v.22 Statistics. Results. We include stage I (N= 34) and stage II (N=63) patients. The median follow-up period was 42 months. We found statistical difference between SOX9 expression and age (p=0.003), tumor localization (p=0.048) and stage (p&lt0.001) for whole patients group. Twelve patients (12.3%) relapsed, 7 (at locally level) and 5 with distant metastasis: liver (N=3) and retroperitoneal nodes (N=2), all had low SOX9 expression; one patient with local relapse had died (because of surgical complications). In the relapse group, we found a significant correlation between SOX9 expression and stage (p=0.038) but not with relapse free survival (RFS). Conclusion. We found interesting data even though this is a retrospective study with limitations. This study could not associated SOX9 and RFS, because there are few events. Nevertheless, we observe that 41% patients had a systemic relapse. In the other hand, the youngest patients (&lt40 y) and the right colon had the highest SOX9 expression. With this result, we have decided to increase the sample size before any assumption, and perform NGS to the five systemic relapses. Citation Format: Erika Ruiz-Garcia, Tatiana Galicia, Edith Fernandez Figueroa, Saul Lino-Silva, Cesar Lopez-Camarillo, Laurence Marchat, German Calderillo, Juan Zinser, Abelardo Meneses-Garcia, Horacio Astudillo-de la Vega. Association between SOX9 expression in Mexican patients with early colon cancer stage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4221.

Increase of SOX9 promotes hepatic ischemia/reperfusion (IR) injury by activating TGF-β1

Ischemia/reperfusion (IR) injury causes damage in aerobically metabolizing organs or tissues, which is an essential injury mechanism in various clinical settings. SRY-related high mobility group-Box gene 9 (SOX9) is a transcription factor of the SRY family, modulating various cellular processes, including fibrosis formation and tumor growth. However, the effects of SOX9 on hepatic IR injury have not been explored. In the present study, a hepatic IR injury model was established, supported by a significant histological alteration with high Suzuki scores, and a remarkable up-regulation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Importantly, we found that SOX9 was over-expressed in liver of mice after IR operation. Suppressing SOX9 markedly reduced inflammatory response, as evidenced by the reduced mRNA expressions of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1β and inactivation of inhibitor of κBα (IκBα)/nuclear factor (NF)-κB pathway. In addition, SOX9 suppression alleviated apoptosis in liver of mice after IR injury, as supported by the reduced number of terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL)-staining cells and decreased expression of Caspase-3 in liver tissue sections. The role of SOX9 in accelerating hepatic IR injury was further confirmed in primary hepatocytes under hypoxiaand reoxygenation (HR) treatment by enhancing inflammatory response and apoptosis. Of note, we found that transforming growth factor (TGF)-β1 was highly induced in liver of mice after IR injury. HR treatment also stimulated TGF-β1 expressions in vitro. Significantly, SOX9 over-expression-induced inflammation and apoptosis were obviously reduced by pirfenidone (Pirf), TGF-β1 inhibitor. In contrast, TGF-β1 exposure to cells further enhanced inflammation and apoptosis in HR-operated cells either with SOX9 knockdown or over-expression. Therefore, we identified a novel SOX9-dependent pathway that contributed to hepatic IR injury through enhancing inflammation and apoptosis by activating TGF-β1.

A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte

Sox9 is a member of the gene family of SOX transcription factors, which is highly conserved among vertebrates. It is involved in different developmental processes including gonadogenesis. In all amniote species examined thus far, Sox9 is expressed in the Sertoli cells of the male gonad, suggesting an evolutionarily conserved role in testis development. However, in the anamniotes, fishes and amphibians, it is also expressed in the oocyte but the significance of such an expression remains to be elucidated. Here, we have investigated the nuclear localization of the SOX9 protein in the oocyte of three amphibian species, the urodelan Pleurodeles waltl, and two anurans, Xenopus laevis and Xenopus tropicalis. We demonstrate that SOX9 is associated with ribonucleoprotein (RNP) transcripts of lampbrush chromosomes in an RNA-dependent manner. This association can be visualized by Super-resolution Structured Illumination Microscopy (SIM). Our results suggest that SOX9, known to bind DNA, also carries an additional function in the posttranscriptional processes. We also discuss the significance of the acquisition or loss of Sox9 expression in the oocyte during evolution at the transition between anamniotes and amniotes.

Stem cell transcription factor SOX2 in synovial sarcoma and other soft tissue tumors

BackgroundSOX2 has gained considerable interest as a pluripotency inducing gene. Co-transfection of SOX2 together with NANOG, KLF4 and c-MYC into adult fibroblasts was able to generate pluripotent stem cells. SOX2 has been reported to be expressed in synovial sarcoma, a tumor being characterized by the SS18-SSX gene fusion forming part of the SWI/SNF chromatin remodeling complex that affects histone methylation. The role of SOX2 in this tumor type as well as other soft tissue tumor entities however is still poorly characterized. We analyzed SOX2 protein expression in soft tissue tumors. Alongside we tested Histone H3 expression (H3K27me3) in SOX2 positive cases to investigate this epigenetic mark and its correlation with the SOX2 status and clinicopathological parameters. MethodologyIn total, 60 samples of synovial sarcomas from the reference center for soft tissue tumors at the institute of pathology of the Jena University hospital were included into the study along with 343 other tissue tumors. Protein analysis was done by immunohistochemistry of tissue microarrays. All synovial sarcoma cases were confirmed by molecular testing using SS18 FISH break apart probes. ResultsSOX2 reactivity was detectable in 35 synovial sarcoma cases (58.3%) while 25 (41.7%) were negative. Only 13 cases of the other 343 soft tissue tumors, varying from nodular fasciitis to undifferentiated pleomorphic sarcoma, revealed a SOX2 expression, 12 out of these were undifferentiated high grade sarcoma. There was no obvious correlation with the clinicopathological data. H3K27me3 immunohistochemistry of the synovial sarcoma cases revealed a high statistically significant correlation between SOX2 and H3K27me3 expression (p < 0,0005, Chi square test). Similar to SOX2, there was no correlation between H3K27me3 expression and tumor grade. Six SOX2 positive synovial sarcoma cases were analyzed by FISH using a SOX2/CEN3 dual color FISH probe. None of these cases revealed an amplification of the SOX2 gene. ConclusionThe data confirms previous studies reporting SOX2 and H3K27me3 expression in synovial sarcoma and reveals that both biomarkers are related to each other. It strengthens the notion that the tumor type is driven by epigenetic processes similar to those that are operating in pluripotent stem cells. The relevance of these parameters in the pathway pathology of synovial sarcoma, i.e. the timing and dosing of SOX2 and H3K27me3 expression initiated by the SS18-SSX driver mutation together with the interplay of these events with other signaling pathways, cellular mechanisms and additional mutations in tumor progression, will require further studies.

The Stem Cell Factor Sox2 Is a Positive Timer of Oligodendrocyte Development in the Postnatal Murine Spinal Cord

Myelination in the central nervous system takes place predominantly during the postnatal development of humans and rodents by myelinating oligodendrocytes (OLs), which are differentiated from oligodendrocyte progenitor cells (OPCs). We recently reported that Sox2 is essential for developmental myelination in the murine brain and spinal cord. It is still controversial regarding the role of Sox2 in oligodendroglial lineage progression in the postnatal murine spinal cord. Analyses of a series of cell- and stage-specific Sox2 mutants reveal that Sox2 plays a biphasic role in regulating oligodendroglial lineage progression in the postnatal murine spinal cord. Sox2 controls the number of OPCs for subsequent differentiation through regulating their proliferation. In addition, Sox2 regulates the timing of OL differentiation and modulates the rate of oligodendrogenesis. Our experimental data prove that Sox2 is an intrinsic positive timer of oligodendroglial lineage progression and suggest that interventions affecting oligodendroglial Sox2 expression may be therapeutic for overcoming OPC differentiation arrest in dysmyelinating and demyelinating disorders.

A Novel Role for Sox6 in Renin Expression Control During Renal Artery Stenosis Induced Hypertension

Study ObjectiveWe wanted to determine if the transcription factor Sox6 played a role in renin expression control and in the hypertension induced by renal artery stenosis. In renal artery stenosis, renin is a key disease driver. Renin catalyzes the rate limiting step in the Renin Angiotensin Aldosterone System (RAAS); converting angiotensinogen to angiotensin I. We used a novel transgenic mouse (Ren1dCre/Sox6fl/fl) to determine the impact of specific Sox6 ablation in renin expression during JG cell expansion and renal artery stenosis renal induced hypertension.MethodsIn vivo: JG cell expansion was induced by 10 days of low sodium diet (0.01% Na) and furosemide (0.1 mg/g body weight). Renal artery stenosis was induced by the 2 Kidney 1 clip (2K1C) Goldblatt model; renin is overexpressed in the clipped kidney with renin expression being repressed in contralateral kidney. In vitro: primary renal Mesenchymal Stromal Cells (MSC) were isolated from wild‐type mice. MSC were differentiated into renin expressing cells by 7 days I&amp;F (3‐isobutyl‐methyl‐xanthine (IBMX)‐100 uM and Forskolin‐10 uM) treatment.ResultsI&amp;F induced renin expression in MSCs (Renin relative expression to gapdh 0.0153±0.005, n=3, P= 0.05, no renin expression in control MSCs). In the Ren1dCre/Sox6fl/fl there was no difference in blood pressure or heart rate at base line. However, under conditions that promote JG cell expansion, Sox6 ablation halted the increase in the number of JG cells (8.75 fold decrease, n= 6 to 9, P= 0.001). In our preliminary study we found that specific knock‐out of Sox6 in renin expressing cells protected the kidney against renal artery stenosis injury when compared to control mice (two mice per group). After renal artery stenosis, wild‐type kidneys were one‐third the size of Ren1dCre/Sox6fl/fl kidneys. In addition, renin expression was ~50% higher in the wild‐type group.ConclusionOur data indicates that Sox6 plays a novel role in renal physiology; modulating renin expression during pathological conditions. These results will aid in the discovery of a novel transcriptional regulatory network controlled by Sox6, involved in blood pressure regulation. Identification of this novel pathway and its regulators may lead to new therapies for blood pressure control aiming at decreasing cardiovascular disease in hypertensive patients.Support or Funding InformationResearch was supported by American Heart Association Scientist Development Award to JAG (16SDG29880007), the Vanderbilt University Medical Center Faculty Research Scholars Program, and NHLBI Research Scientist Development Grant 1K01HL135461‐01 to JAGThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Role for Sox2 in the Adult Cerebellum.

The cerebellum, a derivative of the hindbrain, plays a crucial role in balance and posture as well as in higher cognitive and locomotive processes. Cerebellar development is initiated during the segmental phase of hindbrain formation. Here, we describe the phenotype, of a single surviving adult conditional mouse mutant mouse, in which Sox2 function is ablated in embryonic radial glial cells by means of hGFAP-CRE. The single Sox2RGINV/mosaic adult mutant mouse displays motor disability, microsomia, reduced Central Nervous System (CNS) size and cerebellar defects associated with human genetically related congenital abnormalities.

Abstract 30: SOX9 enhancer regulator may play an oncogenic role in B-cell lymphomas

Abstract A majority of B-cell lymphomas, including diffuse large B-cell lymphomas (DLBCLs) and follicular lymphomas (FLs), arise from germinal center (GC) B cells. GC B cells are highly proliferative and manifest genomic instability as a byproduct of immunoglobulin affinity maturation. These characteristics make GC B cells particularly prone to malignant transformation such that DLBCLs and FLs inherit and are dependent on mutations in proteins that are also required for normal GC B cells. For example, the BCL6 transcriptional repressor is required to establish the GC phenotype in both normal B-cell development and GC B-cell malignancy through its effect on silencing expression of plasma cell differentiation and checkpoint genes. GC B cells feature a unique transcriptional program featuring upregulation of genes involved in cell proliferation and other growth pathways. Cell context-specific gene expression is largely mediated through gene enhancers. Hence we hypothesized that transcription factors that activate GC specific gene enhancers would play a critical role in formation of these cells, and would likely be required in turn to maintain the survival of GC-derived lymphoma cells. To address this question we first mapped gene enhancers in primary human resting B cells and purified GC B cells by performing ChIP-seq to map the distribution of H3K27Acetyl, H3K4me1, and H3K4me3. This procedure allowed us to identify several thousand GC-specific enhancers. We next examined these enhancers using bioinformatic approaches to identify transcription factor binding sites and identified a list of fifteen putative GC enhancer regulators. Of these potential GC enhancer regulators, only SOX9 was highly induced in GC B cells as compared to resting B cells, pointing to SOX9 as a candidate master enhancer regulator of the GC transcriptome with potential relevance to lymphomas. Indeed, in addition to the well-known role of SOX9 as a stem cell self-renewal and cell-differentiation regulator during development, SOX9 has also been described as playing a role in colon, breast, and prostate carcinogenesis through inhibition of apoptosis, and promoting proliferation, invasion, and metastasis. We confirmed that SOX9 is highly induced in GC cells using QPCR and Western blots. To determine whether SOX9 would indeed bind to these enhancer sites, we next performed SOX9 ChIP-seq in GC B-cell derived lymphoma cell lines. These experiments validated and showed that SOX9 binds to 1,668 upstream distal enhancer regions (-5 to -100 kb upstream from TSS) associated with 963 genes. These target genes were significantly enriched in cell cycle regulation (CCND2, CDC25B, CDK1), transcription regulation (BCOR, NCOR2), epigenetic regulation (BMI1, DNMT3A, MLL2, SUZ12, TET3), and MAPK signaling (MAP2K3, MAP3K7) and also for B-cell activation and BCL6 repressed pathways (p&amp;lt;0.001). We next wished to determine whether SOX9 was also expressed in GC-derived lymphoma clinical samples. Therefore we next examined RNA-seq gene expression profiles derived from cohorts of FL and DLBCL patients. We found that SOX9 is expressed at higher levels in FL patient samples, but lower levels in most DLBCLs compared to naïve B cells. These data raise the possibility that SOX9 might be differentially relevant to FL more than DLBCLs, perhaps contributing to the biologic distinction between these two GC-derived lymphomas. To determine if SOX9 protein was also expressed in these tumors, we examined protein expression by immunohistochemistry using tissue microarrays (TMAs) containing both FLs and DLBCLs. We found that 88 of the 242 FL TMA samples (36.4%) were positive for SOX9 staining. Eleven of the 114 DLBCL TMA samples (10%) were positive for SOX9 staining, among which 80% (9/11) were GC-type DLBCL and 45% (5/11) had a prior FL history. To determine if any of the currently available GC derived lymphoma cell lines manifest high SOX9 levels, we performed a series of Western blot experiments. These studies show high levels of SOX9 protein in the GC-type DLBCL cell lines HT, Karpas422, DB, and Farage, but not in any of the eight ABC-DLBCLs subtypes. Ongoing studies are assessing the biologic relevance of SOX9 for the GC reaction and possible contribution to lymphomagenesis using gain- and loss-of-function studies in human cells and mouse models. These data will determine whether this apparently key enhancer regulatory protein is a novel lineage factor for follicular lymphoma and potentially suitable as a therapeutic target. Citation Format: Angela A. Fachel, Yanwen Jiang, Wayne Tam, Ashlesha S. Muley, Cem Meydan, Xabier Agirre, Ari M. Melnick, Kristy L. Richards. SOX9 enhancer regulator may play an oncogenic role in B-cell lymphomas [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 30.

Sox2 regulates astrocytic and vascular development in the retina.

Sox2 is a transcriptional regulator that is highly expressed in retinal astrocytes, yet its function in these cells has not previously been examined. To understand its role, we conditionally deleted Sox2 from the population of astrocytes and examined the consequences on retinal development. We found that Sox2 deletion does not alter the migration of astrocytes, but it impairs their maturation, evidenced by the delayed upregulation of glial fibrillary acidic protein (GFAP) across the retina. The centro-peripheral gradient of angiogenesis is also delayed in Sox2-CKO retinas. In the mature retina, we observed lasting abnormalities in the astrocytic population evidenced by the sporadic loss of GFAP immunoreactivity in the peripheral retina as well as by the aberrant extension of processes into the inner retina. Blood vessels in the adult retina are also under-developed and show a decrease in the frequency of branch points and in total vessel length. The developmental relationship between maturing astrocytes and angiogenesis suggests a causal relationship between the astrocytic loss of Sox2 and the vascular architecture in maturity. We suggest that the delay in astrocytic maturation and vascular invasion may render the retina hypoxic, thereby causing the abnormalities we observe in adulthood. These studies uncover a novel role for Sox2 in the development of retinal astrocytes and indicate that its removal can lead to lasting changes to retinal homeostasis.

SOX4 gene and lung development

SOX4 is a member of the group C subfamily of the SOX transcription factors and has a critical role during embryogenesis and in controlling cell fate.SOX4 can participate in TGF-β, Wnt signaling ways to mediate cell differentiation, proliferation, migration.SOX4 can facilitate epithelial-mesenchymal transition by directly regulating EZH2 expression.It is one of the 64 genes that constitute a general signature in all human cancers.SOX4 is widely expressed in lung and establishes the lung structure in lung development.Here, we provide an overview of role of SOX4 in cell and lung development. Key words: SOX4; TGF-β; Wnt; EZH2; lung development

Mass Spectrometric Analysis of SOX11-Binding Proteins in Head and Neck Cancer Cells Demonstrates the Interaction of SOX11 and HSP90α.

Deregulated expression of SOX11 has been shown to be involved in the progression of various types of cancer. However, the role of SOX11 in head and neck cancer remains largely unknown. In this study, coimmunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were performed to identify the proteins that bind to SOX11 at significantly higher levels in head and neck cancer cells than in normal human oral keratinocytes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that many potential SOX11-binding partners were associated with protein synthesis, cell metabolism, and cell-cell adhesion. One of the identified proteins, heat shock protein 90 alpha (HSP90α), was selected for further investigation. The binding of HSP90α with SOX11 in head and neck cancer cells was validated by Co-IP with western blotting. In addition, HSP90α was found to be remarkably overexpressed in head and neck cancer cell lines when compared to its level in normal human oral keratinocytes, and knockdown of HSP90α inhibited the proliferation and invasion capacity of these cancer cells. On the basis of The Cancer Genome Atlas (TCGA) data analysis, HSP90AA1 gene was overexpressed in head and neck cancer tissues compared to normal controls and increased HSP90AA1 gene expression was positively associated with extracapsular spread and clinical stage. Head and neck cancer patients with higher HSP90AA1 expression had significantly poorer long-term overall and disease-free survival rates than those with lower HSP90AA1 expression. Collectively, our studies indicate that SOX11 binds to HSP90α, a highly overexpressed protein that may promote invasion and progression of head and neck cancer cells.

Abstract B15: Comprehensive cistromic characterization of SOX10, a lineage-specific genetic dependency in melanoma, via the integration of functional genomic approaches

Abstract Melanoma accounts for less than 5% of skin cancers, but a majority of skin cancer deaths. Genomic studies and drug discovery programs have led to the development of targeted therapies, including BRAF and MEK inhibitors, but the inevitable resistance to these targeted agents necessitates a greater understanding of melanoma biology and genetics. By integrating genome-wide functional genomic and transcriptomic data, we have identified SOX10 as a lineage-specific genetic dependency in melanoma independent of BRAF or NRAS mutation status. Subsequent analyses indicate that SOX10 dependency is highly correlated with SOX10 gene expression and functional in vitro experiments have confirmed the role of SOX10 in the proliferation and growth of melanoma cell lines regardless of their sensitivity to MAPK pathway inhibitors. Chromatin immunoprecipitation of endogenous SOX10 followed by sequencing (SOX10 ChIP-seq) combined with transcriptomic profiling (RNA-seq) following SOX10 depletion by shRNA in a panel of melanoma cell lines determined the SOX10 cistrome, the set of SOX10 target genes across the melanoma genome. Integration of the SOX10 cistrome with genetic dependency data may lead to novel therapeutic approaches in melanoma. Citation Format: Terence Wong, Cory Johannessen, Jingyu Fan, Henry Long, Shirley Liu, Levi Garraway. Comprehensive cistromic characterization of SOX10, a lineage-specific genetic dependency in melanoma, via the integration of functional genomic approaches [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B15.

SOX9 Regulates Cancer Stem-Like Properties and Metastatic Potential of Single-Walled Carbon Nanotube-Exposed Cells

Engineered nanomaterials hold great promise for the future development of innovative products but their adverse health effects are a major concern. Recent studies have indicated that certain nanomaterials, including carbon nanotubes (CNTs), may be carcinogenic. However, the underlying mechanisms behind their potential malignant properties remain unclear. In this study, we linked SOX9, a stem cell associated transcription factor, to the neoplastic-like properties of human lung epithelial cells chronically exposed to a low-dose of single-walled carbon nanotubes (SWCNTs). We found that SOX9 is upregulated in SWCNT-exposed cells, which is consistent with their abilities to induce tumor formation and metastasis in vivo. We therefore hypothesized that SOX9 overexpression may be responsible for the neoplastic-like phenotype observed in our model. Indeed, SOX9 knockdown inhibited anchorage-independent cell growth in vitro and lung colonization in vivo in a mouse xenograft model. SOX9 depletion also suppressed the formation of cancer stem-like cells (CSCs), as determined by tumor sphere formation and aldehyde dehydrogenase (ALDH) activity (Aldefluor) assays. Furthermore, SOX9 knockdown suppressed tumor metastasis and the expression of the stem cell marker ALDH1A1. Taken together, our findings provide a mechanistic insight into SWCNT-induced carcinogenesis and the role of SOX9 in CSC regulation and metastasis.

Abstract 1913: SOX9 regulates cancer stem-like cells in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer death worldwide in both men and women, every year killing more people than breast, prostate and colon cancers combined. Drug/radiation resistance and tumor relapse contribute to low patient survival, which has largely been attributed to the acquisition of stem-like cells (CSCs), or tumor initiating cells. Moreover, conventional therapies are not effective against CSCs. This signifies the need to identify mechanisms of CSC regulation which could lead to the discovery of CSC-specific drug targets and the development of more effective drug therapies. An embryonic transcription factor SOX9 has been implicated in CSC regulation in a number of cancer types, but little is known about its role in non-small cell lung cancer (NSCLC). We demonstrated that high SOX9 expression correlates with poor survival in lung cancer patients by analyzing The Cancer Genome Atlas (TCGA) data. We hypothesized that SOX9 plays a key role in tumor progression and chemoresistance by upregulating CSCs in NSCLC. To test this hypothesis, SOX9 expression was stably or transiently knocked down in NSCLC cells, and their effects on CSC formation and biomarkers, and on drug resistance were investigated. Our results showed that SOX9 knockdown decreased the number of tumor spheres in NSCLC cell lines. Furthermore, SOX9 knockdown downregulated the expression and activity of stem cell marker ALDH1A1 as determined by Western blot and flow cytometric assays. Consistent with this finding, overexpression of SOX9 in non-cancer lung epithelial Beas-2B cells elevated tumor sphere formation, supporting the role of SOX9 in CSC regulation. Analysis of the soft agar colony formation assay further showed that SOX9 knockdown inhibited anchorage-independent growth in NSCLC cells. More importantly, SOX9 knockdown resulted in an increased sensitivity of the cells to a chemotherapeutic drug cisplatin, whereas SOX9 overexpression decreased it. Together, our results indicate an important role of SOX9 in the regulation of CSCs in NSCLC and suggest its potential utility as a drug target for chemoresistant lung cancer. Citation Format: Maria Voronkova, Sudjit Luanpitpong, Heimo Riedel, Yon Rojanasakul. SOX9 regulates cancer stem-like cells in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1913. doi:10.1158/1538-7445.AM2017-1913

The role of Sox6 and Netrin-1 in ovarian cancer cell growth, invasiveness, and angiogenesis.

SOX6 plays important roles in cell proliferation, differentiation, and cell fate determination. It has been confirmed that SOX6 is a tumor suppressor and downregulated in various cancers, including esophageal squamous cell carcinoma, hepatocellular carcinoma, and chronic myeloid leukemia. Netrin-1 is highly expressed in various human cancers and acts as an anti-apoptotic and proangiogenic factor to drive tumorigenesis. The role of SOX6 and netrin-1 in regulating the growth of ovarian tumor cells still remains unclear. Real-time polymerase chain reaction and western blot were used to determine the SOX6 messenger RNA and protein levels, respectively, in ovarian cancer cells and tumor tissues. Stable transfection of SOX6 was conducted to overexpress SOX6 in PA-1 and SW626 cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Invasion of ovarian cancer cells and migration of human umbilical vein endothelial cells were confirmed by Transwell assays. To overexpress netrin-1, ovarian cancer cells with SOX6 restoration was transduced with netrin-1 lentiviral particles. PA-1 xenografts in a nude mice model were used to conduct in vivo evaluation of the role of SOX6 and its relationship with netrin-1 in tumor growth and angiogenesis. In this study, we found significantly reduced SOX6 levels in PA-1, SW626, SK-OV-3, and CaoV-3 ovarian cancer cell lines and human tumor tissues in comparison with normal human ovarian epithelial cells or matched non-tumor tissues. SOX6 overexpression by stable transfection dramatically inhibited proliferation and invasion of PA-1 and SW626 cells. Also, conditioned medium from PA-1 and SW626 cells with SOX6 restoration exhibited reduced ability to induce human umbilical vein endothelial cells migration and tube formation compared with conditioned medium from the cells with transfection control. Furthermore, an inverse relationship between SOX6 and netrin-1 expression was observed in PA-1 and SW626 cells. Overexpression of netrin-1 in ovarian cancer cells with forced SOX6 expression remarkably abrogated the inhibitory effect of SOX6 on proliferation, invasion of the cells, and tumor xenograft growth and vascularity in vivo. Human umbilical vein endothelial cell migration and tube formation were enhanced in the conditioned medium from the ovarian cancer cells transduced with netrin-1 lentivirus particles. Our observations revealed that SOX6 is a tumor suppressor in ovarian cancer cells, and SOX6 exerts an inhibitory effect on the proliferation, invasion, and tumor cell-induced angiogenesis of ovarian cancer cells, whereas nerin-1 plays an opposite role and its expression is inversely correlated with SOX6. Moreover, our findings suggest a new role of SOX6 and netrin-1 for understanding the progression of ovarian cancer and have the potential for the development of new diagnosis and treatment strategies for ovarian cancer.

SOX17 regulates cholangiocyte differentiation and acts as a tumor suppressor in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/β-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker.

Abstract A35: SOX9 induces chemo-resistance in pancreatic cancer cells and its high expression predicts poor prognosis

Abstract Introduction: Although surgical resection still remains the most important treatment for pancreatic cancer, there is a growing body of evidence showing the importance of chemotherapy. On the other hand, despite the advances in chemotherapy, the outcome of patients with pancreatic cancer remains far from satisfactory. These facts suggest that the understanding of the molecular mechanisms underlying the chemo-resistance of pancreatic cancer is urgently needed for developing more effective chemotherapies. To identify the factors that regulate chemo-resistance in pancreatic cancer, we focused on a transcription factor, Sex determining region Y box 9 (Sox9), that maintains progenitor cells during pancreatic organogenesis and carcinogenesis. The purpose of this study is to evaluate the roles of Sox9 in pancreatic cancer. Methods: Pancreatic cancer tissue were obtained from 106 consecutive patients who underwent curative surgical resection in the Department of General Surgery, Chiba University Hospital, Japan, from June 2006 to October 2011. We analyzed these primary invasive pancreatic ductal adenocarcinoma (PDAC) tissues by immunohistochemistry of Sox9. Human pancreatic cancer cell lines, PANC-1, Sox9 high expressing cell, and BxPC-3, Sox9 low expressing cell were used for in vitro analysis. Sox9 was inhibited by siRNAs. Cell cytotoxicity assay was done with Gemcitabine and 5-FU by PANC-1 and BxPC-3 cells. To elucidate the mechanism of chemo-resistance, we focused on the stemness of Sox9 expressing cells and performed sphere formation assays and flow cytometry analysis. Gene expression was determined by quantitative RT-PCR and Western blotting. The expression levels of CD44 and CD24, which are thought to be stem cell markers of pancreatic cancer, were examined by flow cytometry analysis. Tet-On Inducible Sox9 shRNA transfected PANC-1 cells were established and injected subcutaneously into the bilateral flanks of mouse. Xenograft tumors were assessed four weeks after injection. Results: Expression of Sox9 predicts poor survival in PDAC. Sox9 high expressing patients showed poorer prognosis than Sox9 low expressing patients. Median overall survival time and 3-year survival rate was 25.3 months and 21.1% for the Sox9 high group, and not reached and 62.9% for the low group, respectively (p=0.011). The multivariate overall survival time analysis showed Sox9 expression level as an independent prognostic factor (HR 0.344; 95%CI 0.183-0.647; p=0.0009), along with histological grade, distant metastasis, arterial invasion, residual tumor, and adjuvant chemotherapy. Sox9 high expressing PANC-1 cells showed chemo-resistance against GEM and 5-FU treatments. PANC-1 cells with suppressed Sox9 expression by siRNA showed decreased cell numbers when they were treated with GEM. These data implicated that Sox9 induced chemo-resistance in pancreatic cancer cells. PANC-1 cells, which have strong Sox9 expression, successfully formed spheres during culture on ultra-low attachment plates. The knockdown of Sox9 expression in PANC-1 cells significantly decreased sphere formation rates, compared with cells treated with negative siRNA. In PANC-1 cells, knockdown of Sox9 by siRNA significantly decreased the proportion of the CD44highCD24high cells compared with cells treated with negative control siRNA (p=0.0005). Sox9 knockdown can reduce tumor initiation capacity in vivo analysis. Conclusions: These data indicate that Sox9 plays an important role in chemo-resistance by the induction of stemness in pancreatic cancer cells. Citation Format: Shingo Kagawa, Taku Higasihara, Hideyuki Yoshitomi, Shigetsugu Takano, Hiroaki Shimizu, Masayuki Ohtsuka, Atsushi Kato, Katsunori Furukawa, Masaru Miyazaki.{Authors}. SOX9 induces chemo-resistance in pancreatic cancer cells and its high expression predicts poor prognosis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A35.

Clinicopathological evaluation of Sox10 expression in diffuse-type gastric adenocarcinoma.

Sox10, one of the transcription factors, regulates Wnt/β-catenin signaling in diverse developmental processes in normal tissues. Sox10 is also expressed in variable solid tumors such as breast cancer, salivary tumor, hepatocellular carcinoma, ovarian tumor, nasopharyngeal carcinoma, prostate cancer, and digestive cancer. The role of Sox10 during tumorigenesis is still controversial, especially in digestive cancers; thus, we performed clinicopathological evaluation of Sox10 expression in 41 cases of diffuse-type gastric adenocarcinoma (DGA). We examined the expression of Sox10 by immunohistochemical staining and real-time quantitative reverse transcriptase PCR and evaluated the correlation between Sox10 expression and clinicopathological factors. A low-level expression of Sox10 was significantly associated with high-level venous invasion by immunohistochemical evaluation, while it was significantly associated with high-level lymphatic permeation when analyzed by real-time PCR assay. Survival analysis of 41 cases indicated that high level of vascular permeation was a statistically poor prognostic factor, suggesting that derogation of Sox10 would lead to unfavorable patients' outcome through the acceleration of vascular invasion. In this study, we revealed the clinical benefit of evaluation of Sox10 expression to predict the risk of vascular permeation which yields patients' poor prognosis in DGA.