Stromal Antigen-1 Research Articles

A cohesin-associated gene score may predict immune checkpoint blockade in hepatocellular carcinoma.

Stromal antigen 1 (STAG1), a component of cohesion, is overexpressed in various cancers, but it is unclear whether it has a role in the transcriptional regulation of hepatocellular carcinoma (HCC). To test this hypothesis, here, we screened global HCC datasets and performed multiscale embedded gene co‐expression network analysis to identify the potential functional modules of differentially expressed STAG1 co‐expressed genes. The putative transcriptional targets of STAG1 were identified using chromatin immunoprecipitation followed by high‐throughput DNA sequencing. The cohesin‐associated gene score (CAGS) was quantified using the The Cancer Genome Atlas HCC cohort and single‐sample gene set enrichment analysis. Distinct cohesin‐associated gene patterns were identified by calculating the euclidean distance of each patient. We assessed the potential ability of the CAGS in predicting immune checkpoint blockade (ICB) treatment response using IMvigor210 and GSE78220 cohorts. STAG1 was upregulated in 3313 HCC tissue samples compared with 2692 normal liver tissue samples (standard mean difference = 0.54). A total of three cohesin‐associated gene patterns were identified, where cluster 2 had a high TP53 mutated rate and a poor survival outcome. Low CAGS predicted a significant survival advantage but presaged poor immunotherapy response. Differentially expressed STAG1 co‐expression genes were enriched in the mitotic cell cycle, lymphocyte activation, and blood vessel development. PDS5A and PDGFRA were predicted as the downstream transcriptional targets of STAG1. In summary, STAG1 is significantly upregulated in global HCC tissue samples and may participate in blood vessel development and the mitotic cell cycle. A cohesin‐associated gene scoring system may have potential to predict the ICB response.

PO-503 The cohesin stromal antigen 1 (SA-1) modulates colonic and colorectal cancer (CRC) stem cells: mechanism for racial disparities

IntroductionCRC disproportionately impacts African-Americans (incidence and mortality increased by ~25% and~50%, respectively). While mechanisms remain unclear, Vogelstein posited that the number of stem cell divisions determine CRC risk Science 2015. CRC stem cells may impact mortality via chemoresistance. LGR5, aldehyde dehydrogenase (ALDH1a3) and DCAMKL1 are markers of both intestinal and CRC stem cells. We have noted loss of SA-1 (a chromatin remodeler) occurred during colonic field carcinogenesis was markedly accentuated in Blacks (Cancer Prev Res 2016) via specific SNPs (Neoplasia 2018). SA-1 loss was also associated with poorer CRC prognosis. We hypothesised that SA-1 loss leads to stem cell induction and hence CRC disparities.Material and methodsRectal biopsies were obtained from endoscopically normal mucosa from ~200 patients undergoing screening colonoscopy with an IRB approved protocol. SA-1 was assessed by RT-PCR normalised to β-actin. We modulated SA-1 in human CRC cell line HT29 and tested efficacy of chemotherapy 5 fluorouracil (5-FU) and oxaliplatin via annexin V apoptosis assay.Results and discussionsAdenoma-harbouring subjects had ~50% increase in LGR5, ALD1a3 and DCAMKL1 (p<0.05) with concomitant suppression of SA-1. Causality was indicated by demonstrating that SiRNA SA-1 knockdowns (KD) in HT29 cells caused stem cell marker induction (LGR5=380%, ALD1a3=30% and DCAMKL1=85%, p<0.05). SA-1 overexpression resulted in reciprocal effects downregulation of all 3 stem cell markers. Functionally, SA-1 KD suppressed 5-FU and oxaliplatin induced apoptosis by 56% and 72% respectively versus scramble vector (p<0.0001). Underscoring racial disparities relevance was the observation that Blacks have a 31% greater SA1 loss vs. Whites (p<0.0004) which mirrored a 35% higher upregulation in LGR5 and ALDH1a3 (p<0.05). CRISPR editing of RKO cells to have the SNP rs34149860 (found only in Blacks) resulted in SA1 loss (41% loss, p<0.005) and concomitant ~45% LGR5 and ALDH1a3 upregulation (p<0.05).ConclusionThis novel finding that the proneoplastic effects of SA-1 loss may be transduced through intestinal/colonic stem cell (CRC incidence) and also augmenting CRC stem cells resulting in chemotherapy resistance (CRC mortality). Future studies may mitigate CRC disparities in Blacks through development of effective biomarkers and therapeutic.

Single Nucleotide Polymorphism Facilitated Down-Regulation of the Cohesin Stromal Antigen-1: Implications for Colorectal Cancer Racial Disparities

The biological underpinnings for racial disparities in colorectal cancer (CRC) incidence remain to be elucidated. We have previously reported that the cohesin SA-1 down-regulation is an early event in colon carcinogenesis which is dramatically accentuated in African-Americans. In order to investigate the mechanism, we evaluated single nucleotide polymorphisms (SNPs) for association with SA-1-related outcomes followed by gene editing of candidate SNP. We observed that rs34149860 SNP was significantly associated with a lower colonic mucosal SA-1 expression and evaluation of public databases showed striking racial discordance. Given that the predicted SNP would alter miR-29b binding site, we used CRISPR knock-in in CRC cells and demonstrated that the SNP but not wild-type had profound alterations in SA-1 expression with miR-29b inhibitor. This is the first demonstration of high-order chromatin regulators as a modulator of racial differences, risk alteration with SNPs and finally specific modulation by microRNAs.

Abstract 2562: Stromal antigen 1 (SA1) as a potential pro-neoplastic factor in non-small-cell lung cancer (NSCLC)

Abstract Introduction: SA1, encoded by the STAG1 gene, is a subunit of the higher-order chromatin remodeler cohesin. SA1 deficiency has been implicated as a driver of aneuploidy and tumorigenesis (Remeseiro et al, EMBO J 2012). Furthermore, our lab has recently shown that SA1 functions as a tumor suppressor protein in early colon cancer (Wali et al, Cancer Prev Res 2016). The aim of this project was to investigate the role of SA1 in NSCLC. Combining data from The Cancer Genome Atlas (TCGA), human lung tissue, and in vitro gene knockdown, we have surprisingly identified SA1 as a potential pro-neoplastic factor in NSCLC. Methods: Data was extracted from TCGA and plotted to characterize STAG1 gene alteration in NSCLC. Immunohistochemistry (IHC) was performed on human lung tissue microarray assay (TMA) to assess SA1 protein expression. Transient knockdowns were performed on A549 human lung adenocarcinoma cells using STAG1 siRNA (Dharmacon) with an incubation time of 48 hours. Following this, quantitative real-time polymerase chain reaction (qPCR, Life Technologies) and WST-1 cell proliferation assays (Promega) were performed as per protocol. Results: STAG1 alterations occur in about 3% of lung adenocarcinoma and 17% of lung squamous cell carcinoma. Of these alterations, the majority were amplifications. Furthermore, although not reaching statistical significance, there was a trend towards decreased survival with STAG1 alteration in adenocarcinoma patients (11.6 months vs. 46 months). IHC demonstrated a 1.7-fold increase in SA1 protein expression in NSCLC when compared with non-malignant lung tissue (p&amp;lt;0.001). Transfected A549 cells showed a 78% decrease in SA1 RNA expression (qPCR, p=0.0002) and a 50% decrease in cell proliferation (WST-1, p&amp;lt;0.0001). Proliferating cell nuclear antigen (PCNA), a marker of cell proliferation, was also decreased by 37% in transfected cells (p&amp;lt;0.001). Conclusions: Using TCGA data, we found that STAG1 alteration occurs with some frequency in NSCLC and that many of these alterations are amplifications of the gene. Furthermore, there is a trend towards decreased survival with a STAG1 gene alteration. Our IHC data shows robust over-expression of SA1 in NSCLC when compared with non-malignant lung tissue. Our qPCR data showed a profound decrease in SA1 mRNA expression following transfection; accordingly, our WST-1 proliferation data suggests that knockdown of the STAG1 gene significantly decreases proliferation of A549 lung adenocarcinoma cells. Decreased PCNA confirms a true anti-proliferative effect, rather than a pro-apoptotic effect. This novel data suggests a link between SA1 and NSCLC, of which there are no prior published accounts. Even more compelling is the implication that in NSCLC, SA1 may be pro-neoplastic rather than anti-neoplastic, which introduces a new potential target for future gene therapy. Citation Format: Michelle Zhang, Mart Dela Cruz, Navneet Momi, Sanjib Chowdhury, Hemant Roy, Adam Lerner. Stromal antigen 1 (SA1) as a potential pro-neoplastic factor in non-small-cell lung cancer (NSCLC) [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 2562. doi:10.1158/1538-7445.AM2017-2562

P2.01-094 Stromal Antigen 1 (SA-1), a Cohesin, is a Novel Proto-Oncogene Regulating Chromatin in Non-Small Cell Lung Cancer (NSCLC)

The molecular composition NSCLC is heterogeneous and clinically manifested as differential therapeutic responsiveness. It is increasingly appreciated that changes in high order chromatin (HOC) structure may play an important role in controlling gene expression and may be one of the fundamental events in carcinogenesis. However, while several HOC regulators are altered in lung cancer (e.g. Arid1a) from the cancer genome atlas work (TCGA), these occur in a minority of tumors suggesting involvement of other modulators. Recently, SA-1 (Stag-1), a member of the cohesin family, has been shown to be a HOC regulator in cancer via controlling chromatin looping and hence gene expression. Since no previous reports on cohesin in lung cancer, we therefore, focused on the role of SA-1 in NSCLC. We performed immunohistochemical analysis (IHC) of 190 cancers and compared to benign tissue through standard techniques. We also extracted SA-1 data from the TCGA databases (Nature 2012 & 2014). SA-1 was markedly (∼2-3 fold) overexpressed in all types of NSCLC (p<0.01) versus benign tissue (Figure 1). This increase was striking at stage 1 NSCLCs with minimal further increase noted at higher stages. TCGA data demonstrated amplification/mutation in ∼17% of squamous but only 3% of adenocarcinomas. This suggested that epigenetic regulations was paramount. Kaplan-Meier analysis showed major impact of SA-1 alterations on survival. For instance, in squamous cancers, median disease-free survival with versus without SA-1 amplification was 8 vs 38 months, respectively. We show for the first time that SA-1 is overexpressed early in NSCLC consonant with status as a proto-oncogene. This upregulation occurs predominantly epigenetic with some contributions with genetic factors. Moreover, SA-1 may have important prognostic markers underscoring its importance of HOC regulation in NSCLC. Further studies are ongoing to elucidate the precise role of SA-1 in the pathogenesis and natural history of NSCLC.

Abstract B04: Cohesin complex subunit (stromal antigen1): Potential molecular determinant of racial disparities in prostate cancer

Abstract Purpose: The overarching goal is to determine the molecular cause of racial disparities in Prostate Cancer (PCa) and exploit its clinical implications. Background: African Americans (AA) have two-thirds higher risk of being diagnosed with PCa with mortality rate twice as high compared to Whites. Evidence supports an earlier transformation to clinically significant PCa in AA. While PCa incidence has declined in the last decade, the racial disparity has not narrowed. Even the improved healthcare, socio-economic/lifestyle factors does not seem to benefit this disparity greatly. Thus, it is evocative that certain biological factors may hold crucial links, therefore necessitating emerging biomarkers for valuable risk stratification. We have recently provided a sensitive/specific optical biomarker as an indicator of field carcinogenesis, segregating patients with indolent vs aggressive PCa. A susceptible genetic background or a field effect is very vital for tumor initiation, allowing accumulation of mutations in neoplastic lesions leading to genomic instability. On these lines, recent studies have demonstrated novel role of cohesion complex genes driving aneuploidy and tumorigenesis. Intriguingly, our investigations in human bio-specimens revealed a consistent downregulation of a putative cohesion complex subunit, Stromal Antigen1 (SA1), in PCa progression. We hypothesized that “SA1 aberrations may serve as an indicator of genomic filed carcinogenesis, providing insights into the biological mechanisms for racial disparities in PCa”. For this, we aimed to characterize SA1 expression in PCa initiation/progression in AA vs Whites, primarily focusing on its relevance with the widely recognized signaling axis in PCa, (pAKT/PI3K), and androgen responsiveness, since both are disproportionately prevalent among AA. Methods: To characterize SA1 levels in PCa field carcinogenesis for AA vs Whites, immunohistochemistry (IHC) for SA1 and PI3K was performed on histologically normal biopsies collected from racially-distinct Gleason6-7 PCa patients undergoing prostactomy. Further, we investigated by IHC the SA1 expression/sub-cellular localization in PTEN (tumor suppressor gene that negatively regulates AKT/PI3K) PCa transgenic mice model. The findings were corroborated in cell-line models using PCa cells (PC3 and DU145) and prostate epithelial cells (RWPE1) by inhibitor studies (for PI3K(LY-294002), nuclear exportin CRM1(LeptomycinB)) via immunofluorescence/immunoblotting. Results: Interestingly, prostate biopsies from AA demonstrated amplified PI3K levels and attenuated SA1 expression with significantly higher cytoplasmic/nuclear ratio compared to Whites, indicating a localization-allied role of SA1. Additionally, SA1 demonstrated altered expression/localization wrt PTEN milieu: nuclear in benign prostatic tissues of PTEN+/+ mice, whereas relatively less and predominantly cytoplasmic in tumor tissues of PTEN-/- mice. Further, PC3 and DU145 PCa cells demonstrated lesser overall SA1 expression with cytoplasmic co-localization with CRM1, whereas in RWPE1 cells SA1 was localized to the nucleus/nuclear membrane. The mechanistic substantiation by antagonist studies revealed significant blocking (~2fold) of CRM1-mediated nuclear-cytoplasmic SA1 shuttling in PC3 cells with LY+LeptomycinB treatment. Moreover, by transiently downregulating SA1, substantial induction of androgen receptor (AR) was noted in AR-negative PC3 cells. Conclusions: Overall, our studies provide a greater role of pAKT/PI3K/PTEN and CRM1 in SA1 mislocalization, facilitating PCa field carcinogenesis, with potential implications in PCa aggressiveness and therapeutic resistance, the two major aspects of poor clinical outcome in AA patients. Our findings may lead to the discovery of novel biomarkers to identify high-risk AA men potentially providing druggable targets. Citation Format: Navneet Momi, Charles B. Brendler, Hemant K. Roy. Cohesin complex subunit (stromal antigen1): Potential molecular determinant of racial disparities in prostate cancer. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B04.

Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres

Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis.