Xue Gong

Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene (MSANTD3) in salivary gland acinic cell carcinoma

Pathogenic gene fusions have been identified in several histologic types of salivary gland neoplasia, but not previously in acinic cell carcinoma (AcCC). To discover novel gene fusions, we performed whole-transcriptome sequencing surveys of three AcCC archival cases. In one specimen we identified a novel HTN3-MSANTD3 gene fusion, and in another a novel PRB3-ZNF217 gene fusion. The structure of both fusions was consistent with the promoter of the 5’ partner (HTN3 or PRB3), both highly expressed salivary gland genes, driving overexpression of full-length MSANTD3 or ZNF217. By fluorescence in situ hybridization of an expanded AcCC case series, we observed MSANTD3 rearrangements altogether in 3 of 20 evaluable cases (15%), but found no additional ZNF217 rearrangements. MSANTD3 encodes a previously uncharacterized Myb/SANT domain-containing protein. Immunohistochemical staining demonstrated diffuse nuclear MSANTD3 expression in 8 of 27 AcCC cases (30%), including the three cases with MSANTD3 rearrangement. MSANTD3 displayed heterogeneous expression in normal salivary ductal epithelium, as well as among other histologic types of salivary gland cancer though without evidence of translocation. In a broader survey, MSANTD3 showed variable expression across a wide range of normal and neoplastic human tissue specimens. In preliminary functional studies, engineered MSANTD3 overexpression in rodent salivary gland epithelial cells did not enhance cell proliferation, but led to significant upregulation of gene sets involved in protein synthesis. Our findings newly identify MSANTD3 rearrangement as a recurrent event in salivary gland AcCC, providing new insight into disease pathogenesis, and identifying a putative novel human oncogene.

Integrative Genomics Implicates EGFR as a Downstream Mediator in NKX2-1 Amplified Non-Small Cell Lung Cancer.

NKX2-1, encoding a homeobox transcription factor, is amplified in approximately 15% of non-small cell lung cancers (NSCLC), where it is thought to drive cancer cell proliferation and survival. However, its mechanism of action remains largely unknown. To identify relevant downstream transcriptional targets, here we carried out a combined NKX2-1 transcriptome (NKX2-1 knockdown followed by RNAseq) and cistrome (NKX2-1 binding sites by ChIPseq) analysis in four NKX2-1-amplified human NSCLC cell lines. While NKX2-1 regulated genes differed among the four cell lines assayed, cell proliferation emerged as a common theme. Moreover, in 3 of the 4 cell lines, epidermal growth factor receptor (EGFR) was among the top NKX2-1 upregulated targets, which we confirmed at the protein level by western blot. Interestingly, EGFR knockdown led to upregulation of NKX2-1, suggesting a negative feedback loop. Consistent with this finding, combined knockdown of NKX2-1 and EGFR in NCI-H1819 lung cancer cells reduced cell proliferation (as well as MAP-kinase and PI3-kinase signaling) more than knockdown of either alone. Likewise, NKX2-1 knockdown enhanced the growth-inhibitory effect of the EGFR-inhibitor erlotinib. Taken together, our findings implicate EGFR as a downstream effector of NKX2-1 in NKX2-1 amplified NSCLC, with possible clinical implications, and provide a rich dataset for investigating additional mediators of NKX2-1 driven oncogenesis.

NUSAP1 promotes invasion and metastasis of prostate cancer

We have previously identified nucleolar and spindle associated protein 1 (NUSAP1) as a prognostic biomarker in early stage prostate cancer. To better understand the role of NUSAP1 in prostate cancer progression, we tested the effects of increased and decreased NUSAP1 expression in cell lines, in vivo models, and patient samples. NUSAP1 promotes invasion, migration, and metastasis, possibly by modulating family with sequence similarity 101 member B (FAM101B), a transforming growth factor beta 1 (TGFβ1) signaling effector involved in the epithelial to mesenchymal transition. Our findings provide insights into the importance of NUSAP1 in prostate cancer progression and provide a rationale for further study of NUSAP1 function, regulation, and clinical utility.

SWI/SNF aberrations sensitize pancreatic cancer cells to DNA crosslinking agents

While gemcitabine has been the mainstay therapy for advanced pancreatic cancer, newer combination regimens (e.g. FOLFIRINOX) have extended patient survival, though carry greater toxicity. Biomarkers are needed to better stratify patients for appropriate therapy. Previously, we reported that one-third of pancreatic cancers harbor deletions or deleterious mutations in key subunits of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. The SWI/SNF complex mobilizes nucleosomes on DNA, and plays a key role in modulating DNA transcription and repair. Thus, we hypothesized that pancreatic cancers with SWI/SNF aberrations might exhibit compromised DNA repair, and show increased sensitivity to DNA damaging agents. Here, we studied human pancreatic cancer cell lines with deficient (or else exogenously reconstituted) SWI/SNF subunits, as well as normal pancreatic epithelial cells following SWI/SNF subunit knockdown. Cells were challenged with DNA damaging agents, including those used in current combination regimens, and then cell viability assayed. We found that pancreatic cells with SWI/SNF dysfunction showed markedly increased sensitivity to DNA damaging agents, and in particular DNA crosslinking agents (cisplatin and oxaliplatin). Assaying clearance of γH2AX confirmed that SWI/SNF dysfunction impaired DNA damage response/repair. Finally, by analyzing pancreatic cancer patient data from The Cancer Genome Atlas, we found that pancreatic cancers with SWI/SNF deficiency (subunit mutation and/or decreased expression) were associated with extended patient survival specifically when treated with platinum containing regimens. Thus, SWI/SNF dysfunction sensitizes pancreatic cancer cells to DNA crosslinking agents, and SWI/SNF mutation status may provide a useful biomarker to predict which patients are likely to benefit from platinum-containing chemotherapy regimens.

Novel lincRNA SLINKY is a prognostic biomarker in kidney cancer

Clear cell renal cell carcinomas (ccRCC) show a broad range of clinical behavior, and prognostic biomarkers are needed to stratify patients for appropriate management. We sought to determine whether long intergenic non-coding RNAs (lincRNAs) might predict patient survival. Candidate prognostic lincRNAs were identified by mining The Cancer Genome Atlas (TCGA) transcriptome (RNA-seq) data on 466 ccRCC cases (randomized into discovery and validation sets) annotated for ~21,000 lncRNAs. A previously uncharacterized lincRNA, SLINKY (Survival-predictive LINcRNA in KidneY cancer), was the top-ranked prognostic lincRNA, and validated in an independent University of Tokyo cohort (P=0.004). In multivariable analysis, SLINKY expression predicted overall survival independent of tumor stage and grade [TCGA HR=3.5 (CI, 2.2-5.7), P < 0.001; Tokyo HR=8.4 (CI, 1.8-40.2), P = 0.007], and by decision tree, ROC and decision curve analysis, added independent prognostic value. In ccRCC cell lines, SLINKY knockdown reduced cancer cell proliferation (with cell-cycle G1 arrest) and induced transcriptome changes enriched for cell proliferation and survival processes. Notably, the genes affected by SLINKY knockdown in cell lines were themselves prognostic and correlated with SLINKY expression in the ccRCC patient samples. From a screen for binding partners, we identified direct binding of SLINKY to Heterogeneous Nuclear Ribonucleoprotein K (HNRNPK), whose knockdown recapitulated SLINKY knockdown phenotypes. Thus, SLINKY is a robust prognostic biomarker in ccRCC, where it functions possibly together with HNRNPK in cancer cell proliferation.

Identification of transcripts associated with renal damage due to ureteral obstruction as candidate urinary biomarkers

Renal obstruction is a common cause of renal failure in adults and children and is suspected when hydronephrosis is detected on imaging. Because not all cases of hydronephrosis are associated with renal damage, biomarkers are needed to guide intervention to relieve obstruction. We performed gene expression profiling on the kidneys from adult mice over a detailed time course after obstruction and compared these data with a neonatal model of bilateral high-grade obstruction induced by conditional deletion of the calcineurin β1 gene. Having identified a set of 143 transcripts modulated in both adult and neonatal obstruction, we tested their expression in a model of short-term obstruction (1 day), where renal damage is transient and reversible, and long-term obstruction (5 days), where significant renal damage is permanent. A significant number of transcripts increased early after obstruction, and later normalized, while 26 transcripts remained elevated 10 and 28 days after relief of 5 days of ureteral obstruction. With the use of qPCR, elevated levels of several of these candidate RNA biomarkers of renal damage were detected in urine from obstructed mice. In addition, several of these candidate RNA biomarkers of damage resulting from obstruction were detectable in catheterized urine samples from children undergoing surgery for ureteropelvic junction obstruction. Measurement of urinary transcripts modulated in response to renal obstruction could serve as biomarkers of renal damage with important clinical applications.

Abstract 4721: Novel lincRNA SLINKY is a prognostic biomarker in kidney cancer

Clear cell renal cell carcinomas (ccRCC) show a broad range of clinical behavior, and prognostic biomarkers are needed to stratify patients for appropriate management. We sought to determine whether long intergenic non-coding RNAs (lincRNAs) might predict patient survival. Candidate prognostic lincRNAs were identified by mining The Cancer Genome Atlas (TCGA) transcriptome (RNA-seq) data on 466 ccRCC cases (randomized into discovery and validation sets) annotated for ~21,000 lncRNAs. A previously uncharacterized lincRNA, SLINKY (Survival-predictive LINcRNA in KidneY cancer), was the top-ranked prognostic lincRNA, and validated in an independent University of Tokyo cohort (P=0.004). In multivariable analysis, SLINKY expression predicted overall survival independent of tumor stage and grade [TCGA HR=3.4 (CI, 2.1-5.4), P Citation Format: Xue Gong, Zurab Siprashvili, Okyaz Eminaga, Zhewei Shen, Yusuke Sato, Haruki Kume, Yukio Homma, Seishi Ogawa, Paul A. Khavari, Jonathan R. Pollack, James D. Brooks. Novel lincRNA SLINKY is a prognostic biomarker in kidney 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 4721. doi:10.1158/1538-7445.AM2017-4721

Abstract A1-70: Klip1 lincRNA is a prognostic biomarker for clear cell renal cell carcinoma

Background: Large intergenic non-coding RNAs (lincRNAs) are pervasively transcribed in the human genome, and are involved in various biological functions such as cell proliferation, apoptosis and cell signaling pathways. Dysregulation of lincRNAs has been found to underlie human diseases including cancer. As lincRNAs are detectable in blood and urine, they may serve as useful disease biomarkers (as has been shown for PCA3 in prostate cancer). Here, we set out to study lincRNAs in clear cell renal cell carcinoma (ccRCC), the most common tumor of the kidney, accounting for approximately 60,000 cases each year in the United States. In particular, we set out to identify lincRNAs for improved prognostication, in order to better select appropriately aggressive treatments for personalized medicine. Methods and Results: In this study, we mined RNAseq data from The Cancer Genome Atlas (TCGA) on over 500 ccRCC cases. We first re-annotated the TCGA data to identify lincRNAs present from either of two lincRNA compendia. To identify robust prognostic lincRNAs, we split the entire dataset into two even sub-sets and performed survival analysis for each lincRNA in each sub-set. If the P value of the lincRNA was less than 0.001 in both sub-sets, we considered it a candidate “hit”. After 1000 such random sample splits, for each lincRNA we calculated the frequency of “hits”. The top lincRNA, which we named Klip1 (Kidney LincRNA Involved in Proliferation 1) was significant in 90% of random sample splits, which meant its survival prediction power was highly reproduced across the sub-sets of the samples. Klip1 expression correlated with tumor stage, but in multivariate analysis was a significant outcome predictor independent of tumor stage and grade. It was also a significant prognostic marker in an independent Japanese patient cohort (P Discussion and Conclusions: Our findings indicate that lincRNAs have active roles in ccRCC cell proliferation, and may provide useful biomarkers for prognostication, and possibly targets for therapy. Further studies of Klip1 are warranted to translate this biomarker to clinical utility, and to uncover the mechanisms linking Klip1 to cancer cell proliferation. Citation Format: Xue Gong, Jonathan Pollack, James D. Brooks. Klip1 lincRNA is a prognostic biomarker for clear cell renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-70.

Abstract 3436: Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Rare cancer types are not only understudied, but are typically represented by formalin-fixed paraffin-embedded (FFPE) (rather than freshly-frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non-surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole-transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 21 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog pathway mutation confirmed increased basal pathway activity, and defined the response profile to various pharmacologic Hedgehog inhibitors. Together, our results define new ameloblastoma drivers and nominate new molecularly-directed therapies for this rare but disfiguring disease. More generally, our findings validate a robust approach for discovering driver mutations in rare cancers. Citation Format: Andrew C. McClary, Robert T. Sweeney, Jewison Biscocho, Benjamin R. Myers, Lila Neahring, Kevin A. Kwei, Kunbin Qu, Xue Gong, Tony Ng, Carol D. Jones, Sushama Varma, Justin I. Odegaard, Brian Rubin, Megan L. Troxell, Robert J. Pelham, James L. Zehnder, Philip A. Beachy, Jonathan R. Pollack, Robert B. West. Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2014-3436

Abstract B15: NuSAP is regulated by RB1 and modulates prostate cancer progression

We have recently shown that nucleolar and spindle-associated protein ( NUSAP1 ; encoding NuSAP) is overexpressed in recurrent prostate cancer tumors. Correlation between NUSAP1 transcript levels and prognosis has been seen in melanomas, glioblastomas, and breast cancers, indicating that NuSAP plays an important role in cancer aggressiveness. Although NuSAP is known to be essential for cell cycle progression, faithfully binding to and stabilizing microtubules during mitosis, little is known about its role in cancer progression. Hence, in this study we performed an extensive analysis to understand the role of NuSAP in aggressive cancer, focusing on prostate cancer. We first aimed to identify a mechanism leading to NuSAP upregulation in prostate cancer. In our previous study we found that E2F transcription factor-1 (E2F1) directly binds to the promoter of NUSAP1 , enhancing NUSAP1 expression. Since E2F1 is known to be negatively regulated by the retinoblastoma protein (RB1), and RB1 is frequently lost in aggressive prostate cancer, we hypothesized that the expression of NuSAP is regulated, at least in part, by the RB1/E2F1 axis. We confirmed that NUSAP1 transcripts are anti-correlated with RB1 transcripts and positively correlated with E2F1 transcripts in prostate cancer microarray datasets, and then used lentiviral-based technology to knockdown RB1 in human prostate cancer cell lines (PC3 and LNCaP). RT-qPCR and western blot revealed that NuSAP expression increased upon knockdown of RB1, and was reduced upon further knockdown of E2F1, supporting the notion that NuSAP is regulated by the RB1/E2F1 axis. We next aimed to determine the function of NuSAP in prostate cancer progression. Using lentiviral-based technology we knocked-down and overexpressed NuSAP in prostate cancer cell lines, and examined proliferation, invasion, apoptosis, cell cycle stages, migration, and global gene expression. Proliferation, invasion, and scratch assays revealed that knockdown of NuSAP consistently reduced proliferation, invasion, and migration, respectively, while overexpression of NuSAP significantly increased invasion. Flow cytometry analyses revealed that knockdown of NuSAP led to a significant increase in the number of apoptotic cells and number of cells in the G2/M phase of the cell cycle. RNA-Seq analysis further revealed genes significantly differentially expressed upon knockdown or overexpression of NuSAP. Of note, overexpression led to an activation of genes involved in invasion, cellular movement, angiogenesis, and metastasis. Examination of microarray datasets additionally revealed increased NUSAP1 transcripts in metastatic prostate tumors. Taken together, our work provides a mechanism accounting for NuSAP overexpression in prostate cancer, and identifies novel functions of NuSAP in aggressive cancers. Understanding the role of NuSAP in prostate cancer progression will provide insights into the inner workings of aggressive cancer cells, and may ultimately pave the way for new routes to prognosticate and treat prostate and other cancers. Citation Format: Catherine A. Gordon, Xue Gong, Zulfiqar G. Gulzar, James D. Brooks. NuSAP is regulated by RB1 and modulates prostate cancer progression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B15.