Whole-exome sequencing combined with functional genomics reveals novel candidate driver cancer genes in endometrial cancer

Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
Genome Research (Impact Factor: 14.63). 10/2012; 22(11). DOI: 10.1101/gr.137596.112
Source: PubMed

ABSTRACT Endometrial cancer is the most common gynecological malignancy, with more than 280,000 cases occurring annually worldwide. Although previous studies have identified important common somatic mutations in endometrial cancer, they have primarily focused on a small set of known cancer genes and have thus provided a limited view of the molecular basis underlying this disease. Here we have developed an integrated systems-biology approach to identifying novel cancer genes contributing to endometrial tumorigenesis. We first performed whole-exome sequencing on 13 endometrial cancers and matched normal samples, systematically identifying somatic alterations with high precision and sensitivity. We then combined bioinformatics prioritization with high-throughput screening (including both shRNA-mediated knockdown and expression of wild-type and mutant constructs) in a highly sensitive cell viability assay. Our results revealed 12 potential driver cancer genes including 10 tumor-suppressor candidates (ARID1A, INHBA, KMO, TTLL5, GRM8, IGFBP3, AKTIP, PHKA2, TRPS1, and WNT11) and two oncogene candidates (ERBB3 and RPS6KC1). The results in the "sensor" cell line were recapitulated by siRNA-mediated knockdown in endometrial cancer cell lines. Focusing on ARID1A, we integrated mutation profiles with functional proteomics in 222 endometrial cancer samples, demonstrating that ARID1A mutations frequently co-occur with mutations in the phosphatidylinositol 3-kinase (PI3K) pathway and are associated with PI3K pathway activation. siRNA knockdown in endometrial cancer cell lines increased AKT phosphorylation supporting ARID1A as a novel regulator of PI3K pathway activity. Our study presents the first unbiased view of somatic coding mutations in endometrial cancer and provides functional evidence for diverse driver genes and mutations in this disease.

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Available from: Han Liang, Sep 28, 2015
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    • "MCF7 is a well-established breast carcinoma cell line and MRC5 a commonly used human primary lung fibroblast cell line. Although MCF7 contains a moderately activating mutation of PIK3CA [24, 25], depletion of ARID1A by siRNA considerably increased phosphorylation of AKT at Ser-473 in this cell line, as described previously in endometrial carcinoma cell lines [21]. The normal primary fibroblast cell line, MRC5, that does not contain any known mutations [26] confirmed our results, thus minimizing the likelihood of secondary effects by other mutations in the ARID1A knockdown experiments. "
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    ABSTRACT: ARID1A mutations are observed in various tumors, including ovarian clear cell (OCCC) and endometrioid carcinomas, endometrial, and breast carcinomas. They commonly result in loss of ARID1A-protein expression and frequently co-occur with PI3K/AKT-pathway activating mechanisms. The aim of this study was to test the hypothesis as to whether PI3K/AKT-pathway activation is a critical mechanism in ARID1A-mutated tumors and if consequently ARID1A-deficient tumors show increased sensitivity to treatment with PI3K- and AKT-inhibitors. Upon ARID1A knockdown, MCF7 breast cancer cells and primary MRC5 cells exhibited a significantly increased sensitivity towards the AKT-inhibitors MK-2206 and perifosine, as well as the PI3K-inhibitor buparlisib. Knockdown of ARID1A in MCF7 led to an increase of pAKT-Ser473. AKT-inhibition with MK-2206 led to increased apoptosis and to a decrease of pS6K in ARID1A-depleted MCF7 cells but not in the controls. In five OCCC cell lines ARID1A-deficiency correlated with increased pAKT-Ser473 levels and with sensitivity towards treatment with the AKT-inhibitor MK-2206. In conclusion, ARID1A-deficient cancer cells demonstrate an increased sensitivity to treatment with small molecule inhibitors of the PI3K/AKT-pathway. These findings suggest a specific requirement of the PI3K/AKT pathway in ARID1A-deficient tumors and reveal a synthetic lethal interaction between loss of ARID1A expression and inhibition of the PI3K/AKT pathway.
    Oncotarget 06/2014; 5(14). DOI:10.5167/uzh-106117 · 6.36 Impact Factor
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    • "Unlike previously reported observations in endometrial cancers cell lines [20], we have been unable to demonstrate a mechanism by which AKT phosphorylation (pAKT) occurs in tumors using cell line models (RMG1, ES2, and JHOC5). Despite good knockdown of BAF250a in these lines, we could not demonstrate clear increases in pAKT, or downstream signaling. "
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    ABSTRACT: Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC. Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A. Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser 473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three CCC cell lines wild type for ARID1A showed no increase in either pAKT-Thr308 or pAKT-S473 suggesting that pAKT in tumor tissues is indirectly regulated by BAF250a expression. Proteomic assessment of CCC and EC demonstrates remarkable differences in protein expression that are dependent on histotype, thereby further characterizing these cancers. AKT phosphorylation is associated with ARID1A/BAF250a deficient tumors, however in ovarian cancers the mechanism remains to be elucidated.
    BMC Cancer 02/2014; 14(1):120. DOI:10.1186/1471-2407-14-120 · 3.36 Impact Factor
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    • "microsatellite instability (20%), while TP53 mutations appear to be the key driver in serous lesions (∼90% of nonendometrioid lesions) [6] [7] [8] [9]. It is critically important to note that varying types of p53 mutant proteins exist, with different implications for chemosensitivity. "
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    ABSTRACT: Serous uterine endometrial carcinomas are aggressive type II cancers with poor outcomes for which new treatment strategies are urgently needed, in particular, strategies that augment sensitivity to established chemotherapy regimens. The tumor suppressor gene TP53 is dysregulated in more than 90% of serous tumors, altering master regulators of the G2/M cell cycle checkpoint in unique and predictable ways and desensitizing cells to chemotherapy. We hypothesized that synthetic lethality can be achieved in endometrial cancer cells with mutant p53 by combining paclitaxel with agents to overcome G2/M arrest and induce mitotic catastrophe. The combination of BIBF1120, an investigational VEGFR, PDGFR, and FGFR multityrosine kinase inhibitor with established anti-angiogenic activity, with paclitaxel abrogated the G2/M checkpoint in p53-null endometrial cancer cells via modulation of G2/M checkpoint regulators followed by induction of mitotic cell death. In endometrial cancer cells harboring an oncogenic gain-of-function p53 mutation, synthetic lethality was created by combining paclitaxel with BIBF1120 and a histone deacetylase inhibitor, which serves to destabilize mutant p53. These cells were also sensitive to an inhibitor of the G2/M kinase Wee1 in combination with paclitaxel. These findings reveal that, in addition to antiangiogenic activity, the angiokinase inhibitor BIBF1120 can be used to restore sensitivity to paclitaxel and induce mitotic cell death in endometrial cancer cells with non-functional p53. These preclinical data serve as a critical platform for the creative design of future clinical trials utilizing molecularly enhanced chemotherapy to achieve synthetic lethality based on the mutational landscape.
    Obstetrics and Gynecology International 12/2013; 2013:828165. DOI:10.1155/2013/828165
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