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Supplementary Figure 1 from Development of a Multiplex Quantitative PCR Signature to Predict Progression in Non–Muscle-Invasive Bladder Cancer

Supplementary Methods, Figures 1-7 from Development of a Multiplex Quantitative PCR Signature to Predict Progression in Non–Muscle-Invasive Bladder Cancer

In bladder cancer, clinical grade and stage fail to capture outcome. We developed a clinically applicable quantitative PCR (QPCR) gene signature to predict progression in non–muscle-invasive bladder cancer. Comparative metaprofiling of 12 DNA microarray data sets (comprising 631 samples and 241,298 probe sets) identified 96 genes, which showed differential expression in seven clinical outcome categories, or were identified as outliers, historic markers, or housekeeping genes. QPCR was done to determine mRNA expression from 96 bladder tumors. Fifty-seven genes differentiated T2 from non-T2 tumors (P < 0.05). Principal components analysis and Cox regression models were used to predict probability of T2 progression for non-T2 patients, placing them into high- and low-risk groups based on their gene expression. At 2 years, high-risk patients exhibited greater T2 progression (45% for high-risk patients versus 12% for low-risk patients; P = 0.003, log-rank test). This difference remained significant within T1 tumors (61% for high-risk patients versus 22% for low-risk patients; P = 0.02) and Ta tumors (29% for high-risk patients versus 0% for low-risk patients; P = 0.03). The best multivariate Cox model included stage and gender, and this signature provided predictive improvement over both (P = 0.002, likelihood ratio test). Immunohistochemistry was done for two genes in the signature not previously described in bladder cancer, ACTN1 and CDC25B, corroborating their up-regulation at the protein level with disease progression. Thus, we identified a 57-gene QPCR panel to help predict progression of non–muscle-invasive bladder cancers and delineate a systematic, generalizable approach to converting microarray data into a multiplex assay for cancer progression. [Cancer Res 2009;69(9):3810–8]

Supplementary Figure 2 from Development of a Multiplex Quantitative PCR Signature to Predict Progression in Non–Muscle-Invasive Bladder Cancer

Bladder cancer is a common and deadly malignancy, but incomplete understanding of the molecular events driving tumor development and metastasis has limited development of effective treatments. Ataxia-Telangiectasia Group D Complementing (ATDC; also named TRIM29) is highly expressed in many cancer types, including bladder and pancreas. To determine the role of ATDC in tumorigenesis, we developed a novel transgenic mouse model in which ATDC was overexpressed in multiple tissue types. The dominant phenotype in these mice was the development of both papillary and invasive bladder carcinoma beginning at approximately 8 months of age. In this model, invasive bladder tumors were histologically indistinguishable and shared a similar gene expression signature with human invasive bladder cancers. We also found that ATDC was highly expressed in ~70% human bladder cancers where it marked invasive tumors and correlated with worse survival after chemotherapy (p = 0.002). Furthermore, ATDC knockdown decreased proliferation, invasion and tumor growth in multiple human bladder cancer cell lines and in an in vivo orthotopic bladder tumor model. ATDC-induced bladder tumorigenesis was mediated by upregulation of DNA methyltransferase 3A (DNMT3A), which drove PTEN promoter methylation and silencing, events which promoted invasion and proliferation in both transgenic mice and human bladder cancers. These findings establish ATDC as a novel driver and therapeutic target in bladder cancer. In addition, the ATDC transgenic mouse model should serve as an important and unique new model to study bladder cancer development, progression and therapeutic responses. Citation Format: Phillip Palmbos, Lidong Wang, Huibin Yang, Jake LeFlein, John E. Wilkinson, Chandan Kumar-Sinha, L. Priya Kunju, Stephanie Daignault, Xue-Ru Wu, Yair Lotan, Monica Liebert, Mats Ljungman, Diane Simeone. ATDC (Trim29) drives invasive bladder cancer formation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B24.

Bladder cancer is the 5th most common malignancy and a significant cause of morbidity and mortality, but the molecular events leading to its development are incompletely understood. We have identified an oncogene, Ataxia-Telangiectasia Group D Complementing (ATDC) gene, which drives formation and progression of pancreatic and bladder tumors. To better characterize the oncogenic function of ATDC, we generated transgenic (tg) mice which overexpress ATDC and the predominant phenotype of these mice was the development of non-invasive and muscle-invasive urothelial carcinomas. Tg bladder tumors were histologically identical to human tumors and displayed a similar gene expression signature to human invasive bladder tumors. ATDC expression was assessed in a tissue microarray and was highly up-regulated in 70% of human invasive bladder tumors (173/252). High expression of ATDC correlated with more advanced stage disease and worse survival after chemotherapy (p = 0.002). ATDC knockdown in human bladder cancer cell lines correlated with increased sensitivity to chemotherapy in vitro and decreased proliferation and invasion both in vitro and in vivo in a human bladder cancer xenograft model. To understand the mechanism of ATDC-mediated tumor formation, we modulated ATDC expression in normal and bladder cancer cells lines. ATDC overexpression down-regulated PTEN levels via DNA promoter methylation by DNMT3A in both tg mouse tumors and human cell lines. IHC analysis of ATDC, PTEN and DNMT3A expression in human bladder tumors specimens demonstrated similar findings. ATDC was also found to bind to p53 and inhibit p53 mediated functions. These findings establish ATDC as a novel determinant of bladder cancer initiation, progression and resistance to treatment which mediates tumorigenesis by down-regulation of PTEN expression and inhibition of p53. Citation Format: Phillip Palmbos, Lidong Wang, Huibin Yang, Taylor Detzler, Gina Ney, Justin Hart, Stephanie Daignault-Newton, L. Priya Kunju, Chandan Kumar-Sinha, Monica Liebert, Mats Ljungman, Diane Simeone. ATDC drives bladder cancer formation by promoting methylation of the PTEN promoter and inhibition of p53 function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2013-313