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Chromatin-Associated Protein SIN3B Prevents Prostate Cancer Progression by Inducing Senescence Anthony J

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Chromatin-Associated Protein SIN3B Prevents Prostate Cancer Progression by Inducing Senescence Anthony J Published OnlineFirst August 14, 2017; DOI: 10.1158/0008-5472.CAN-16-3410 Cancer Tumor and Stem Cell Biology Research Chromatin-Associated Protein SIN3B Prevents Prostate Cancer Progression by Inducing Senescence Anthony J. Bainor1, Fang-Ming Deng2, Yu Wang1, Peng Lee2,4, David J. Cantor1, Susan K. Logan1,3,4, and Gregory David1,3,4 Abstract Distinguishing between indolent and aggressive prostate ade- cinoma. Furthermore, SIN3B was downregulated in human pros- nocarcinoma remains a priority to accurately identify patients tate adenocarcinoma correlating with upregulation of its target who need therapeutic intervention. SIN3B has been implicated in genes. Our results suggest a tumor suppressor function for the initiation of senescence in vitro. Here we show that in a mouse SIN3B that limits prostate adenocarcinoma progression, with model of prostate cancer, SIN3B provides a barrier to malignant potential implications for the use of SIN3B and its target genes progression. SIN3B was required for PTEN-induced cellular senes- as candidate diagnostic markers to distinguish indolent from cence and prevented progression to invasive prostate adenocar- aggressive disease. Cancer Res; 77(19); 1–10. Ó2017 AACR. Introduction damage, activation of oncogenes, or loss of a tumor suppres- sor (5, 6). Senescent cells have been identified in preneoplastic Prostate adenocarcinoma is the second most common cancer lesions of several solid tumor types, including prostatic intrae- type in American men with approximately 230,000 new pithelial neoplasias (PIN), but are rarely found in normal patients diagnosed each year, equating to about 1 in 7 men prostate or prostate adenocarcinoma (7). On the basis of these being diagnosed with prostate adenocarcinoma in his lifetime findings, cellular senescence has been hypothesized to prevent (1). Despite the prevalence of prostate adenocarcinoma, the 5- cancer progression, through its ability to trigger stable cell- year survival rate for local or regional disease (stages I–early IV) cycle exit and prevent the proliferation of potentially delete- is nearly 100%. However, men with invasive or metastatic rious cells. disease (stage IV) have a significantly reduced survival rate of Mouse models of cancer have enabled a greater understanding about 28% (2). Therefore, defining the molecular mechanisms of the biology of tumor initiation and progression, leading to that restrict prostate cancer progression remains a priority to improved diagnostic and therapeutic strategies (8, 9). Various improve the identification of patients in need of therapeutic attempts have been made to recapitulate prostate adenocarcino- intervention. Clinical monitoring of prostate cancer currently ma in the mouse. Among these, somatic inactivation of PTEN in relies on histopathologic and chemical indicators, such as prostate epithelium most accurately models the initiation of Gleason scoring and PSA levels. Unfortunately, the use of these prostate cancer. Importantly, approximately 70% of human pri- parameters to manage prostate cancer treatment has proven mary prostate tumors harbor loss or alteration of at least one largely inadequate (3, 4). PTEN allele (10). In mice, prostate-specific inactivation of Pten As a potential mechanism that restricts cancer progression, causes the generation of hyperplasia early in life, followed by the cellular senescence has generated much interest. Cellular generation of PINs that rarely progress to prostate adenocarcino- senescence is a stable form of cell-cycle arrest that can be ma. More recently, it was demonstrated that Pten-deletion in the triggered by different stimuli including serial passaging, DNA prostate leads to the activation of a TRP53-dependent senescence response, termed PTEN-loss–induced cellular senescence or PICS (11). Importantly, combined inactivation of Pten and Trp53 in the 1Department of Biochemistry and Molecular Pharmacology, NYU Langone prostate alleviates PICS and accelerates the progression to invasive Medical Center, New York, New York. 2Department of Pathology, NYU Langone prostate adenocarcinoma (7). However, due to the wide range of Medical Center, New York, New York. 3Department of Urology, NYU Langone TRP53 targets in vivo, it has been difficult to precisely delineate 4 Medical Center, New York, New York. NYU Cancer Institute, NYU Langone the specific effector programs engaged upon loss of PTEN that Medical Center, New York, New York. trigger senescence and restrict cancer progression in early prostate Note: Supplementary data for this article are available at Cancer Research lesions. Online (http://cancerres.aacrjournals.org/). In mammals, the SIN3 family consists of two proteins, SIN3A Corresponding Author: Gregory David, Department of Biochemistry and Molec- and SIN3B, which are ubiquitously expressed. SIN3 proteins are ular Pharmacology, MSB417, NYU Langone Medical Center, 550 First Ave., New evolutionary conserved noncatalytic scaffold components of large York, NY10016. Phone: 212-263-2926; Fax: 212-263-7133; E-mail: transcriptional repressive complexes that are recruited to genomic [email protected] loci through their interaction with sequence specific transcription doi: 10.1158/0008-5472.CAN-16-3410 factors (12, 13). Repression of gene transcription is achieved at Ó2017 American Association for Cancer Research. least in part through the association of SIN3 proteins with the www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst August 14, 2017; DOI: 10.1158/0008-5472.CAN-16-3410 Bainor et al. histone deacetylases HDAC1/2. While genetic inactivation of Histology and IHC Sin3a leads to cell death in a wide range of cell types (14), Mouse prostates were fixed in 10% formalin (Thermo Fisher SIN3B is dispensable for cellular viability and proliferation. Scientific) and processed for paraffin embedding. Histology was However, Sin3b-null mice die during late embryogenesis, likely performed at the NYU School of Medicine Histopathology Core due to the inability of different cellular compartments to Facility. Five-micron sections were deparaffinized, stained with differentiate appropriately (14, 15). Recently, we have demon- Gill hematoxylin and eosin Y, followed by an alcohol dehydration strated that the genetic inactivation of Sin3b renders mouse series and mounting (Permount; Thermo Fisher Scientific). For embryonic fibroblasts refractory to senescence triggered by IHC, deparaffinized five-micron sections were rehydrated and ectopic expression of oncogenic RAS (15). In addition, using quenched in 3% hydrogen peroxide for 15 minutes. Antigen a mouse model for pancreatic cancer, we showed that SIN3B retrieval was performed in 10 mmol/L sodium citrate and expression is upregulated in PanINs (pancreatic intraepithelial 0.1% Tween-20 (pH 6.0) for 15 minutes in a microwave oven. neoplasia) and that the genetic deletion of Sin3b in the pancreas Blocking was done in 5% serum, 1% BSA, and 0.1% Tween-20 for prevents KRAS-induced cellular senescence (12, 16). Surpris- 1 hour at room temperature, followed by incubation with primary ingly, the inactivation of a SIN3B-dependent senescence pro- antibodies diluted in 1% BSA overnight at 4C. The following gram in a mouse model of pancreatic cancer protected against primary antibodies were used: rabbit anti-SIN3B (Novus Biolo- cancer progression, likely due to the decrease of senescence- gicals); rabbit anti-HP1g (phospho S93; Abcam); rabbit anti- associated inflammation. The paradoxical outcome resulting PTEN (Cell Signaling Technology; D4.3 XP), rabbit anti-pAKT from inactivation of senescence in this model warrants further (phospho S473; Cell Signaling Technology; D9E), rabbit anti- investigation of the relationship between senescence and cancer KI67 (Lab Vision; SP6), rabbit anti-P21 (Santa Cruz Biotechnol- progressioninspecificcontextsin vivo. On the basis of the ogy; C-19), rabbit anti-alpha smooth muscle actin (Abcam; requirement of SIN3B for the induction of OIS both in vitro and ab5694), mouse anti-CK18 (Abcam; C-04), rabbit anti-AR (Santa in vivo and as PICS is believed to prevent the progression of PIN Cruz Biotechnology; N-20), rabbit anti-CK5 (BioLegend; lesions, we sought to investigate the necessity of SIN3B for PICS Poly19055), and rabbit anti-IL6 (Santa Cruz Biotechnology; M- and elucidate its contribution to prostate cancer initiation and 19). After incubating with secondary biotinylated antibodies and progression. Using genetic inactivation of Pten to model pros- solution T.U. horseradish peroxidase streptavidin (both from tate cancer in the mouse, we demonstrate here that SIN3B Vector Laboratories), sections were developed with DAB Perox- protects against prostate cancer progression by promoting PICS idase Substrate Kit (Vector Laboratories). After counterstaining and limiting cellular proliferation in preneoplastic lesions. with Gill hematoxylin (Sigma-Aldrich), slides were subjected to Furthermore, we identify that downregulation of SIN3B expres- an alcohol dehydration series and mounted with Permount sion is a common event in human prostate adenocarcinoma. (Thermo Fisher Scientific). Slides were examined on a Zeiss This study identifies the chromatin-associated SIN3B as a AxioImager A2 microscope. critical suppressor of prostate cancer progression through the induction of the senescence process. Infection of prostate cancer cells A PTRIPZ lentiviral doxycycline-inducible short hairpin RNA Materials and Methods (shRNA)
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