Tumor Suppressor Genes RB1 Research Articles

Abstract GMM-061: MOLECULAR CHARACTERIZATION OF MOUSE MODELS OF HIGH-GRADE SEROUS CARCINOMA ARISING IN THE OVIDUCT

Abstract We recently developed genetically engineered mouse models (GEMMs) of oviductal high-grade serous carcinoma (HGSC) based on conditional inactivation of several different combinations of tumor suppressor genes (Brca1, Trp53, Rb1, and Nf1) that are recurrently mutated in human HGSCs. The histopathological features of the mouse tumors closely mimic their human tumor counterparts. In order to characterize how well the mouse tumors recapitulate the molecular characteristics of human HGSCs, targeted exome sequencing was used to analyze the 32 most commonly mutated genes in human HGSC, in 60 mouse tumors arising in the context of Brca1, Trp53, Rb1, and/or Nf1 inactivation. We employed the sequence data to assess DNA copy number alterations (CNAs) and single nucleotide variants (SNVs) in the mouse tumors. Compared to 14 normal tissues and 8 oviductal tumors arising in the context of Apc, Pten, ± Arid1a inactivation, the mouse HGSCs showed a high level of genomic instability, with many widely distributed CNAs – very similar to the widespread CNAs observed in human HGSCs. Targeted exome sequencing also showed that a subset of the mouse tumors acquired alterations observed in human HGSCs, including amplification of cMyc, and deletion of Pten. Variant analysis identified nonsynonymous SNVs in Csmd3, Crebbp, Pten, Mettl17, and Zymynd8 and a frameshift deletion of Pten. Sanger sequencing confirmed the presence of these somatic mutations in the mouse tumors and their absence in matched normal tissues. Loss of PTEN expression was observed in those tumors that acquired somatic Pten alterations. These data show that HGSCs arising in our GEMMs have very similar molecular characteristics to their human tumor counterparts. The somatic alterations are likely acquired during the relatively lengthy period (several months) between tumor initiation and progression to overt malignancy. These features render the models particularly well suited for studying the early phases of HGSC development and for translational applications aimed at identifying effective strategies for HGSC prevention and early detection. Citation Format: Yali Zhai, Yisheng Wang, Stephanie M. Schulman, Kevin Hu, Albert Liu, Scott A. Tomlins, Eric R. Fearon, and Kathleen R. Cho. MOLECULAR CHARACTERIZATION OF MOUSE MODELS OF HIGH-GRADE SEROUS CARCINOMA ARISING IN THE OVIDUCT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-061.

The KDM5A/RBP2 histone demethylase represses NOTCH signaling to sustain neuroendocrine differentiation and promote small cell lung cancer tumorigenesis.

More than 90% of small cell lung cancers (SCLCs) harbor loss-of-function mutations in the tumor suppressor gene RB1 The canonical function of the RB1 gene product, pRB, is to repress the E2F transcription factor family, but pRB also functions to regulate cellular differentiation in part through its binding to the histone demethylase KDM5A (also known as RBP2 or JARID1A). We show that KDM5A promotes SCLC proliferation and SCLC's neuroendocrine differentiation phenotype in part by sustaining expression of the neuroendocrine transcription factor ASCL1. Mechanistically, we found that KDM5A sustains ASCL1 levels and neuroendocrine differentiation by repressing NOTCH2 and NOTCH target genes. To test the role of KDM5A in SCLC tumorigenesis in vivo, we developed a CRISPR/Cas9-based mouse model of SCLC by delivering an adenovirus (or an adeno-associated virus [AAV]) that expresses Cre recombinase and sgRNAs targeting Rb1, Tp53, and Rbl2 into the lungs of Lox-Stop-Lox Cas9 mice. Coinclusion of a KDM5A sgRNA decreased SCLC tumorigenesis and metastasis, and the SCLCs that formed despite the absence of KDM5A had higher NOTCH activity compared to KDM5A+/+ SCLCs. This work establishes a role for KDM5A in SCLC tumorigenesis and suggests that KDM5 inhibitors should be explored as treatments for SCLC.

Promoting regulated gene diagnosis for retinoblastoma in clinical work

Retinoblastoma, the most frequent malignant intraocular tumor in childhood, is caused by oncogenic mutations in the RB1 tumor suppressor gene. Identification of these mutations in patients is important for genetic counseling and clinical management of relatives at risk, and thus probands are conventionally applied gene detection in developed countries. However, gene diagnosis is still in the elementary period in China. This article reviews the characteristics of retinoblastoma genetics and the current status of genetic testing in China, so as to attract more attention from ophthalmologists and to promote regulated gene diagnosis in clinical work. Not only does good understanding of retinoblastoma genetics support optimal care for retinoblastoma children and their families, but also promotes the development in foundational research. (Chin J Ophthalmol, 2019, 55: 806-810).

1170P - A parent-of-origin effect of the RB1 mutations in retinoblastoma with low penetrance and variable expressivity

BackgroundRetinoblastoma (RB) is the most common pediatric intraocular neoplasm caused by the biallelic inactivation of the RB1 tumor suppressor gene. In 40% of cases, the development of RB is mediated by a germline mutation in one of the alleles of RB1. Patients with germline mutation develop bilateral tumor with penetrance of more than 90%. However, some families demonstrate cases of RB with low penetrance (unaffected carries) and variable expressivity (carries develop either bi- or uni- RB).It is believed that the phenotypic manifestation of hereditary retinoblastoma depends on the functional type of the germinal mutation in the RB1. The molecular mechanisms underlying the variable phenotypic manifestation of the same mutation in different family members are currently explained by the parent-of-origin effect of RB1 mutation. MethodsUsing NGS of the RB1 we have analyzed DNA from blood of 331 unrelated patients with RB (226 patients with uni- RB and 105 with bi- RB). DNA samples of available family members were also examined for the presence of an identified mutation using Sanger sequencing. ResultsWe identified 11 germline mutations in the RB1 that led to the RB with low penetrance and/or variable expressivity in 12 families. Among the identified mutations:, 25.0% - are missense mutations, 58.3% - are splice mutations and 16.7% - are frame shift mutations. In 91,7% of cases, probands inherited the mutant allele from their fathers, who were either clinically healthy carriers (7 families) or had the uni-/bilateral form of RB (3 families).Table: 1170PTable: 1170PMutationsCarrier (Proband, P)Form (uni, U; bi-lateral, B; no symptoms, -)c.1364G>C; c.1573G>A; c.1981C>T; с.607+1G-T (2 families); c.45_76del; с.83delP ♂U -c.861G>CP ♀U -c.939G>AP ♂ Grandfather UncleU - - Uc.380+1G-A; с.1695+5G-T; c.1696-2A-GP ♂B U ConclusionsThe identification of mutations in the RB1 leading to the development of RB with low penetrance and variable expressivity, is necessary for adequate treatment and competent determination of the risk of developing the disease in other family members. Legal entity responsible for the studyState assignment of Ministry of Science and Higher Education of the Russian Federation. FundingThe state assignment of Ministry of Science and Higher Education of the Russian Federation. DisclosureAll authors have declared no conflicts of interest.

DKK1 promotes hepatocellular carcinoma inflammation, migration and invasion: Implication of TGF-β1.

Dickkopf-1 (DKK1), an inhibitor of the most frequently impaired signaling pathway in hepatocellular carcinoma (HCC), the Wnt/beta-catenin pathway, seems to fulfill contradictory functions in the process of tumorigenesis, acting either as an oncogenic promoter of metastasis or as a tumor suppressor. Elevated serum levels of DKK1 have been reported in HCC; however, little is known about its functional significance. In the current study, we treated HepG2/C3A and PLC/PRF/5 with the recombinant protein DKK1. Cytotoxicity was first determined by the WST-8 assay. AFP expression was measured at both the mRNA and protein levels. Expression of the oncogenes MYC, CCND1, hTERT, and MDM2 and the tumor suppressor genes TP53, P21 and RB was assessed. Western blot analysis of non-phosphorylated ẞ-catenin and Sanger sequencing were performed to explain the functional differences between the two cell lines. Subsequently, inflammation, migration and invasion were evaluated by qPCR, ELISA, the Boyden chamber assay, zymography, and MMP-2 and MMP-9 western blot analysis. Knockdown of DKK1 and TGF-β1 were also performed. Our results suggest that DKK1 exerts an oncogenic effect on HepG2/C3A cell line by upregulating the expression of oncogenes and downregulating that of tumor suppressor genes, whereas the opposite effect was demonstrated in PLC/PRF/5 cells. This differential impact of DKK1 can be explained by the mutations that affect the canonical Wnt pathway that were detected in exon 3 of the CTNNB1 gene in the HepG2 cell line. We further confirmed that DKK1 promotes inflammation, tumor invasion and migration in both cell types. The canonical pathway was not responsible for the DKK1 proinvasive effect, as indicated by the active ẞ-catenin levels in the two cell lines upon DKK1 treatment. Interestingly, knockdown of TGF-β1 negatively affected the DKK1 proinvasive effect. Taken together, DKK1 appears to facilitate tumor invasion and migration through TGF- β1 by remodeling the tumor microenvironment and inducing inflammation. This finding endorses the relevance of TGF-β1 as a therapeutic target.

Integrative genomic analysis of matched primary and metastatic pediatric osteosarcoma.

Despite being the most common childhood bone tumor, the genomic characterization of osteosarcoma remains incomplete. In particular, very few osteosarcoma metastases have been sequenced to date, critical to better understand mechanisms of progression and evolution in this tumor. We performed an integrated whole genome and exome sequencing analysis of paired primary and metastatic pediatric osteosarcoma specimens to identify recurrent genomic alterations. Sequencing of 13 osteosarcoma patients including 13 primary, 10 metastatic, and 3 locally recurring tumors revealed a highly heterogeneous mutational landscape, including cases of hypermutation and microsatellite instability positivity, but with virtually no recurrent alterations except for mutations involving the tumor suppressor genes RB1 and TP53. At the germline level, we detected alterations in multiple cancer related genes in the majority of the cohort, including those potentially disrupting DNA damage response pathways. Metastases retained only a minimal number of short variants from their corresponding primary tumors, while copy number alterations showed higher conservation. One recurrently amplified gene, KDR, was highly expressed in advanced cases and associated with poor prognosis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 748: Genomic characterization of recurrent small cell lung cancer through research autopsy reveals clonal diversity and candidate driver of chemoresistance

Abstract Genomic characterization of recurrent small cell lung cancer (SCLC) is needed to dissect the molecular mechanisms of relapse, metastatic dissemination, and chemoresistance. SCLC is a highly aggressive, lethal neuroendocrine cancer arising from the bronchial epithelium that develops in heavy smokers. Although many SCLC patients demonstrate sensitivity to first-line platinum-doublet chemotherapy, the response is short-lived and nearly all patients progress during therapy or relapse within several months of completing treatment. Treatment options beyond first-line chemotherapy are limited and ineffective. Metastatic SCLC has a poor prognosis and is associated with a 5-year overall survival rate of 5-10%. A well-characterized genetic hallmark of SCLC is the concurrent inactivation of tumor suppressor genes TP53 and RB1. Additional SCLC candidate driver genes have been reported and include CREBBP, MLL, NOTCH1-4, SOX2 and MYC. However, most SCLC tumors profiled in these studies were obtained from treatment-naïve patients. Therefore, genomic alterations that are acquired during therapy and drive the development of chemoresistance in recurrent SCLC remain undefined. We present results from whole exome sequencing (WES) of multiple metastatic tumors procured from five treatment-refractory SCLC patients who underwent research autopsy. WES was also performed on pre-treatment samples from three of these patients. Our results showed universal TP53 alterations and to a lesser extent RB1 mutations in our SCLC cohort. Next, we utilized bioinformatics methods to analyze clonal heterogeneity and evolution in these SCLC patients. This analysis demonstrated the existence of multiple clones of tumor cells in both the pre-treatment and treatment-resistant autopsy SCLC samples, suggesting that tumor heterogeneity occurs early in SCLC development and is subsequently maintained. Furthermore, allele-specific analysis of copy number variations in our SCLC cohort demonstrated near universal deletion of a region on chromosome 5q containing the tumor suppressor APC, consistent with a recently reported role for deregulated WNT signaling in chemoresistance development in relapsed SCLC. Finally, in one SCLC patient, we identified PTEN deletion (loss of heterozygosity) as a potential mechanism of metastasis specifically to the brain. In conclusion, we have performed genomic characterization of recurrent SCLC, defined by significant clonal diversity, through research autopsy and identified candidate genetic drivers of treatment resistance and organ-specific metastasis. Citation Format: Hui-Zi Chen, Russell Bonneville, Melanie A. Krook, Michele R. Wing, Julie W. Reeser, Jharna Miya, Anoosha Paruchuri, Eric Samorodnitsky, Lianbo Yu, Amy M. Smith, Thuy Dao, Dorrelyn Martin, Qishan Guo, Hailey Magenheim, Aharon G. Freud, Sharon Cole, Gregory Otterson, Peter Shields, David P. Carbone, Patricia Allenby, Sameek Roychowdhury. Genomic characterization of recurrent small cell lung cancer through research autopsy reveals clonal diversity and candidate driver of chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 748.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Tumor Suppressors RB1 and CDKN2a Cooperatively Regulate Cell-Cycle Progression and Differentiation During Cardiomyocyte Development and Repair.

Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 (retinoblastoma) and CDKN2a (cyclin-dependent kinase inhibitor 2a) are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear. We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes, and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle reentry. Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes revealed that RB1 and CDKN2a are upregulated at the onset of cardiac precursor specification, simultaneously with GATA4 (GATA-binding protein 4) homeobox genes PBX1 (pre-B-cell leukemia transcription factor 1) and MEIS1 (myeloid ecotropic viral integration site 1 homolog), and remain so until terminal cardiomyocyte differentiation. In both GATA4+ hPSC cardiac precursors and postmitotic hPSC-cardiomyocytes, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the cardiac precursors stage, but not in terminally differentiated hPSC-cardiomyocytes, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in postmitotic hPSC-cardiomyocytes can be induced with transient, dual RB1, and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4+ cells correlates with the degree of scar size reduction and endogenous cardiomyocyte mitosis, particularly in response to combined transendocardial injection of adult human hMSCs (bone marrow-derived mesenchymal stromal cells) and cKit+ cardiac cells. Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4+ cells and cardiomyocytes mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications.

Flax seed oil reduced tumor growth, modulated immune responses and decreased HPV E6 and E7 oncoprotein expression in a murine model of ectopic cervical cancer

Cervical cancer is the second leading cause of cancer death in women in India. Previously, we have reported that alpha linolenic acid (ALA), induced apoptosis in cervical cancer cell lines and reduced expression of E6 and E7 oncoproteins with simultaneous decrease in Cox2/VEGF/MAP kinase proteins. Here, we investigated the tumor retardation potential of flax oil (FO), rich in ALA, in mouse papilloma model. Flax oil significantly reduced tumor volume and weight in mice compared to the Tumor control (TC) group. Interestingly, compared to cisplatin (Cis) alone, there was slightly enhanced decrease in tumor weight when FO was given together with Cis (Cis + FO). A marked increase in plasma antioxidant levels in mice, and increase in lipid peroxidation in tumors with simultaneous decrease in liver tissues was observed in Cis + FO group compared to either TC or Cis groups. FO and Cis + FO significantly modulated immune response in mice by increasing CD8α and IFNγ and decreasing IL-4 expression. Interestingly, when given together with cisplatin, flax oil reduced HPV E6 and E7 oncoprotein expression with concomitant increase in the relative mRNA expression of tumor suppressor genes p53 and Rb. Thus, flax oil could be explored for its therapeutic potential in cervical cancer.

MEG3: an Oncogenic Long Non-coding RNA in Different Cancers.

Long noncoding RNAs (lncRNAs) have recently considered as central regulators in diverse biological processes and emerged as vital players controlling tumorigenesis. Several lncRNAs can be classified into oncogenes and tumor suppressor genes depending on their function in cancer. A maternally expressed gene 3 (MEG3) gene transcripts a 1.6kb lncRNA whose act as an antitumorcomponent in different cancer cells, such as breast, liver, glioma, colorectal, cervical, gastric, lung, ovarian and osteosarcoma cancer cells. The present review highlights biological function of MEG3 to repress tumor through regulating the major tumor suppressor genes p53 and Rb, inhibiting angiogenesis-related factor, or controlling miRNAs. On the other hand, previous studies have also suggested that MEG3 mediates epithelial-mesenchymal transition (EMT). However, deregulation of MEG3 is associated with the development and progression of cancer, suggesting that MEG3 may function as a potential biomarker and therapeutic target for human cancers.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract P5-04-26: Identification of preclinical mechanisms driving acquired resistance to endocrine therapy in estrogen-receptor positive (ER+) breast cancer cells

Abstract Estrogen Receptor positive (ER+) breast cancer accounts for the majority of breast cancer cases and standard of care for these tumors is treatment with endocrine therapy, including the blockade of estrogen production (i.e. aromatase inhibitors; AIs) as well as the use of antagonists of ER function, i.e. selective estrogen receptor modulators (SERMs, i.e. tamoxifen) and selective estrogen receptor degraders (SERDs, i.e. fulvestrant). Despite the initial dependency of ER+ breast tumors on estrogen and ER for their survival and proliferation, treatment in the metastatic setting invariably leads to therapeutic resistance. While mechanisms of resistance to AIs include mutations in the estrogen receptor gene ESR1, less is known about mechanisms of resistance to SERMs and SERDs, thus it is essential to further investigate the latter, in order to successfully treat relapsed patients. To pre-clinically model cell-autonomous acquired resistance to these agents, we used T47D, an ER+ and p53- estrogen-responsive cell line treated with increasing concentrations of the SERM/SERD hybrid (SSH) ER-targeting agent GDC-0810 over the period of several months during which individual clones with acquired resistance to GDC-0810 were selected. GDC-0810-resistant clones were cross-resistant to other endocrine agents, including SERMs (tamoxifen) and SERDs (fulvestrant), consistent with general loss of dependency on ER. Surprisingly, the cells also lost sensitivity to palbociclib, the latter likely linked to their loss of one copy of the retinoblastoma (Rb) tumor suppressor gene. Comprehensive genetic and phenotypic characterization of the resistant clones relative to the parental cells revealed multiple mutations and deletions in DNA repair and cell cycle genes, and associated defects in DNA repair and cell cycle checkpoints. Cell cycle, proteomic, and mRNA expression analysis of parental versus resistant clones at baseline and upon DNA damage, identified a distinct cell cycle profile in the GDC-0810-resistant clones, characterized by accumulation of cells in the mitotic phase. A broad chemical screen identified pharmacologic inhibitors of cell cycle regulators and chemotherapeutic drug classes that preferentially target the ER-independent, GDC-0810 resistant clones compared to the parental cells. Our work provides novel insights into mechanisms and biomarkers of acquired resistant to estrogen therapies in ER+ breast cancer and reveals the acquisition of actionable dependencies that may potentially be exploited in resistant tumors. Furthermore, our studies provide rationale for testing specific chemotherapy regimens upon endocrine resistance accompanied by cell cycle and DNA repair checkpoint dysfunction in ER+ breast cancer. Citation Format: Ong C, Daemen A, Merrick K, O'Brien T, Friedman L, Hatzivassiliou G. Identification of preclinical mechanisms driving acquired resistance to endocrine therapy in estrogen-receptor positive (ER+) breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-26.

Compound Genomic Alterations of TP53, PTEN, and RB1 Tumor Suppressors in Localized and Metastatic Prostate Cancer

BackgroundTP53, PTEN, and RB1 tumor suppressor genes (TSGs) are recurrently altered in treatment-resistant prostate cancer. Cooperative loss of two or more TSGs may drive more aggressive disease. ObjectiveTo determine clinical outcomes of single and compound TSG alterations across the spectrum of prostate cancer. Design, setting, and participantsMassively parallel targeted sequencing using castration-sensitive prostate cancer (CSPC; localized [L] and metastatic [M1]) and castration-resistant prostate cancer (CRPC) specimens (n=285). TSG altered (TSG-alt) was any copy number loss or deleterious mutation of one or more TSGs (TP53, PTEN, and RB1). Outcome measurements and statistical analysisFor L-CSPC, event-free survival (EFS) and time to CRPC were estimated. For M1-CSPC and M1-CRPC, overall survival (OS) was estimated. Cox regression models assessed the association between cumulative TSG hits (zero hits vs one hit vs two to three hits) and outcomes with multivariable analyses adjusted for clinicopathological factors. Results and limitationsTSG variants increased with advanced disease (L-CSPC: 39%; M1-CSPC: 63%, M1-CRPC: 92%). TSG-alt L-CSPC had shorter EFS (median 2.6yr, hazard ratio [HR] 1.95, 95% confidence interval [CI] 1.22–3.13) and time to CRPC (median 9.5mo, HR 3.36, 95% CI 1.01–11.16). Cumulative gene hits led to an incremental risk of relapse (EFS: one gene, HR 1.69, 95% CI 0.99–2.87; two to three genes, HR 2.70, 95% CI 1.43–5.08; both versus zero genes, p=0.004). There was evidence of inferior OS with increasing TSG hits in the metastatic cohorts. Only four (8%) patients in the M1-CRPC cohort were TSG-neg, one of whom died after 5.2yr. Multivariable analyses adjusting for mutational and copy number burden did not demonstrate a significant independent association of increasing gene hits and poorer outcomes. ConclusionsDeleterious TSG variants are associated with an increased risk of relapse (L) and death (M1) in CSPC. Poorer outcomes are seen with compound gene hits in both early and advanced disease, and this may in part reflect increasing global genomic instability. Patient summaryMen with prostate tumors with compound tumor suppressor gene mutations have poorer outcomes. These findings help identify patients with aggressive features who may benefit from intensified treatment.

Generation of heterozygous and homozygous hESC H9 sublines carrying inactivating mutations in RB1

Inactivation of the tumor suppressor gene RB1 is causal for development of retinoblastoma, a tumor of the neural retina arising in children under the age of five. In addition, secondary RB1 mutations are found in many other tumor types. To investigate retinoblastoma formation in vitro, stem cells with inactivated RB1 can be differentiated into neural retina. To enable such studies, two sublines of hESC line H9 carrying mutations in RB1 exon 3 in heterozygous or homozygous state were generated and characterized. Homozygous mutation led to loss of RB1 protein expression.Resource tableUnlabelled TableUnique stem cell lines identifierWAe009-A-12WAe009-A-13Alternative names of stem cell linesC7 (homozygous deletion, WAe009-A-12)G12LS (heterozygous deletion, WAe009-A-13)InstitutionUniversity Hospital Essen, University Duisburg-Essen, Essen, GermanyContact information of distributorDr. Laura Steenpass, laura.steenpass@uni-due.deDr. Deniz Kanber, deniz.kanber@uni-due.deType of cell linesESCOriginHumanCell SourceHuman ESC line H9 purchased from WiCellClonalityClonalMethod of reprogrammingN/AMultiline rationaleclones selected for deletion in heterozygous and homozygous stateGene modificationYESType of modificationIndels in RB1 exon 3Associated diseaseRetinoblastomaGene/locusRB1, chromosome 13q14.2Method of modificationCRISPR/Cas9 nucleaseName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateC7 12.05.2018G12LS 12.05.2018Cell line repository/bankN/AEthical approvalApproval obtained from the Robert-Koch Institute, Berlin, Germany (Az.3.04.02/0101) and from the local Ethical Review Board University Duisburg-Essen (16–7215-BO)

Biallelic tumour suppressor loss and DNA repair defects in de novo small-cell prostate carcinoma.

Small-cell prostate carcinoma (SCPC) is an aggressive malignancy that is managed similarly to small-cell lung cancer. SCPC can evolve from prostate adenocarcinoma in response to androgen deprivation therapy, but, in rare cases, is present at initial cancer diagnosis. The molecular aetiology of de novo SCPC is incompletely understood, owing to the scarcity of tumour tissue and the short life-expectancy of patients. Through a retrospective search of our regional oncology pharmacy database, we identified 18 patients diagnosed with de novo SCPC between 2004 and 2017. Ten patients had pure SCPC pathology, and the remainder had some admixed adenocarcinoma foci, but all were treated with first-line platinum-based chemotherapy. The median overall survival was 28 months. We performed targeted DNA sequencing, whole exome sequencing and mRNA profiling on formalin-fixed paraffin-embedded archival tumour tissue. We observed frequent biallelic deletion and/or mutation of the tumour suppressor genes TP53, RB1, and PTEN, similarly to what was found in treatment-related SCPC. Indeed, at the RNA level, pure de novo SCPC closely resembled treatment-related SCPC. However, five patients had biallelic loss of DNA repair genes, including BRCA1, BRCA2, ATM, and MSH2/6, potentially underlying the high genomic instability of this rare disease variant. Two patients with pure de novo SCPC harboured ETS gene rearrangements involving androgen-driven promoters, consistent with the evolution of de novo SCPC from an androgen-driven ancestor. Overall, our results reveal a highly aggressive molecular landscape that underlies this unusual pathological variant, and suggest opportunities for targeted therapy strategies in a disease with few treatment options. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical outcomes with cumulative tumor suppressor gene (TSG) alterations in castration sensitive (CSPC) and resistant (CRPC) prostate cancer.

5055Background: Alterations (alt) in TP53, PTEN and RB1 TSGs have been identified in some prostate cancers. Preclinical data suggest that cooperative loss of 2 or more TSGs drives development of mo...

Clinical and molecular features of treatment-related neuroendocrine prostate cancer.

Treatment-related neuroendocrine prostate cancer is a lethal form of prostate cancer that emerges in the later stages of castration-resistant prostate cancer treatment. Treatment-related neuroendocrine prostate cancer transdifferentiates from adenocarcinoma as an adaptive response to androgen receptor pathway inhibition. The incidence of treatment-related neuroendocrine prostate cancer has been rising due to the increasing use of potent androgen receptor pathway inhibitors. Typically, treatment-related neuroendocrine prostate cancer is characterized by either low or absent androgen receptor expression, small cell carcinoma morphology and expression of neuroendocrine markers. Clinically, it manifests with predominantly visceral or lytic bone metastases, bulky tumor masses, low prostate-specific antigen levels or a short response duration to androgen deprivation therapy. Furthermore, although the tumor initially responds to platinum-based chemotherapy, the duration of the response is short. Based on the poor prognosis, it is imperative to identify novel molecular targets for treatment-related neuroendocrine prostate cancer. Recent advances in genomic and molecular research, supported by novel invivo models, have identified some of the key molecular characteristics of treatment-related neuroendocrine prostate cancer. The gain of MYCN and AURKA oncogenes, along with the loss of tumor suppressor genes TP53 and RB1 are key genomic alterations associated with treatment-related neuroendocrine prostate cancer. Androgen receptor repressed genes, such as BRN2 and PEG10, are also necessary for treatment-related neuroendocrine prostate cancer. These genetic changes converge on pathways upregulating genes, such as SOX2 and EZH2, that facilitate lineage plasticity and neuroendocrine differentiation. As a result, on potent androgen receptor pathway inhibition, castration-resistant prostate cancer transdifferentiates to treatment-related neuroendocrine prostate cancer in a clonally divergent manner. Further understanding of the disease biology is required to develop novel drugs and biomarkers that would help treat this aggressive prostate cancer variant.

Next-Generation Sequencing to Detect Deletion of RB1 and ERBB4 Genes in Chromophobe Renal Cell Carcinoma: A Potential Role in Distinguishing Chromophobe Renal Cell Carcinoma from Renal Oncocytoma

Overlapping morphologic, immunohistochemical, and ultrastructural features make it difficult to diagnose chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Because ChRCC is a malignant tumor, whereas RO is a tumor with benign behavior, it is important to distinguish these two entities. We aimed to identify genetic markers that distinguish ChRCC from RO by using next-generation sequencing (NGS). NGS for hotspot mutations or gene copy number changes was performed on 12 renal neoplasms, including seven ChRCC and five RO cases. Matched normal tissues from the same patients were used to exclude germline variants. Rare hotspot mutations were found in cancer-critical genes (TP53 and PIK3CA) in ChRCC but not RO. The NGS gene copy number analysis revealed multiple abnormalities. The two most common deletions were tumor-suppressor genes RB1 and ERBB4 in ChRCC but not RO. Fluorescence in situ hybridization was performed on 65 cases (ChRCC, n=33; RO, n=32) to verify hemizygous deletion of RB1 (17/33, 52%) or ERBB4 (11/33, 33%) in ChRCC, but not in RO (0/32, 0%). In total, ChRCCs (23/33, 70%) carry either a hemizygous deletion of RB1 or ERBB4. The combined use of RB1 and ERBB4 fluorescence in situ hybridization to detect deletion of these genes may offer a highly sensitive and specific assay to distinguish ChRCC from RO.

MiR-93-5p up-regulation is involved in non-small cell lung cancer cells proliferation and migration and poor prognosis

Background/aimMicroRNA-93-5p (miR-93-5p) dysregulation has been reported in many types of human cancer. However, the collective effect of miR-93-5p in both lung adenocarcinoma and squamous cell carcinoma and the mechanism underlying miR-93-5p involvement in non-small cell lung cancer cells (NSCLC) is unknown. Herein, our purpose was to reveal the role and explain this mechanism, with the goal of contributing to the development of new diagnostic biomarkers and individualized therapeutic targets. Materials and methodsWe examined miR-93-5p expression in NSCLC specimens (including lung adenocarcinoma and squamous cell carcinoma) by qPCR. The effects of miR-93-5p inhibitor on proliferation, migration, and invasion of NSCLC cells were determined by MTT assay, colony formation assays, apoptosis assay, cell cycle assay and transwell chamber assays, respectively. The molecular mechanisms underlying these effects were investigated via dual luciferase reporter assay and western blotting. ResultsMiR-93-5p expression levels were significantly correlated with NSCLC patients overall survival rate. Cell proliferation, migration, and invasion were significantly inhibited by miR-93-5p down-regulation. Dual luciferase reporter assay demonstrated that miR-93-5p directly bound with the 3′-untranslated region of the tumor suppressor gene PTEN and RB1. ConclusionMiR-93-5p is up-regulated in NSCLC and plays an oncogenic role by inhibiting PTEN and RB1, suggesting miR-93-5p may be a novel prognostic indicator and a therapeutic target in NSCLC.