Loss Of Tumor Suppressor Genes Research Articles

CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.

Abstract I06: Clonal evolution and progression of pancreatic cancer precursor lesions

Abstract Pancreatic cancer arises from noninvasive precursor lesions that are curable if detected and treated early enough. These precursor lesions include microscopic pancreatic intraepithelial neoplasia (PanIN) and cystic intraductal papillary mucinous neoplasm (IPMN). Interrogation of these lesions can provide critical insights into the earliest stages of pancreatic tumorigenesis and provide a rational foundation for early detection approaches. We have performed comprehensive genomic analyses of PanINs, IPMNs, and associated invasive carcinomas, highlighting several unique features of initiation and progression of pancreatic neoplasms. We show that early-stage precursor lesions contain multiple independent clones, each with distinct mutations, indicating their polyclonal origin. In addition, we identify convergent evolution of tumor suppressor gene loss at later stages of tumorigenesis, as well as subclonal complexity of driver gene mutations and mutations in specific genes limited to invasive cancers. Taken together, these data challenge the traditional monoclonal origin of pancreatic tumors, highlight distinct evolutionary features of precancerous lesions, and transform our understanding of the clonal evolution of pancreatic neoplasia. Citation Format: Laura D. Wood. Clonal evolution and progression of pancreatic cancer precursor lesions [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I06.

888P - Circulating tumour cell (CTC) genomic landscape in neuroendocrine prostate cancer (NEPC) by single cell copy number analysis

BackgroundCell-to-cell heterogeneity is a major driver of cancer evolution, progression, and drug resistance. NEPC is an aggressive variant of prostate cancer that may develop from pre-existing prostate adenocarcinoma as a mechanism of treatment resistance. Loss of tumour suppressors RB1, TP53, PTEN are frequent in NEPC but also present in a subset of prostate adenocarcinoma. Through single-cell CTC genomics of men with castration resistant adenocarcinoma (CRPC-Adeno) and NEPC, we aimed to characterize the clonal heterogeneity that underlies these resistance scenarios. MethodsCTCs were assessed using Epic Sciences platform for morphology and expression of adenocarcinoma (AR) and NEPC (DLL3) markers. Low pass whole genome sequencing was performed on individual cells/clusters. CTC copy number alterations (CNAs) were compared with whole exome sequencing data of patient-matched tumor biopsies. ResultsA total of 182 CTCs from 13 pts (5 CRPC, 8 NEPC) with matched biopsies were analyzed for 87 commonly altered prostate cancer genes. 11 pts (7 NEPC and 4 CRPC-Adeno, 177 CTCs) had at least 3 CTCs with one or more gene CNA. Concordance of these alterations with matched biopsy was 90.9%. All 7 NEPC pts had at least one tumor RB1,TP53, or PTEN loss; 71.4% (5/7) pts had loss of > two tumor suppressors, of which 40.6% (43/106) of CTCs harbored alterations in the same cell (vs. different cells). In contrast, only 2/4 CRPC-Adeno pts had one RB1/TP53/PTEN loss and loss of>2 occurred in none. Combined loss of RB1, TP53 and/or PTEN in CTCs was associated with lower AR expression and higher DLL3 expression in CTCs. Other CNAs showed similar trends, with higher concordance of alterations across CTCs in individuals with NEPC. ConclusionsProstate cancer associated CNA profiles of single cell CTCs was consistent with matched metastatic tumor biopsies in our cohort. Combined loss of tumour suppressor genes is more frequent in NEPC than in CRPC-Adeno and more often co-occurs in individual cells. Overall there is higher concordance, or less diversity (heterogeneity), across CTCs in NEPC. Analysis of additional patients including serial CTC assessment is ongoing. Legal entity responsible for the studyThe authors. FundingProstate Cancer Foundation, National Cancer Institute SPORE P50-CA211024, and Epic Sciences. DisclosureV. Conteduca: Advisory / Consultancy, Travel / Accommodation / Expenses: Bayer; Travel / Accommodation / Expenses: Janssen; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Astellas; Travel / Accommodation / Expenses: Sanofi. Y. Wang: Full / Part-time employment: Epic Sciences. L. Fernandez: Full / Part-time employment: Epic Sciences. M. Kearney: Full / Part-time employment: Epic Sciences. J. Lee: Full / Part-time employment: Epic Sciences. R. Jiles: Full / Part-time employment: Epic Sciences. A. Rodriguez: Full / Part-time employment: Epic Sciences. R. Dittamore: Full / Part-time employment, Officer / Board of Directors: Epic Sciences. H. Beltran: Advisory / Consultancy: Janssen; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Astellas. All other authors have declared no conflicts of interest.

Asbestos: A Silent Potent Killer

Asbestos is a general name given to a group of carcinogenic and genotoxic fibrous silicate minerals that have been of commercial values over the years. They include chrysolite, amosite, crocidolite, tremolite, actinolite and anthrophylite. Because of their special properties such as flexibility, tensile length, heat stability and resistivity to chemical, electrical, thermal and biological attacks, asbestos fibres have been used as components of several construction, building and automotive materials. This materials when disturbed, during home redecoration, demolition, renovation and automobiles repairs, release asbestos fibres into the atmosphere leading to both residential and occupational exposure. Measured concentration of fibres has been found to be higher in urban areas (especially those in close proximity with asbestos industries which may include mining and demolition sites) than in rural areas. Several studies have linked asbestos exposure to several serious health challenges such as lung cancer, mesothelioma, asbestosis, and other respiratory diseases. Although the main mechanisms through which asbestos can lead to diseases are not fully understood, three hypotheses have been made. These include alterations at the chromosomal level, activation of oncogenes, loss of tumour suppressor genes, alterations in cellular signal transduction pathways, generation of reactive oxygen and nitrogen species, and direct mechanical damage to cells from asbestos fibres. This article reviews briefly the ways by which the general public (occupational and non-occupational) can be exposed to asbestos and its most recent pathogenic mechanisms leading to malignant and non-malignant disease conditions.

MiR-496 promotes migration and epithelial-mesenchymal transition by targeting RASSF6 in colorectal cancer.

Aberrant loss of tumor-suppressor genes plays a crucial role in tumorigenesis and development of colorectal cancer (CRC). Extensive studies have reported tha hypermethylation of Ras association domain family member 6 (RASSF6) is common in various solid tumors. Another important mode of epigenetic regulation, microRNA (miRNA) regulation of RASSF6, is far from clear. The aim of the present work was to screen out novel miRNA regulating RASSF6, and to explore its underlying mechanism in CRC. With the use of bioinformatics, clinical sample data, and luciferase binding assay, we determined that microRNA-496 (miR-496) could be a novel oncomiR that directly binds to RASSF6. Next, a series of miR-496 mimics or inhibitor, or RASSF6 small interfering RNA (siRNA) introduced into CRC cells were applied to examine the effect of miR-496 on CRC cell viability, migration, and epithelial-mesenchymal transition (EMT). The results demonstrated that miR-496/RASSF6 could promote cell migration and EMT via Wnt signaling activation, but had no effect on cell viability. Our results confirmed that the miR-496/RASSF6 axis is involved in Wnt pathway-mediated tumor metastasis, highlighting its potential as a therapeutic target for CRC.

Abstract 4665: Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis

Abstract Oral cavity squamous cell carcinomas (OCSCCs) account for over 450,000 cases per year worldwide, present a high metastatic potential, and have a survival rate ≤ 50%. There is a high incidence of OCSCC, but the mechanisms of carcinogenesis remain unclear. Therefore, the aim of this research was to delineate the mutational landscape in developing tumors and OCSCCs by combining a lineage tracing approach and a mouse model of tongue carcinogenesis, previously established by our group. We used K14-CreERTAM; Rosa26LacZ mice, which allowed the long term basal stem cells of the epithelium to be permanently marked at the time of a two-day tamoxifen treatment. We then induced carcinogenesis by treating mice for 10 weeks with the carcinogen 4-nitroquinoline 1-oxide (4-NQO), which recapitulates key phenotypic and molecular features observed in human OCSCC. We performed Laser Capture Microdissection (LCM) to isolate and retrieve high-quality genomic DNA exclusively from each clone of permanently labeled, long-lived LacZ+ stem cells; each clone of cells arises from one LacZ+ stem cell. We then performed exome sequencing of 3 to 5 mice per group, sacrificed immediately after the 10-week treatment with 4-NQO (pre-neoplastic), and mice sacrificed >19 weeks after the end of 4-NQO treatment (SCC). As a reference genome for alignment and germline mutation exclusion, we included 3 mice of the same genetic background that did not receive 4-NQO. After 10 weeks of 4-NQO treatment the tongues were hyperplastic, with occasional necrosis and mild atypia, whereas all mice evaluated >19 weeks post 4-NQO treatment showed tongue SCCs. The total numbers of somatic mutations and LOH (loss of heterozygosity) identified using the mutation calling tool VarScan were 75% (4615±2170 vs 1148±76) higher in SCC compared to pre-neoplastic tongue samples. Among the mutations predicted as high and moderate impact, the levels of SNVs (single nucleotide variants) vs. indels were similar between SCCs and pre-neoplastic samples, 98.4% and 96.5%, respectively. By detailed analysis of the mutation types we showed that the pre-neoplastic tissues exhibited a much higher proportion of LOH events than the SCCs, 34.4% and 7.6%, respectively. Some of the genes affected by LOH in pre-neoplastic tissues were identified as tumor suppressors. Our findings suggest that a predominance of LOH in pre-neoplastic tongue tissue may result in loss of tumor suppressor genes, potentially defining the early steps of carcinogenesis in tongue SCCs. In conclusion, following the fate of long term tongue epithelial stem cells has allowed us to identify genomic perturbations occurring in the tongues of 4-NQO-treated mice and to find potential early biomarkers for OCSCC. Grant ID: R01CA205258; Funding Agency: NIH/NCI. Citation Format: Marta Melis, Tuo Zhang, Theresa Scognamiglio, Lorraine J. Gudas. Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis [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 4665.

Abstract 3913: PDK1 drives susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition

Abstract Head and neck squamous cell carcinoma (HNSCC) is primarily driven by the loss of tumor suppressor genes. Although approaches to target driver oncogenes have been clinically successful, targeting tumor suppressors has been challenging. To address this translational gap, we previously demonstrated that HNSCC cell lines with loss of function (LOF) NOTCH1 mutations (NOTCH1MUT n=14) were more sensitive to 6 PI3K pathway inhibitors (Omipalisib, Alpelisib, Bimiralisib, Dactolisib, Copanlisib, Apitolisib) than NOTCH1WT cells (n=45). In contrast to PIK3CAMUT cell lines, NOTCH1MUT lines underwent significant apoptosis after PI3K/mTOR pathway inhibition. NOTCH1MUT lines also showed significantly reduced clonogenic growth and significant tumor growth inhibition in both oral orthotopic and subcutaneous xenograft models. We employed CRISPR-Cas9 gene editing technology to knock out NOTCH1 gene in two NOTCH1WT lines, PJ34 and UMSCC49. After Omipalisib treatment, NOTCH1-KO lines showed decreased cell viability compared to parental lines. The NOTCH1 knock out lines showed significantly increased apoptosis after Omipalisib treatment compared to parental lines (PJ34 KO: 1.62-fld, P<0.003; UM49 KO: 1.37-fld, P=0.002). Both NOTCH1 KO lines also showed increased cleaved PARP and cleaved caspase 3 by western blot analysis. With NOTCH1 knock out lines, treatment significantly decreased number of colonies (PJ34 KO: 1.35-fld, P<0.005; UM49 KO: 1.62-fld, P<0.0001). As no canonical pathways account for the underlying mechanism of sensitivity, we measured 301 proteins by reverse phase protein array (RPPA) in NOTCH1MUT and NOTCH1WT lines after Omipalisib treatment. Several proteins were differentially regulated in NOTCH1MUT cells compared with NOTCH1WT lines including PDK1, FoxM1 and p-ERK. We then investigated PDK1 because it is activated by PI3K, and can regulate FOXM1 and ERK which were also inhibited more robustly in NOTCH1MUT HNSCC. To test the role of PDK1 in PI3K/mTOR inhibition, we over expressed PDK1 in NOTCH1MUT cells and it successfully led to decreased apoptosis after Omipalisib treatment as compared to the parental lines. The combination of AKT and PDK1 inhibition led to decreased cell viability in all four NOTCH1WT lines tested as compared to single agents. The combination also led to significantly increased apoptosis in all NOTCH1WT cell lines tested as demonstrated by cleaved PARP and Caspase 3 levels by western blot analysis. Our research is the first to establish a therapeutic vulnerability of NOTCH1MUT HNSCC to any class of drugs and may inform the development of the first biomarker-driven targeted therapy for HNSCC. Also, we may uncover combination therapies and define a heretofore-unknown mechanism of PDK1 regulation. Because NOTCH1 LOF mutations are common in other squamous carcinomas, including skin, esophagus and lung, our findings will likely have implications beyond HNSCC. Citation Format: Vaishnavi Sambandam, Li Shen, Shaohua Peng, Tuhina Mazumdar, Curtis Pickering, Jeffrey Myers, Jing Wang, Mitchell Frederick, Faye Johnson. PDK1 drives susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition [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 3913.

Abstract 425: Development and validation of liquid biopsy assay for copy number loss in cancers

Abstract Introduction: Copy number loss (CNL) of tumor suppressor genes, such as PTEN, RB1 and BRCA1/2, are common and key characteristics of many cancers. However, non-invasive detection of CNL has represented a strong demand and practical challenge in the clinic. Here we report a high-sensitive liquid biopsy NGS assay coupled with a proprietary bioinformatics pipeline to profile and detect CNL using cell-free circulating DNA (cfDNA). Method: We developed PrediSeq, a proprietary liquid biopsy NGS assay to detect CNL, in addition to SNV, indels, rearrangement, and copy number gain. We leveraged unique molecular identifier (UMI) and proprietary bioinformatics algorithm to suppress background noise caused by PCR pre-amplification and sequencing steps. In-house developed bioinformatics pipeline, DeepSea, is used for ultra-sensitive detection of CNL and other genomic alterations. Reference materials, matched tissue and plasma samples were used for assay validation. Results: PrediSeq CNL assay was validated using serially diluted reference materials with known copy number status. The method was evaluated with the orthogonal whole-genome sequencing using a set of clinical plasma samples with matched tissues. About 82% of CNL calls (ATM, BRCA1, BRCA2, CDKN2A, RB1) in were confirmed between plasma PrediSeq and tissue WGS. In alignment with literature, CNL of ATM, CDKN2A, BRCA1, BRCA2, was observed in plasma samples of a cohort of Her2+, HR+, and triple-negative breast cancer patients. Similarly, we observed the dynamic changes of plasma-based CNL of BRCA2, PTEN, RB1, CDKN2A, ATM genes in patients of metastatic castration-resistant prostate cancer (mCRPC) and metastatic hormone sensitive prostate cancer (mHSPC). Conclusion: PrediSeq CNL assay has been successfully developed and validated to detect copy number loss in circulation, providing valuable molecular biomarkers to guide patient selection and treatment monitoring in precision oncology and clinical drug development. Citation Format: Simo Zhang, Feng Xie, Amy Chang, Zhixin Zhao, Carlos Montesinos, Xiaohong Wang, Kemin Zhou, Shidong Jia, Jianjun Yu, Pan Du. Development and validation of liquid biopsy assay for copy number loss in cancers [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 425.

PDK1 Mediates NOTCH1-Mutated Head and Neck Squamous Carcinoma Vulnerability to Therapeutic PI3K/mTOR Inhibition.

Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression. PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1 MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1 MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1 WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1 MUT but not NOTCH1 WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1 MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1 WT HNSCC but had little effect as single agents. Our findings suggest that NOTCH1 MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1 WT HNSCC to PI3K/mTOR pathway inhibitors.

PTEN loss in prostatic adenocarcinoma correlates with specific adverse histologic features (intraductal carcinoma, cribriform Gleason pattern 4 and stromogenic carcinoma)

The loss of PTEN tumor suppressor gene is one of the most common somatic genetic aberrations in prostate cancer (PCa) and is frequently associated with high-risk disease. Deletion or mutation of at least one PTEN allele has been reported to occur in 20% to 40% of localized PCa and up to 60% of metastases. The goal of this study was to determine if somatic alteration detected by PTEN immunohistochemical loss of expression is associated with specific histologic features. Two hundred sixty prostate core needle biopsies with PCa were assessed for PTEN loss using an analytically validated immunohistochemical assay. Blinded to PTEN status, each tumor was assessed for the Grade Group (GG) and the presence or absence of nine epithelial features. Presence of stromogenic PCa was also assessed and defined as grade 3 reactive tumor stroma as previously described: the presence of carcinoma associated stromal response with epithelial to stroma ratio of greater than 50% reactive stroma. Eight-eight (34%) cases exhibited PTEN loss while 172 (66%) had intact PTEN. PTEN loss was significantly (P < 0.05) associated with increasing GG, poorly formed glands (74% of total cases with loss vs 49% of intact), and three well-validated unfavorable pathological features: intraductal carcinoma of the prostate (IDC-P) (69% of total cases with loss vs 12% of intact), cribriform Gleason pattern 4 (38% of total cases with loss vs 10% of intact) and stromogenic PCa (23% of total cases with loss vs 6% of intact). IDC-P had the highest relative risk (4.993, 95% confidence interval, 3.451-7.223, P < 0.001) for PTEN loss. At least one of these three unfavorable pathological features were present in 67% of PCa exhibiting PTEN loss, while only 11% of PCa exhibited PTEN loss when none of these three unfavorable pathological features were present. PCa with PTEN loss demonstrates a strong correlation with known unfavorable histologic features, particularly IDC-P. This is the first study showing the association of PTEN loss with stromogenic PCa.

Copy number analysis to identify tumor suppressor genes associated with enzalutamide (Enza) resistance and poor prognosis in metastatic castration-resistant prostate cancer (mCRPC) patients.

5011 Background: Although enza prolongs life in mCRPC pts, the development of drug resistance and subsequent disease progression is nearly universal. Seeking to clarify molecular mechanisms that underlie enza resistance, we analyzed whole genome sequencing (WGS) and RNA sequencing (seq) of tumors obtained from patients with enza-naive or -resistant mCRPC. Methods: One hundred and one men with mCRPC who underwent image-guided biopsy and subsequent WGS were included (n = 64 with enza-naive and n = 37 with enza-resistant mCRPC). The differential copy number alteration (CNA) events enriched in enza-resistant vs. naïve samples were determined, and the prognostic significance of differential CNAs was assessed. RNA-seq data were evaluated to confirm that CNAs correlated with changes in gene expression of relevant loci and to identify potentially druggable targets selectively activated in tumors with specific CNAs. Results: Copy number loss was more common than gain in enza-resistant tumors. Specifically, we identified 123 protein-coding genes that were more commonly lost in enza-resistant samples—eight of which were previously described tumor suppressor genes. There was a strong concordance of copy number loss and reduced mRNA expression of these genes. We identified one gene from this list of eight genes whose copy number loss was associated with poor overall survival (median overall survival from date of CRPC was 19.1 months in tumors with gene loss vs. 42.0 months in intact tumors, hazard ratio 3.8 [1.46–9.8], log-rank p = 0.003). Finally, Master Regulator analysis determined that tumors with copy number loss of this poor prognosis gene had activation of several potentially targetable factors, including the kinases Akt and PLK1. Conclusions: Copy number loss of specific tumor suppressor genes is associated with enza resistance in mCRPC patients. Previously unappreciated molecular subsets of enza-resistant CRPC were identified, including one subset associated with poor clinical outcome.

Intrinsic apoptosis shapes the tumor spectrum linked to inactivation of the deubiquitinase BAP1.

Malignancies arising from mutation of tumor suppressors have unexplained tissue proclivity. For example, BAP1 encodes a widely expressed deubiquitinase for histone H2A, but germline mutations are predominantly associated with uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver, and pancreatic tissue but not in melanocytes and mesothelial cells. Ubiquitin ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing expression of the prosurvival genes Bcl2 and Mcl1. In contrast, BAP1 loss in melanocytes had little impact on expression of prosurvival genes, instead inducing Mitf Thus, BAP1 appears to modulate gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program.

PTEN-deficient prostate cancer is associated with an immunosuppressive tumor microenvironment mediated by increased expression of IDO1 and infiltrating FoxP3+ T regulatory cells.

Accumulating evidence shows that tumor cell-specific genomic changes can influence the cross talk between cancer cells and the surrounding tumor microenvironment (TME). Loss of the PTEN tumor suppressor gene is observed in 20% to 30% of prostate cancers (PCa) when first detected and the rate increases with PCa progression and advanced disease. Recent findings implicate a role for PTEN in cellular type I interferon response and immunosuppression in PCa. However, the way that PTEN inactivation alters antitumor immune response in PCa is poorly understood. To investigate the changes associated with PTEN loss and an immunosuppressive TME in PCa, we used CIBERSORT to estimate the relative abundance of 22 immune-cell types from 741 primary and 96 metastatic tumors. Our in silico findings were then validated by immunohistochemical analysis of immune cells and IDO1 and PDL1 checkpoint proteins in a cohort of 94 radical prostatectomy specimens. FoxP3+ T regulatory cells (Tregs) were significantly increased in PTEN-deficient PCa in all three public domain cohorts. Loss of PTEN in bone metastases was associated with lower CD8+ T-cell abundance, but in liver metastasis, FoxP3+ Tregs were present at higher levels. PTEN-deficient lymph node metastasis had a distinct profile, with high levels of CD8+ T cells. Moreover, we found that metastatic PCa presents higher abundance of FoxP3+ Treg when compared to primary lesions. Since PTEN-deficient tumors are likely to be immunosuppressed as a consequence of increased FoxP3+ Tregs, we then evaluated the localization and expression of IDO1, PDL1 immune checkpoints, and the corresponding density of FoxP3+ Treg and CD8+ T cells using our validation cohort (n = 94). We found that IDO1 protein expression and FoxP3+ Treg density were higher in neoplastic glands compared with benign adjacent tissue. Moreover, higher densities of FoxP3+ Treg cells in both stromal (P = 0.04) and tumor (P = 0.006) compartments were observed in PTEN-deficient tumors compared to tumors that retained PTEN activity. Similarly, IDO1 protein expression was significantly increased in the tumor glands of PTEN-deficient PCa (P < 0.0001). Spearman correlation analysis showed that IDO1 expression was significantly associated with FoxP3+ Treg and CD8+ T-cell density (P < 0.01). Our findings imply that PTEN deficiency is linked to an immunosuppressive state in PCa with distinct changes in the frequency of immune cell types in tumors from different metastatic sites. Our data suggest that determining PTEN status may also help guide the selection of patients for future immunotherapy trials in localized and metastatic PCa.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Genetic drivers of oncogenic pathways in molecular subgroups of peripheral T-cell lymphoma

AbstractPeripheral T-cell lymphoma (PTCL) is a group of complex clinicopathological entities, often associated with an aggressive clinical course. Angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specified (PTCL-NOS) are the 2 most frequent categories, accounting for >50% of PTCLs. Gene expression profiling (GEP) defined molecular signatures for AITL and delineated biological and prognostic subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21). Genomic copy number (CN) analysis and targeted sequencing of these molecular subgroups revealed unique CN abnormalities (CNAs) and oncogenic pathways, indicating distinct oncogenic evolution. PTCL-GATA3 exhibited greater genomic complexity that was characterized by frequent loss or mutation of tumor suppressor genes targeting the CDKN2A/B-TP53 axis and PTEN-PI3K pathways. Co-occurring gains/amplifications of STAT3 and MYC occurred in PTCL-GATA3. Several CNAs, in particular loss of CDKN2A, exhibited prognostic significance in PTCL-NOS as a single entity and in the PTCL-GATA3 subgroup. The PTCL-TBX21 subgroup had fewer CNAs, primarily targeting cytotoxic effector genes, and was enriched in mutations of genes regulating DNA methylation. CNAs affecting metabolic processes regulating RNA/protein degradation and T-cell receptor signaling were common in both subgroups. AITL showed lower genomic complexity compared with other PTCL entities, with frequent co-occurring gains of chromosome 5 (chr5) and chr21 that were significantly associated with IDH2R172 mutation. CN losses were enriched in genes regulating PI3K–AKT–mTOR signaling in cases without IDH2 mutation. Overall, we demonstrated that novel GEP-defined PTCL subgroups likely evolve by distinct genetic pathways and provided biological rationale for therapies that may be investigated in future clinical trials.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Colorectal Cancer and Metabolism.

Metabolic reprogramming is essential for the rapid proliferation of cancer cells and is thus recognized as a hallmark of cancer. In this review, we will discuss the etiologies and effects of metabolic reprogramming in colorectal cancer. Changes in cellular metabolism may precede the acquisition of driver mutations ultimately leading to colonocyte transformation. Oncogenic mutations and loss of tumor suppressor genes further reprogram CRC cells to upregulate glycolysis, glutaminolysis, one-carbon metabolism, and fatty acid synthesis. These metabolic changes are not uniform throughout tumors, as subpopulations of tumor cells may rely on different pathways to adapt to nutrient availability in the local tumor microenvironment. Finally, metabolic cross-communication between stromal cells, immune cells, and the gut microbiota enable CRC growth, invasion, and metastasis. Altered cellular metabolism occurs in CRC at multiple levels, including in the cells that make up the bulk of CRC tumors, cancer stem cells, the tumor microenvironment, and host-microbiome interactions. This knowledge may inform the development of improved screening and therapeutics for CRC.

Chromosome 3p Loss-Orchestrated VHL, HIF, and Epigenetic Deregulation in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cell carcinoma subtype, and metastatic ccRCC is associated with 5-year survival rates of 10% to 20%. Genetically, ccRCC originates from sequential losses of multiple tumor suppressor genes. Remarkably, chromosome 3p loss occurs in more than 90% of sporadic ccRCCs. This results in concurrent one-copy loss of four tumor suppressor genes that are also mutated individually at high frequency in ccRCC (ie, VHL, 80%; PBRM1, 29% to 46%; BAP1, 6% to 19%; and SETD2, 8% to 30%). Pathogenically, 3p loss probably represents the first genetic event that occurs in sporadic ccRCC and the second genetic event in VHL-mutated hereditary ccRCC. VHL constitutes the substrate recognition module of the VCB-Cul2 E3 ligase that degrades HIF1/2α, whereas PBRM1, BAP1, and SETD2 are epigenetic modulators that regulate gene transcription. Because 3p loss and VHL inactivation are nearly universal truncal events in ccRCC, the resulting HIF1/2 signaling overdrive and accompanied tumor hypervascularization probably underlie the therapeutic benefits observed with vascular endothelial growth factor receptor inhibitors, including sorafenib, sunitinib, pazopanib, axitinib, bevacizumab, cabozantinib, and lenvatinib. Furthermore, recent marked advances in ccRCC genomics, transcriptomics, proteomics, metabolomics, molecular mechanisms, mouse models, prognostic and predictive biomarkers, and clinical trials have rendered invaluable translational insights concerning precision kidney cancer therapeutics. With an armamentarium encompassing 13 drugs that exploit seven unique therapeutic mechanisms (ie, cytokines, vascular endothelial growth factor receptor, mTORC1, cMET/AXL, fibroblast growth factor receptor, programmed cell death-1 and programmed death-ligand 1, and cytotoxic T-cell lymphocyte associated-4) to treat metastatic renal cell carcinoma, one of the imminent clinical questions concerning care of patients with metastatic ccRCC is how a personalized treatment strategy, through rationally combining and sequencing different therapeutic modalities, can be formulated to offer the best clinical outcome for individual patients. Here, we attempt to integrate recent discoveries of immediate translational impacts and discuss future translational challenges and opportunities.

Genomic and Transcriptomic Characterization Links Cell Lines with Aggressive Head and Neck Cancers

SUMMARYCell lines are important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations in tumors could accelerate functional and therapeutic discoveries. We conducted integrated analyses of genomic and transcriptomic profiles of 15 human papillomavirus (HPV)-negative and 11 HPV-positive head and neck squamous cell carcinoma (HNSCC) lines to compare with 279 tumors from The Cancer Genome Atlas (TCGA). We identified recurrent amplifications on chromosomes 3q22–29, 5p15, 11q13/22, and 8p11 that drive increased expression of more than 100 genes in cell lines and tumors. These alterations, together with loss or mutations of tumor suppressor genes, converge on important signaling pathways, recapitulating the genomic landscape of aggressive HNSCCs. Among these, concurrent 3q26.3 amplification and TP53 mutation in most HPV(–) cell lines reflect tumors with worse survival. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with HNSCC lines and provide valuable models for future studies.

A dual-type L2 11–88 peptide from HPV types 16/18 induced strong immune responses and broad spectrum cross-reactive antibodies in mice

The fusion between human tumorigenic cells and normal human diploid fibroblasts results in non-tumorigenic hybrid cells, suggesting a dominant role for tumor suppressor genes in the generated hybrid cells. After long-term cultivation in vitro, tumorigenic segregants may arise. The loss of tumor suppressor genes on chromosome 11q13 has been postulated to be involved in the induction of the tumorigenic phenotype of human papillomavirus (HPV)18-positive cervical carcinoma cells and their derived tumorigenic hybrid cells after subcutaneous injection in immunocompromised mice. The aim of this study was the identification of novel cellular genes that may contribute to the suppression of the tumorigenic phenotype of non-tumorigenic hybrid cells in vivo. We used cDNA microarray technology to identify differentially expressed cellular genes in tumorigenic HPV18-positive hybrid and parental HeLa cells compared to non-tumorigenic HPV18-positive hybrid cells. We detected several as yet unknown cellular genes that play a role in cell differentiation, cell cycle progression, cell−cell communication, metastasis formation, angiogenesis, antigen presentation, and immune response. Apart from the known differentially expressed genes on 11q13 (e.g., phosphofurin acidic cluster sorting protein 1 (PACS1) and FOS ligand 1 (FOSL1 or Fra-1)), we detected novel differentially expressed cellular genes located within the tumor suppressor gene region (e.g., EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) and leucine rich repeat containing 32 (LRRC32) (also known as glycoprotein-A repetitions predominant (GARP)) that may have potential tumor suppressor functions in this model system of non-tumorigenic and tumorigenic HeLa x fibroblast hybrid cells.