Major Tumor Suppressor Gene Research Articles

Genomic architecture of lung cancers.

Lung cancer remains the most frequent cancer worldwide and the leading cause of cancer death in most countries. The molecular characteristics of lung tumors play an important role in clinical decisions, which ultimately affect patients' survival. This review aims to summarize the most recent genomic discoveries made on lung cancer. A relatively comprehensive molecular characterization has been achieved for the three major types of lung cancer: adenocarcinoma, squamous-cell carcinoma, and small-cell carcinoma. Little is still known about large-cell neuroendocrine carcinoma and carcinoid tumors. A major finding has been the nonnegligible inter and intratumor heterogeneity of lung cancer and their impact in the clinical management of this disease. The high load of mutations, the frequent inactivation of major tumor suppressor genes, and the huge heterogeneity of lung cancer tumors may complicate long-lasting therapeutic responses. The development of strategies for the early detection of lung cancer might translate into an increase of the number of surgical resectable tumors, and therefore contribute to improve the survival rate of these patients.

CD133 and BMI1 expressions and its prognostic role in primary glioblastoma

Glioblastoma is the most common malignant brain tumour, generated by bulk of malignant cancer stem cells, which express various stem cell factors like CD133, BMI1 and nestin. There are several studies which show the importance of CD133 in cancer, but the function and interaction with other major oncogenes and tumour suppressor genes is still not understood. This study aimed to analyse the expression of CD133 mRNA and its correlations with BMI1 protein expression and TP53 mutations in newly diagnosed glioblastoma patients and its role in prognosis. Overexpression of CD133 mRNA and BMI1 protein was found in 47.6 and 76.2% patients respectively and TP53 mutations was seen in 57.1% of patients in our study.There was no correlation among TP53 mutations and expressions of CD133 and BMI1. We found that high level of BMI1 expression was favourable for the patient survival (P=0.0075) and high CD133 mRNA expression was unfavourable for the patient survival (P=0.0226).CD133 mRNA and BMI1 protein expression could independently predict the glioblastoma patient survival in multivariate analysis. In conclusion, the overexpression of these stem cell markers is a common event in glioblastoma progression and could be used as potential prognostic markers.

Model systems to study the life cycle of human papillomaviruses and HPV-associated cancers.

The prevalent human papillomaviruses (HPVs) infect either cutaneous or mucosal epithelium. Active Infections lead to epithelial hyperprolifeation and are usually cleared in healthy individuals within a year. Persistent infections in the anogenital tracts by certain high-risk genotypes such as HPV-16, HPV-18 and closely related types, can progress to high grade dysplasias and carcinomas in women and men, including cervical, vulva, penile and anal cancers. A significant fraction of the head and neck cancers are also caused by HPV-16. The viral oncogenes responsible for neoplastic conversion are E6 and E7 that disrupt the pathways controlled by the two major tumor suppressor genes, p53 and members of pRB family. Because HPV cannot be propagated in conventional submerged monolayer cell cultures, organotypic epithelial raft cultures that generate a stratified and differentiated epithelium have been used to study the viral life cycle. This article describes several systems to examine aspects of the viral productive phase, along with the advantages and limitations. Animal model systems of HPV carcinogenesis are also briefly described.

The p53 Isoform Δ133p53β Promotes Cancer Stem Cell Potential

SummaryCancer stem cells (CSC) are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53β, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53β stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53β expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53β in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53β supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53β isoform.

Abstract LB-88: The role of PBRM1 as tumor suppressor gene in renal cell carcinomas

Abstract Objective: The polybromodomain containing gene PBRM1 is thought to be the second major tumor suppressor gene in clear cell renal cell carinomas (ccRCCs) next to the VHL gene. The PBRM1 protein BAF180 belongs to the SWI/SNF complex and provides functional specificity to this class of chromatin remodelers by binding to acetylated histones via its bromodomains and to nucleosomal DNA by its HMG domain. Our aim was to study the expression of PBRM1 in RCC in comparison to the mutational status of PBRM1 and VHL as well as the expression of VHL. Methods: To assess the contribution of somatic mutations for PBRM1 downregulation we resequenced 31 exons of 96 tumor tissues from consenting patients who underwent radical tumor nephrectomy. In addition, the 3 exons of VHL were resequenced as well. Expression of PBRM1 and VHL was measured in 96 cases of paired normal and tumor tissue samples of RCC patients by Q-PCR using TaqMan specific probes. By immunohistochemistry BAF180 could be detected using a rabbit anti-BAF180 antibody. Results: Specific truncating mutations in the PBRM1 gene were detected in 13 (13.5%) of 96 tumor samples (15.5% of 84 ccRCC cases). No mutations could be observed in 12 cases of papillary tumors or tumors of the chromophobe type. Mutations in the VHL gene could be detected in 28/84 (33.3%) of the ccRCC samples. A marked downregulation of PBRM1 expression (> 2fold) was measured in 41/81 ccRCC cases (50.6%) whereas in 3/81 ccRCC cases (3.7%) PBRM1 was upregulated (>2fold). In 37/81 cases (45.7%) no expression differences between normal and tumor tissue could be seen. However, all cases with mutations in the PBRM1 gene showed a clear decrease of PBRM1 mRNA expression. Interestingly, in papillary carcinomas PBRM1 was strongly upregulated in comparison to the normal tissue showing a completely different expression behaviour. In addition, there was a significant difference in PBRM1 expression depending on the site of the tumor within the kidney (p=0.001). PBRM1 codes for different major transcripts. In our experiments five different transcripts could be observed although two transcripts gave only faint bands in PCR. Concurrent expression of three major transcripts (var.1, 2, and 4) is seen in all matched normal and tumor samples analyzed so far. In 22/46 samples (47.8%) we detected a dominance of var.1 (NM_018165) in normal tissue when compared to the corresponding tumor sample, that preferentially expresses var.4 (NM_181042) that might alter the properties of the HMG domain. Conclusions: Understanding the contribution of PBRM1 changes, existing at the genomic (mutation) and/or mRNA (differential splicing) and/or protein (cellular localization) level, to clinical disease progression and outcome in kidney cancer is an absolute prerequisite for the better understanding of renal carcinogenesis and the putative development of new therapeutic approaches. Citation Format: Burkhard Jandrig, Odiljon Ikromov, Anica Hoegner, Johann J. Wendler, Martin Schostak, Hans Krause. The role of PBRM1 as tumor suppressor gene in renal cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-88. doi:10.1158/1538-7445.AM2014-LB-88

RASAL1 in thyroid cancer: promise from a new friend.

RASAL1 in thyroid cancer: promise from a new friend.

Reduced expression of PinX1 correlates to progressive features in patients with prostate cancer.

BackgroundPin2/TRF1 binding protein X1 (PinX1) has been identified as an endogenous telomerase inhibitor and a major haploinsufficient tumor suppressor gene. Increasing evidence suggests that reduced expression of PinX1 plays a key role in tumorigenesis. However, the PinX1 expression status and its correlation with the clinicopathological features in prostate cancer (PCa) have not been investigated.MethodsPinX1 mRNA and protein expression in PCa and adjacent normal prostate tissues were evaluated by real-time quantitative RT-PCR (qRT-PCR) and western blotting. The clinicopathological significance of PinX1 was investigated by immunohistochemistry (IHC) analysis on a PCa tissue microarray (TMA). The cut-off score for positive expression of PinX1 was determined by the receiver operating characteristic (ROC) analysis. The correlation between PinX1 expression and clinicopathological features of PCa was analyzed by Chi-square test.ResultsReduced expression of PinX1 mRNA and protein was observed in the majority of PCa, compared with their paired adjacent normal prostate tissues. When PinX1 positive expression percentage was determined to be above 60% (area under ROC curve = 0.833, P = 0.000), positive expression of PinX1 was observed in 100% (8/8) of normal prostate tissues and 32.5% (13/40) of PCa tissues by IHC. Reduced expression of PinX1 in patients was correlated with advanced clinical stage (χ2 = 10.230, p = 0.017), high Gleason score (χ2 = 4.019, p = 0.045), positive regional lymph node metastasis (χ2 = 10.852, p = 0.004) and distant metastasis (χ2 = 7.965, p = 0.005).ConclusionsOur findings suggest that reduced expression of PinX1 is correlates to progressive features in patients with PCa and may serve as a potential marker for diagnosis.

The Role of PinX1 in Growth Control of Breast Cancer Cells and Its Potential Molecular Mechanism by mRNA and lncRNA Expression Profiles Screening

As a major tumor suppressor gene, the role of PinX1 in breast cancer and its molecular mechanism remain unclear. In this study, overexpression of PinX1 was generated in 3 breast cancer cell lines, and knockdown of PinX1 was performed in a nontumorigenic breast cell line. The regulation of PinX1 on cell proliferation and cell cycle was observed. A microarray-based lncRNA and mRNA expression profile screening was also performed. We found a lower growth rate, G0/G1 phase arrest, and S phase inhibition in the PinX1 overexpressed breast cancer cells, while a higher growth rate, decreased G0/G1 phase, and increased S phase rate in the PinX1 knocked-down nontumorigenic breast cell. A total of 977 mRNAs and 631 lncRNAs were identified as differentially expressed transcripts between PinX1 overexpressed and control MCF-7 cells. Further analysis identified the involvement of these mRNAs in 52 cancer related pathways and various other biological processes. 11 enhancer-like lncRNAs and 25 lincRNAs with their adjacent mRNA pairs were identified as coregulated transcripts. Our results confirmed the role of PinX1 as a major tumor suppressor gene in breast cancer cell lines and provided information for further research on the molecular mechanisms of PinX1 in tumorigenesis.

Identification of RASAL1 as a Major Tumor Suppressor Gene in Thyroid Cancer

RAS-coupled MAPK and PI3K pathways play a fundamental role in thyroid tumorigenesis, and classical genetic alterations upregulating these pathways are well characterized. We hypothesized that gene abnormality of negative modulators of these signaling pathways might be an important alternative genetic background for thyroid cancer. By examining gene expression patterns of negative modulators of RAS signaling, we attempted to identify potential tumor suppressor genes. We then analyzed the methylation and mutation patterns of the identified gene in 101 thyroid tumors and tested its functions in vitro and in vivo to establish the tumor suppressor role in thyroid cancer. Among 13 negative modulators of the RAS pathway screened, RASAL1, encoding a RAS GTPase-activating protein, was frequently hypermethylated in thyroid cancers, which was coupled to its silencing in thyroid cancer cells. We also, for the first time, identified the presence of RASAL1 mutations, with a prevalence of 4.88% (n = 2 of 41) in follicular thyroid cancer (FTC) and 16.67% (n = 5 of 30) in anaplastic thyroid cancer (ATC). RASAL1 displayed MAPK- and PI3K-suppressing and thyroid tumor-suppressing activities, which were all impaired by the mutations. Hypermethylation and mutations of RASAL1 were mutually exclusive and collectively found in zero of 20 benign thyroid tumors, 3.22% (n = 1 of 31) of papillary thyroid cancers, 31.70% (n = 13 of 41) of FTCs, and 33.33% (n = 10 of 30) of ATCs. A rate of 20.83% (n = 5 of 24) of tumors carrying RASAL1 mutation or methylation at high levels (>50%) vs 44.16% (n = 34 of 77) of tumors carrying no RASAL1 mutation or methylation at low levels (< 50%) harbored any of the classical mutations (two-sided P = .02, Fisher exact test) in RAS, BRAF, PTEN, and PIK3CA genes in the MAPK and PI3K pathways, revealing a largely mutually exclusive relationship. We identified RASAL1 as a major tumor suppressor gene that is frequently inactivated by hypermethylation and mutations, providing a new alternative genetic background for thyroid cancer, particularly FTC and ATC.

MicroRNAome profiling in benign and malignant neurofibromatosis type 1-associated nerve sheath tumors: evidences of PTEN pathway alterations in early NF1 tumorigenesis

BackgroundNeurofibromatosis type 1 (NF1) is a common dominant tumor predisposition syndrome affecting 1 in 3,500 individuals. The hallmarks of NF1 are the development of peripheral nerve sheath tumors either benign (dermal and plexiform neurofibromas) or malignant (MPNSTs).ResultsTo comprehensively characterize the role of microRNAs in NF1 tumorigenesis, we analyzed 377 miRNAs expression in a large panel of dermal and plexiform neurofibromas, and MPNSTs. The most significantly upregulated miRNA in plexiform neurofibromas was miR-486-3p that targets the major tumor suppressor gene, PTEN. We confirmed PTEN downregulation at mRNA level. In plexiform neurofibromas, we also report aberrant expression of four miRNAs involved in the RAS-MAPK pathway (miR-370, miR-143, miR-181a, and miR-145). In MPNSTs, significant deregulated miRNAs were involved in PTEN repression (miR-301a, miR-19a, and miR-106b), RAS-MAPK pathway regulation (Let-7b, miR-195, and miR-10b), mesenchymal transition (miR-200c, let-7b, miR-135a, miR-135b, and miR-9), HOX genes expression (miR-210, miR-196b, miR-10a, miR-10b, and miR-9), and cell cycle progression (miR-195, let-7b, miR-20a, miR-210, miR-129-3p, miR-449a, and miR-106b).ConclusionWe confirmed the implication of PTEN in genesis of plexiform neurofibromas and MPNSTs in NF1. Markedly deregulated miRNAs might have potential diagnostic or prognostic value and could represent novel strategies for effective pharmacological therapies of NF1 tumors.

Abstract 1992: Is the ubiquitin ligase WWP2 playing a role in PTEN degradation in endometrial cancer.

Abstract Objective: The ubiquitin ligase WWP2 has recently been described to directly interact with PTEN, the major tumor suppressor gene in the PI3K pathway commonly altered in endometrial cancer. Whether alterations of WWP2 contribute to post-translational modifications of the PTEN protein in endometrial cancer are unknown. We sought to determine if WWP2 gene expression levels differ in human endometrial cancer tissues with evidence of post-translational PTEN degradation. We also wished to determine if elevated WWP2 gene expression was associated with clinical prognostic variables. Methods: Twenty-four endometrial cancer specimens collected at hysterectomy were available for analysis. These tumors were stained for PTEN by immunohistochemistry (IHC) and scored using a scale of 0-6. Frozen tissue was then used to determine PTEN and WWP2 expression by Real time PCR (RT PCR). Gene expression was then compared with findings on immunohistochemistry. Patient clinical characteristics including stage, grade, lymph node metastases, recurrence or 2 year disease status were then compared with PTEN and WWP2 expression. Results: Three tumors had elevated PTEN gene expression (at least 2 fold) but low (score of less than 4) PTEN protein on IHC, indicating post-translational modification of PTEN. In these tumors, however, WWP2 expression was not elevated (range 0.004-0.09). There were three tumors with elevation in WWP2 expression (1.9-4.7). PTEN was low in these tumors on IHC, but was also low by RT-PCR indicating genomic, epigenetic or post-transcriptional modification of PTEN. Neither PTEN nor WWP2 expression on RT PCR was associated with stage, grade, lymph node metastases, recurrence or 2 year disease status. Conclusions: In this cohort of tumors, WWP2 did not contribute to post-translational degradation of PTEN. WWP2 expression was not associated with stage, grade, lymph node metastases, recurrence, or 2 year disease status. Citation Format: Aine Clements, Joseph McElroy, Adrian Suarez, Sarmila Majumder, David Cohn, Gustavo Leone. Is the ubiquitin ligase WWP2 playing a role in PTEN degradation in endometrial cancer. [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 1992. doi:10.1158/1538-7445.AM2013-1992

Modeling squamous cell carcinoma development and malignant progression in mice.

Squamous cell carcinoma (SCCs) of the skin and head and neck arise through the accumulation of genetic and epigenetic alterations that contribute to different stages of tumor progression and the metastatic potential of these tumors. Advanced SCCs are refractory to current therapies and exhibit high risk for relapse, and despite significant advances in prevention and in understanding the biology of SCCs, there are currently no effective treatments for patients with metastatic SCCs. Understanding the role of the molecular alterations found in SCCs may facilitate the design of novel therapies to treat these patients. The most frequently mutated gene in SCC of the skin and head and neck is the p53 gene, which is found mutated in up to 50-70% of the SCCs analyzed. The majority of the p53 mutations found in SCCs are missense mutations that result in the expression of altered forms of p53, some of which may promote tumorigenesis, indicating that p53 is not only a major tumor suppressor gene, but it may also be a primary oncogene in SCC. Genetically engineered mouse models are powerful tools to determine the role of genetic alterations found in human cancers. We generated a mouse model for sporadic head and neck cancer based on the focal activation of endogenous K-ras and p53 mutations in the oral epithelium. Using this model we demonstrated that the p53 gain-of-function mutation p53R172H, but not deletion of p53, contributes to oral tumor initiation, accelerates tumor growth and promotes malignant progression. Molecular analysis of tumors and cell lines derived from them determined that mutant p53 promotes the expression of mitotic genes and induces accelerated entry in mitosis, a mechanism that depends on oncogenic K-ras. These findings provide in vivo evidence for the oncogenic potential of mutant p53R172H during SCC development and support clinical observations indicating that certain p53 mutations are associated with poor prognosis in SCC of the head and neck. I will discuss the potential implications of these findings in tumor progression, resistance to therapy and genomic instability associated with p53 mutations. As the oncogenic function of mutant p53 in this mouse model was dependent on K-ras mutations, we speculate that p53 mutants are activated in response to oncogenes. To address this question, I will further discuss the consequences of activating p53 gain- and loss-of-function mutations in the absence of additional genetic alterations in mouse models in which only p53 mutations are induced in stratified epithelia of the skin and the oral mucosa. In addition, novel mouse models generated in our laboratory are shedding new light into the function of genes that cooperate with p53 mutations in the early stages of SCC formation and during malignant progression and metastasis.

The False Paradigm of RUNX3 Function as Tumor Suppressor in Gastric Cancer

Gastric cancer (GC) is a major cause of cancer mortality. GC studies that aim to identify relevant oncogenes and tumor suppressor genes (TSGs) are essential for devising effective new therapies. A decade ago, RUNX3, a gene that resides on human chromosome 1p36.1, was claimed to be a major TSG in GC. Since then, hundreds of studies involving thousands of GC patients have attempted to verify and extend the RUNX3 TSG paradigm. However, RUNX3 is not recognized as TSG and not listed in the “Cancer Gene Census” website. To be a TSG that protects normal cells against malignancy, the gene must be expressed in the normal tissue from which the cancer arose and its loss or inactivation should contribute to cancer development. This review summarizes compelling body of evidence challenging the RUNX3-TSG paradigm. Studies show unequivocally that RUNX3 is not expressed in normal gastric epithelium and that it fails to fulfill all other premises of a TSG. RUNX3 mutations and 1p36 deletions are not frequent in GC and RUNX3 is not associated with familial GC or with increased risk of GC. Accordingly, Runx3-/- mice do not develop tumors. RUNX3 promoter methylation, which has been reported to be a frequent event in GC, is not relevant to its alleged TSG function, since the gene is already silent in normal gastric epithelium. In sharp contrast, overexpression of RUNX3 was found in several types of human cancers, including GC, and the 1p36.1 region is amplified in B-cell lymphoma. Thus, it is possible that RUNX3 actually promotes cancer development rather than being a TSG. The true targets for GC therapy are discussed below. Those are genes frequently lost or amplified in GC and are well known for their tumor suppressive or oncogenic activity, respectively.

Retinoblastoma and Phosphate and Tensin Homolog Tumor Suppressors: Impact on Ductal Carcinoma In Situ Progression

A subset of patients with ductal carcinoma in situ (DCIS) will progress to invasive breast cancer. However, there are currently no markers to differentiate women at high risk from those at lower risk of developing invasive disease. The association of two major tumor suppressor genes, retinoblastoma (RB) and phosphatase and tensin homolog (PTEN), with risk of any ipsilateral breast event (IBE) or progression to invasive breast cancer (IBC) was analyzed using data from 236 DCIS patients treated with breast conserving surgery with long-term follow-up. RB and PTEN expression was assessed with immunohistochemistry. The functional effects of RB and/or PTEN loss were modeled in MCF10A cells. Hazard ratios (HRs) were estimated with univariate and multivariable Cox regression models. All statistical tests were two-sided. Loss of RB immunoreactivity in DCIS was strongly associated with risk of IBE occurrence (HR = 2.64; 95% confidence interval [CI] = 1.64 to 4.25) and IBC recurrence (HR = 4.66; 95% CI = 2.19 to 9.93). The prognostic power of RB loss remained statistically significant in multivariable analyses. PTEN loss occurred frequently in DCIS but was not associated with recurrence or progression. However, patients with DCIS lesions that were both RB and PTEN deficient were at further increased risk for IBEs (HR = 3.39; 95% CI = 1.92 to 5.99) and IBC recurrence (HR = 6.1, 95% CI = 2.5 to 14.76). Preclinical modeling in MCF10A cells demonstrated that loss of RB and PTEN impacted proliferation, motility, and invasive properties. These studies indicate that RB and PTEN together have prognostic utility and could be used to target aggressive treatment for patients with the greatest probability of benefit.

The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite tremendous progress in its molecular characterization. Indeed, PDA tumors harbor four signature somatic mutations1–4, and a plethora of lower frequency genetic events of uncertain significance5. Here, we used Sleeping Beauty (SB) transposon-mediated insertional mutagenesis6,7 in a mouse model of pancreatic ductal preneoplasia8 to identify genes that cooperate with oncogenic KrasG12D to accelerate tumorigenesis and promote progression. Our screen revealed new candidates and confirmed the importance of many genes and pathways previously implicated in human PDA. Interestingly, the most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumors. Although prior work had attributed a pro-survival role to USP9X in human neoplasia9, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and mRNA expression in PDA correlates with poor survival following surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2′-deoxycytidine elevates USP9X expression in human PDA cell lines to suggest a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with KrasG12D to rapidly accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. Therefore, we propose USP9X as a major new tumor suppressor gene with prognostic and therapeutic relevance in PDA.

Tumour suppressor genes in chemotherapeutic drug response

Since cancer is one of the leading causes of death worldwide, there is an urgent need to find better treatments. Currently, the use of chemotherapeutics remains the predominant option for cancer therapy. However, one of the major obstacles for successful cancer therapy using these chemotherapeutics is that patients often do not respond or eventually develop resistance after initial treatment. Therefore identification of genes involved in chemotherapeutic response is critical for predicting tumour response and treating drug-resistant cancer patients. A group of genes commonly lost or inactivated are tumour suppressor genes, which can promote the initiation and progression of cancer through regulation of various biological processes such as cell proliferation, cell death and cell migration/invasion. Recently, mounting evidence suggests that these tumour suppressor genes also play a very important role in the response of cancers to a variety of chemotherapeutic drugs. In the present review, we will provide a comprehensive overview on how major tumour suppressor genes [Rb (retinoblastoma), p53 family, cyclin-dependent kinase inhibitors, BRCA1 (breast-cancer susceptibility gene 1), PTEN (phosphatase and tensin homologue deleted on chromosome 10), Hippo pathway, etc.] are involved in chemotherapeutic drug response and discuss their applications in predicting the clinical outcome of chemotherapy for cancer patients. We also propose that tumour suppressor genes are critical chemotherapeutic targets for the successful treatment of drug-resistant cancer patients in future applications.

Abstract 1360: A mouse model of double heterozygosity for protein kinase A regulatory subunits promotes osteoblastic differentiation of cAMP-induced bone tumors

Abstract Background: Carney Complex (CNC) is a multiple neoplasia syndrome inherited in an autosomal dominant manner and causing various endocrine and bone tumors. The PRKAR1A gene coding for the regulatory subunit type 1A of protein kinase A (PKA) is mutated in CNC patients, but tumorogenesis mechanisms are not well understood. Regulatory subunits suppress activity of catalytic subunits of PKA responsible for cAMP signaling and cell differentiation and maturation. Mice with a deleted prkar1a allele develop a variety of tumors overlapping those of CNC patients. Deletion of a catalytic subunit allele prkaca+/− on the prkar1a+/ background unexpectedly increased the numer and aggressiveness of bone tumors without development of schwannomas or thyroid lesions. Cells from prkar1a+/−prkaca+/ tumors have more type II PKA complexes, indicating that unbalanced expression of type II and type I regulatory subunits may also dysregulates PKA catalytic activity, potentially increasing tumorigenesis. Methods: To explore this hypothesis, single mice with single alleles of prkar1a, prkar2a and prkar2b were crossed to generate heterozygous mice for double heterozygotes prkar1a+/−/prkar2a+/− or prkar1a+/−/prkar2b+/− and double knockouts. The resulting mice of 3,6,9,12,18 month age were phenotyped and compared to prkar1a+/−. Tumor specimens were examined by histology, polarized microscopy and confocal Raman micro-spectroscopy. Results: Mice with both alleles prkar1a+/+ did not develop any tumors. Double heterozygotes developed bone tumors whose onset age was delayed to 9 months in prkar1a+/−/prkar2b+/− mice compared to 3-6 months in the others. However, the mean number of tumors per animal became similar for prkar1a+/− and double heterozygotes by 12 months. Histology of the bone tumors showed abnormal proliferation of a fibroblastoid-like cell with better osteogenic differentiation in the lesions from double heterozygous mice. Polarized microscopy and Raman micro-spectroscopy revealed that bone material growing in tumors was immature and poorly organized in prkar1a+/− mice (disorganized collagen and osteocytes and undermineralized matrix). Prkar2α+/− deletion on the prkar1α+/− background rescued local organization and mineralization but worsened global organization of cortical tumor bone. The prkar1a+/−/prkar2b+/− deletion rescued both global and local organization and mineralization, resulting in formation of mature bone. Conclusion: Prkar1a haploinsufficiency requires both prkar2a and prkar2b for tumorigenesis in most tissues except bone. In bone, however, prkar1a is the major tumor-suppressor gene. Unexpectedly, Prkar2b or prkar2a deficiencies in addition to prkar1a lead to better differentiation of bone tumors, indicating a compensating role for the type II regulatory subunits in bone tumirogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1360. doi:1538-7445.AM2012-1360

DRAM-1 encodes multiple isoforms that regulate autophagy

Macro(autophagy) is a cellular mechanism which delivers cytoplasmic constituents to lysosomes for degradation. Due to its role in maintaining cellular integrity, autophagy protects against various diseases including cancer. p53 is a major tumor suppressor gene which can modulate autophagy both positively and negatively. p53 induces autophagy via transcriptional activation of Damage-Regulated Autophagy Modulator (DRAM-1). We report here that DRAM-1 encodes not just one mRNA, but a series of p53-inducible splice variants which are expressed at varying levels in multiple human and mouse cell lines. Two of these new splice variants, termed SV4 and SV5, result in mature mRNA species. Different to ‘full-length’ DRAM-1 (SV1), SV4 and SV5 do not localise to lysosomes or endosomes, but instead partially localise to peroxisomes and autophagosomes respectively. In addition, SV4 and SV5 can also be found co-localised with certain markers of the endoplasmic reticulum. Similar to SV1, SV4 and SV5 do not appear to be inducers of programmed cell death, but they do modulate autophagy. In summary, these findings identify new autophagy regulators that provide insight into the control of autophagy downstream of p53.

Microsatellite Alterations of Major Tumor Suppressor Genes in Hepatocellular Carcinoma

Purpose : Hepatocellular carcinoma (HCC) shows various molecular and genetic alterations in its development and progression. The major tumor suppressor genes (TSGs) such as p16, PTEN, E-cadherin and p53 on chromosomes 9p21, 10q23, 16q22, 17p13 may play important roles in various regulation pathways as well as tumor suppression. Microsatellite alteration such as loss of heterozygosity (LOH) has been reported is a predominant mechanism of inactivation of TSGs. Also, LOH is known to be related with allelic loss of various TSGs. This study evaluated LOH of 4 TSGs in HCCs and correlated these results with the clinicopathological factors. Methods : LOH analysis was carried out using a polymerase chain reaction with 16 polymorphic microsatellite markers of 4 chromosomes having TSGs in 50 surgically resected tumors and their non-tumorous counterparts. Results : There was no detectable LOH in normal tissue. LOH was detected in 82% of 50 cases of HCCs. LOH of 9p21, 10q23, 16q22, 17p13 was detected in 36%, 54%, 38% and 66%, respectively. Advanced HCCs showed significant LOH results of 9p21, 10q23 & 17p13. According to tumor grade, those of 10q23, 16q22, 17p13 correlated with higher grade III. Vascular invasion & lymphatic emboli showed correlation with those of 9p21, 10q23, 17p13. Cancer recurrence & metastasis significantly correlated with 9p21 LOH. Conclusion : These results suggest that LOH of various chromosomes may be predominantly involved in hepatocarcinogenesis and tumor invasion. Also, those LOH may be useful clinical indicator in determining the poorer prognosis among patients with HCC.

Loss of Heterozygosity at BRCA1 Locus Is Significantly Associated with Aggressiveness and Poor Prognosis in Breast Cancer

BRCA1 and BRCA2 are two major tumor suppressor genes for hereditary breast and ovarian cancer. In sporadic breast cancer, although somatic mutations of these genes are rare, loss of heterozygosity (LOH) at BRCA1 and BRCA2 loci is common. LOH at BRCA1 and BRCA2 loci were investigated in 202 Japanese invasive breast cancer patients. The relationships between LOH at these gene loci and clinicopathologic characteristics were analyzed. Among 166 informative cases for both BRCA1 and BRCA2 loci, 69 (41.6%) and 52 (31.3%) tumors revealed LOH at BRCA1 and BRCA2 loci, respectively. LOH at BRCA1 LOH or BRCA2 locus was associated with higher nuclear grade (P < 0.0001, P = 0.0187). LOH at BRCA1 locus was associated with estrogen receptor and progesterone receptor negativity (P = 0.001 and P = 0.015) and significantly shorter disease-free survival (P < 0.0001), distant metastasis-free survival (P < 0.0001), and overall survival (P < 0.0001). In contrast, LOH at BRCA2 locus had no associations with estrogen receptor or progesterone receptor status and prognosis. LOH at BRCA1 locus was independently associated with poor prognosis in terms of disease-free survival (hazard ratio 3.08, 95% confidence interval [CI] 1.58-6.18, P = 0.0009), distant metastasis-free survival (hazard ratio 5.18, 95% CI 2.35-12.19, P < 0.0001), and overall survival (hazard ratio 4.97, 95% CI 1.84-15.1, P = 0.0013). LOH at BRCA1 locus could be an independent prognostic biomarker useful in identifying a subgroup of patients with poor prognosis.