Spinophilin: a new tumor suppressor at 17q21

The scaffold protein spinophilin (SPN) is a regulatory subunit of phosphatase 1a (PP1a) located at 17q21.33. This region is frequently associated with microsatellite instability and LOH and contains a relatively high density of known tumor suppressor genes, and several unidentified candidate tumor suppressor genes located distal to BRCA1. Spn is located in this locus and proposed to be a new tumor suppressor. Loss of Spn induces a proliferative response by increasing pRb phosphorylation, which in turn activates p53, thereby, neutralizing the proliferative response. The absence of p53 bypasses this barrier and enhances the malignant phenotype. Furthermore, the ectopic expression of SPN in human tumor cells from different types of malignancies greatly reduced cell growth. Spn knock-out mice had decreased lifespan with increased cellular proliferation in tissues such as the mammary ducts and early appearance of tumors. Furthermore, the combined loss of Spn and mutant p53 activity led to increased mammary carcinomas, confirming the functional relationship between p53 and Spn. In human tumors, Spn is absent in 20% and reduced in another 37% of human lung tumors. Spn reduction correlates with malignant grade and p53 mutations. Furthermore, Spn mRNA is lost in a percentage of renal carcinomas and lung adenocarcinomas. Finally, lower levels of Spn mRNA correlate with higher grade of ovarian carcinoma and chronic myelogenous leukemia. Therefore, Spn may be the tumor suppressor gene that is located at 17q21.33 and that its tumor suppressive function is dependent on the absence of p53.     

BK Virus as a Cofactor in the Etiology of Prostate Cancer in Its Early Stages

Prostate cancer has been projected to cause almost 10% of all male cancer deaths in the United States in 2007. The incidence of mutations in the tumor suppressor genes Rb1 and p53, especially in the early stages of the disease, is low compared to those for other cancers. This has led to the hypothesis that a human virus such as BK virus (BKV), which establishes a persistent subclinical infection in the urinary tract and encodes oncoproteins that interfere with these tumor suppressor pathways, is involved. Previously, we detected BKV DNA in the epithelial cells of benign and proliferative inflammatory atrophy ducts of cancerous prostate specimens. In the present report, we demonstrate that BKV is present at a much lower frequency in noncancerous prostates. Additionally, in normal prostates, T-antigen (TAg) expression is observed only in specimens harboring proliferative inflammatory atrophy and prostatic intraepithelial neoplasia. We further demonstrate that the p53 gene from atrophic cells expressing TAg is wild type, whereas tumor cells expressing detectable nuclear p53 contain a mix of wild-type and mutant p53 genes, suggesting that TAg may inactivate p53 in the atrophic cells. Our results point toward a role for BKV in early prostate cancer progression.     

53BP1 ablation rescues genomic instability in mice expressing ‘RING‐less’ BRCA1

BRCA1 mutations strongly predispose affected individuals to breast and ovarian cancer, but the mechanism by which BRCA1 acts as a tumor suppressor is not fully understood. Homozygous deletion of exon 2 of the mouse Brca1 gene normally causes embryonic lethality, but we show that exon 2‐deleted alleles of Brca1 are expressed as a mutant isoform that lacks the N‐terminal RING domain. This “RING‐less” BRCA1 protein is stable and efficiently recruited to the sites of DNA damage. Surprisingly, robust RAD51 foci form in cells expressing RING‐less BRCA1 in response to DNA damage, but the cells nonetheless display the substantial genomic instability. Genomic instability can be rescued by the deletion of Trp53bp1, which encodes the DNA damage response factor 53BP1, and mice expressing RING‐less BRCA1 do not show an increased susceptibility to tumors in the absence of 53BP1. Genomic instability in cells expressing RING‐less BRCA1 correlates with the loss of BARD1 and a defect in restart of replication forks after hydroxyurea treatment, suggesting a role of BRCA1–BARD1 in genomic integrity that is independent of RAD51 loading.     

Asbestos,chromosomal deletions,and tumor suppressor gene alterations in human malignant mesothelioma

Exposure to the carcinogen asbestos is considered to be a major factor contributing to the development of most malignant mesotheliomas (MMs). We highlight the role of asbestos in MM and summarize cytogenetic and molecular genetic findings in this malignancy. The accumulation of numerous clonal chromosomal deletions in most MMs suggests a multistep process of tumorigenesis, characterized by the loss and/or inactivation of multiple tumor suppressor genes (TSGs). Cytogenetic and loss of heterozygosity (LOH) analyses of MMs have demonstrated frequent deletions of specific sites within chromosome arms 1p, 3p, 6q, 9p, 13q, 15q, and 22q. Furthermore, TSGs within two of these regions, i.e., p16/CDKN2A-p14^ARF at 9p21 and NF2 at 22q12, are frequently altered in MMs. Homozygous deletion appears to be the major mechanism affecting p16/CDKN2A-p14^ARF, whereas inactivating mutations coupled with allelic loss occur at the NF2 locus. Finally, recent studies have demonstrated the presence and expression of simian virus 40 (SV40) in many MMs. SV40 large T antigen has been shown to inactivate the TSG products Rb and p53, suggesting the possibility that asbestos and SV40 could act as cocarcinogens in MM. The frequent occurrence of homozygous deletions of p16/CDKN2A-p14^ARF and the ability of SV40 Tag to bind TSG products suggest that perturbations of both Rb- and p53-dependent growth-regulatory pathways are critically involved in the pathogenesis of MM. J. Cell. Physiol. 180:150–157, 1999. © 1999 Wiley-Liss, Inc.     

Microsatellite Alterations and Protein Expression of 5 Major Tumor Suppressor Genes in Gastric Adenocarcinomas

PURPOSE: In gastric adenocarcinoma (GC), the major tumor suppressor genes (TSGs) such as p16, PTEN, Rb, E-cadherin, and p53, may play important roles in various regulatory pathways and in tumor suppression. This study evaluated the loss of heterozygosity (LOH) of microsatellite and protein expression of 5 TSGs and the results were examined for their correlation with clinicopathological factors. METHODS: LOH analysis was carried out using polymerase chain reactions with 15 polymorphic microsatellite markers of 5 chromosomes containing TSGs in 100 surgically resected tumors. Protein expression was evaluated by immunohistochemistry (IHC). RESULTS: LOH was detected in 83% of GCs. LOH of 9p21, 10q23, 13q14, 16q22, and 17p13 were detected in 26%, 31%, 24%, 22%, and 35% of cases, respectively. Protein expression of p16, PTEN, Rb, E-cadherin, and p53 were found to be 31%, 39%, 28%, 32%, and 46% of cases. Advanced GCs showed significantly higher rates of 17p13 LOH and p53 expression. 9p21 LOH and E-cadherin IHC were correlated with higher tumor grade. Lymph node metastasis was correlated with the LOH of 9p21, 16q22, and 17p13 and IHC of the Rb and p53. A higher stage was correlated with 10q23 and 17p13 in LOH and p53 for IHC. CONCLUSION: These results suggest that LOH and protein expression of various TSGs are important in carcinogenesis and tumor invasion. Additionally, LOH and IHC may be useful clinical indicators for determining the prognosis of patients with GCs. In particular, the 17p13 LOH and p53 for IHC can be applied as simple evaluations in the clinic.     

Erratum to: Differential effects of genes of the <Emphasis Type="Italic">Rb1</Emphasis> signalling pathway on osteosarcoma incidence and latency in alpha-particle-irradiated mice

Osteosarcoma is the most frequent secondary malignancy following radiotherapy of patients with bilateral retinoblastoma. This suggests that the Rb1 tumour suppressor gene might confer genetic susceptibility towards radiation-induced osteosarcoma. To define the contribution of the Rb1 pathway in the multistep process of radiation carcinogenesis, we evaluated somatic allelic changes affecting the Rb1 gene itself as well as its upstream regulator p16 in murine osteosarcoma induced by ²²⁷Th incorporation. To distinguish between the contribution of germline predisposition and the effect of a 2-hit allelic loss, two mouse models harbouring heterozygote germline Rb1 and p16 defects were tested for the incidence and latency of osteosarcoma following irradiation. We could show that all tumours arising in BALB/c × CBA/CA hybrid mice (wild-type for Rb1 and for p16) carried a somatic allelic loss of either the Rb1 gene (76.5%) or the p16 gene (59%). In none of the tumours, we found concordant retention of heterozygosity at both loci. Heterozygote knock-out mice for Rb1 exhibit a significant increase in the incidence of osteosarcoma following ²²⁷Th incorporation (22/24 in Rb1+/− vs. 2/18 in Rb1+/+, p = 4 × 10⁻⁵), without affecting tumour latency. In contrast, heterozygote knock-out mice for p16 had no significant change in tumour incidence, but a pronounced reduction of latency (LT_50% = 355 days in p16+/− vs. 445 days in p16+/+, p = 8 × 10⁻³). These data suggest that Rb1 germline defects influence early steps of radiation osteosarcomagenesis, whereas alterations in p16 mainly affect later stages of tumour promotion and growth.     

Ras Regulates Rb via NORE1A

Mutations in the Ras oncogene are one of the most frequent events in human cancer. Although Ras regulates numerous growth-promoting pathways to drive transformation, it can paradoxically promote an irreversible cell cycle arrest known as oncogene-induced senescence. Although senescence has clearly been implicated as a major defense mechanism against tumorigenesis, the mechanisms by which Ras can promote such a senescent phenotype remain poorly defined. We have shown recently that the Ras death effector NORE1A plays a critical role in promoting Ras-induced senescence and connects Ras to the regulation of the p53 tumor suppressor. We now show that NORE1A also connects Ras to the regulation of a second major prosenescent tumor suppressor, the retinoblastoma (Rb) protein. We show that Ras induces the formation of a complex between NORE1A and the phosphatase PP1A, promoting the activation of the Rb tumor suppressor by dephosphorylation. Furthermore, suppression of Rb reduces NORE1A senescence activity. These results, together with our previous findings, suggest that NORE1A acts as a critical tumor suppressor node, linking Ras to both the p53 and the Rb pathways to drive senescence.     

Loss of heterozygosity studies in tumors from families with breast-ovarian cancer syndrome

A gene (BRCA1) predisposing for familial breast and ovarian cancer has been mapped to chromosome band 17q12-21. Based on the observation that ovarian tumors from families with breast and ovarian cancer lose the wild-type allele in the region for the BRCA1 locus, it has been suggested that the gene functions as a tumor suppressor gene. We have studied chromosomal deletions in the BRCA1 region in seven breast tumors, three ovarian tumors, one bladder cancer, and one colon cancer from patients in six families with breast-ovarian cancer, in order to test the hypothesis of the tumor suppressor mechanism at this locus. We have found a low frequency of loss of heterozygosity at this region, and our results do not support the idea that BRCA1 is a tumor suppressor gene. Alternatively, the disease segregating in these families is linked to one or more different loci.     

BRCA1 interacts with dominant negative SWI/SNF enzymes without affecting homologous recombination or radiation-induced gene activation of p21 or Mdm2

BRCA1 is a tumor suppressor gene linked to familial breast and ovarian cancer. The BRCA1 protein has been implicated in a diverse set of cellular functions, including activation of gene expression by the p53 tumor suppressor and control of homologous recombination (HR) during DNA repair. Prior reports have demonstrated that BRCA1 can exist in cells in a complex with the BRG1-based SWI/SNF ATP-dependent chromatin remodeling enzymes and that SWI/SNF components contribute to p53-mediated gene activation. To investigate the link between SWI/SNF function and BRCA1 mediated effects on p53-mediated gene activation and on mechanisms of homologous recombination, we have utilized mammalian cells that inducibly express an ATPase-deficient, dominant negative SWI/SNF enzymes. Mutant SWI/SNF ATPases retain the ability to interact with BRCA1 in cells. We report that expression of dominant negative SWI/SNF enzymes does not affect p53-mediated induction of the p21 cyclin dependent kinase inhibitor or the Mdm2 E3 ubiquitin ligase that regulates p53 in cells exposed to UV or gamma irradiation. Similarly, integration of a reporter that monitors homologous recombination by gene conversion into these cells demonstrated no change in the recombination rate in the absence of functional SWI/SNF enzyme. We conclude that the SWI/SNF chromatin remodeling enzymes may contribute to but are not required for these processes.     

BRCA1-mediated signaling pathways in ovarian carcinogenesis

The link between loss or defect in functional BRCA1 and predisposition for development of ovarian and breast cancer is well established. Germ-line mutations in BRCA1 are responsible for both hereditary breast and ovarian cancer, which is around 5–10% for all breast and 10–15% of all ovarian cancer cases. However, majority of cases of ovarian cancer are sporadic in nature. The inactivation of cellular BRCA1 due to mutations or loss of heterozygosity is one of the most commonly observed events in such cases. Complement-resistant retroviral BRCA1 vector, MFG-BRCA1, is the only approved gene therapy for ovarian cancer patients by the Federal and Drug Administration. Given the limited available information, there is a need to evaluate the effects of BRCA1 on the global gene expression pattern for better understanding the etiology of the disease. Here, we use Ingenuity Pathway Knowledge Base to examine the differential pattern of global gene expression due to stable expression of BRCA1 in the ovarian cancer cell line, SKOV3. The functional analysis detected at least five major pathways that were significantly (p < 0.05) altered. These include: cell to cell signaling and interaction, cellular function and maintenance, cellular growth and proliferation, cell cycle and DNA replication, and recombination repair. In addition, we were able to detect several biologically relevant genes that are central for various signaling networks involved in cellular homeostasis; TGF-β1, TP53, c-MYC, NF-κB and TNF-α. This report provides a comprehensive rationale for tumor suppressor function(s) of BRCA1 in ovarian carcinogenesis.     

ING5 inhibits cancer aggressiveness by inhibiting Akt and activating p53 in prostate cancer

Prostate cancer (PCa) is one of the most common types of cancer in men. In several recent studies, chromosomal deletions in the q arm of chromosome 2, where ING5 resides within, have been identified in various cancer types including PCa. In this study, we investigate the role of ING5 as a tumor suppressor in PCa. We examined the expression level of ING5 in tissue samples and cell lines using quantitative real‐time polymerase chain reaction and western blot analysis. We tested the in vitro tumor suppressor potential of ING5 in PC3 and LNCaP cells stably overexpressing it using cell viability, colony formation, migration, invasion, and apoptosis assays. We then investigated the effects of ING5 on the Akt and p53 signaling using western blot analysis. We show that ING5 is significantly downregulated in PCa tumor tissue samples and cell lines compared with the corresponding controls. In vitro assays demonstrate that ING5 effectively suppresses proliferative, clonogenic, migratory, and invasive potential and induce apoptosis in PCa cells. ING5 may potentially exert its anti‐tumor potential by inhibiting AKT and inducing p53 signaling pathways. Our findings demonstrate that ING5 possesses tumor suppressor roles in vitro, pointing its importance during the prostatic carcinogenesis processes.     

The physiology of p16INK4A-mediated G1 proliferative arrest

Phosphorylation of the product of the retinoblastoma susceptibility gene (Rb) physiologically inactivates its growth-suppressive properties. Rb phosphorylation is mediated by cyclin-dependent kinases (CDKs), whose activity is enhanced by cyclins and inhibited by CDK inhibitors. p16^INK4A is a member of a family of inhibitors specific for CDK4 and CDK6. p16^INK4A is deleted and inactivated in a wide variety of human malignancies, including familial melanomas and pancreatic carcinoma syndromes, indicating that it is an authentic human tumor suppressor. Although one mechanism for its tumor suppression may be prevention of Rb phosphorylation, thereby causing G1 arrest, many normal cell types express p16^INK4A, and are still able to traverse the cell cycle. In a search for other mechanisms, we have found that p16^INK4A is required for p53-independent G1 arrest in response to DNA-damaging agents, including topoisomerase I and II inhibitors. Thus, like other tumor suppressors, p16^INK4A plays an essential role in a DNA-damage checkpoint that leads to cell cycle arrest.