The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells

The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.     

Identification of a putative tumor suppressor gene Rap1GAP in pancreatic cancer

Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and other tumors. We found that expression of rap1GAP, a gene located in this chromosomal region, is significantly down-regulated in pancreatic cancer. Only a small percentage of preneoplastic pancreatic intraductal neoplasia lesions lost rap1GAP expression, whereas loss of rap1GAP expression occurred in 60% of invasive pancreatic cancers, suggesting that rap1GAP contributes to pancreatic cancer progression. In vitro and in vivo studies showed that loss of rap1GAP promotes pancreatic cancer growth, survival, and invasion, and may function through modulation of integrin activity. Furthermore, we showed a high frequency of loss of heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor gene in pancreatic cancer.     

Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway

ARHI, an imprinted putative tumor suppressor gene, encodes a M(r) 26,000 GTP-binding protein that is 60% homologous to ras and rap but has a dramatically different function. ARHI expression is down-regulated in a majority of breast and ovarian cancers. Using a dual adenovirus system, we have reexpressed ARHI in ovarian cancer and breast cancer cells that have lost ARHI expression. Reexpression of ARHI inhibited growth, decreased invasiveness, and induced apoptosis. At 5 days after infection with ARHI adenovirus, 30-45% of MDA-MB-231 breast cancer cells and 5-11% of SKOv3 ovarian cancer cells were apoptotic as judged by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay and by Annexin V staining with flow cytometric analysis. Although poly(ADP-ribose) polymerase could be detected immunohistochemically in the nuclei of apoptotic cells, no activation of the effector caspases (caspase 3, 6, 7, or 12) or the initiator caspases (caspase 8 or 9) could be detected in cell lysates using Western blotting. When gene expression was analyzed on a custom cDNA array that contained 2304 known genes, infection with ARHI adenovirus up-regulated 15 genes relative to control cells infected with LacZ adenovirus. The greatest degree of mRNA up-regulation was observed in a Homo sapiens calpain-like protease. On Western blot analysis, calpain protein was increased 2-3-fold at 3-5 days after infection with ARHI adenovirus. No increase in calpain protein was observed after LacZ adenovirus infection. Calpain cleavage could be detected after ARHI reexpression, and inhibitors of calpain, but not inhibitors of caspase, partially prevented ARHI-induced apoptosis. Consequently, reexpression of ARHI in breast and ovarian cancer cells appears to induce apoptosis through a caspase-independent, calpain-dependent mechanism.     

Frequent silencing of a putative tumor suppressor gene melatonin receptor 1 A (MTNR1A) in oral squamous-cell carcinoma

Array-based comparative genomic hybridization (array-CGH) has good potential for the high-throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of 21 oral squamous-cell carcinoma (OSCC) cell lines for genome-wide copy-number aberrations by array-CGH using our in-house bacterial artificial chromosome arrays, we identified a frequent homozygous deletion at 4q35 loci with approximately 1 Mb in extent. Among the seven genes located within this region, the expression of the melatonin receptor 1 A ( MTNR1A ) messenger RNA (mRNA) was not detected or decreased in 35 out of the 39 (89%) OSCC cell lines, but was detected in immortalized normal oral epithelial cell line, and was restored in gene-silenced OSCC cells without its homozygous loss after treatment with 5-aza-2'-deoxycytidine. The hypermethylation of the CpG (cytosine and guanine separated by phosphate) island in the promoter region of MTNR1A was inversely correlated with its expression in OSCC lines without a homozygous deletion. Methylation of this CpG island was also observed in primary OSCC tissues. In an immunohistochemical analysis of 50 primary OSCC tumors, the absence of immunoreactive MTNR1A was significantly associated with tumor size and a shorter overall survival in patients with OSCC tumors, and seems to be an independent prognosticator in a multivariate analysis. Exogenous restoration of MTNR1A expression inhibited the growth of OSCC cells lacking its expression. Together with the known tumor-suppressive function of melatonin and MTNR1A in various tumors, our results indicate MTNR1A to be the most likely target for epigenetic silencing at 4q35 and to play a pivotal role during oral carcinogenesis. ( Cancer Sci 2008; 99: 1390–1400)     

The p75(NTR) tumor suppressor induces caspase-mediated apoptosis in bladder tumor cells

p75(NTR) was identified as a tumor and metastasis suppressor that functions in part via induction of apoptosis in tumor cells. To examine p75(NTR)-dependent apoptosis in tumor cells, we demonstrated that a dose-dependent increase in p75(NTR) expression was associated with a concomitant increase in the mitochondrial proapoptotic effector proteins Bad, Bax and Bik and a decrease in the mitochondrial prosurvival effector proteins phospho-Bad, Bcl-2 and Bcl-x(L). Significantly, p75(NTR)-dependent induction of cytochrome c release from the mitochondria occurred during CHX potentiation of apoptosis. Furthermore, p75(NTR) expression largely suppressed expression of IAP-1 and induced cleavage of procaspase-9 and procaspase-7 but not of procaspases 2, 3, 6, 8 and 10. A specific peptide inhibitor of procaspase-9 cleavage also inhibited cleavage of procaspase-7, indicating that caspase-7 is downstream of caspase-9. As end points of apoptosis, we observed p75(NTR)-dependent annexin V binding to the plasma membrane, an indicator of early apoptotic events, and Hoechst staining of DNA nuclear fragmentation, an indicator of late apoptotic events, whereas control tumor cells that lack expression of the p75(NTR) protein did not exhibit either of these apoptotic markers. Together, these results delineate the mitochondria-mediated apoptotic pathway of the p75(NTR) tumor-suppressor gene product.     

Role of the putative tumor metastasis suppressor gene Drg-1 in breast cancer progression

The differentiation-related gene-1 (Drg-1) was first identified as a gene strongly upregulated by induction of differentiation in colon carcinoma cells in vitro, and later the same gene was shown to suppress tumorigenicity of human bladder cancer cells in vivo. On the other hand, we and others have demonstrated that the Drg-1 gene suppresses prostate and colon cancer metastases in mouse models. In the context of such potential organ-specific differential function of the Drg-1 gene, the present study was designed to clarify the expression status, regulation and function of Drg-1 in the case of human breast cancer. We found that the expression of the Drg-1 protein was significantly reduced in breast tumor cells, particularly in patients with lymph node or bone metastasis as compared to those with localized breast cancer. Drg-1 expression also exhibited significant inverse correlation with the disease-free survival rate of patients and emerged as an independent prognostic factor. The downregulation of the Drg-1 gene appeared to be largely at the RNA level, and the DNA methylation inhibitor, 5-Azacytidine, significantly elevated the Drg-1 gene expression in various breast tumor cell lines. Furthermore, we found that overexpression of the Drg-1 gene suppresses the invasiveness of breast cancer cells in vitro, and this suppression was also achieved by treatment of cells with 5-Azacytidine. Together, our results strongly suggest functional involvement of the Drg-1 gene in suppressing the metastatic advancement of human breast cancer.     

Limiting the location of a putative human prostate cancer tumor suppressor gene at chromosome 13q14.3

We studied loss of heterozygosity (LOH) on human chromosome 13q in prostate cancer specimens to determine the location of a putative tumor suppressor gene (TSG) and to correlate these losses with the clinicopathological stage of the disease. Overall 13 (21%) of 61 specimens analysed had an allele loss on the long arm of chromosome 13. The most frequent (37%) LOH among the informative cases with allele losses was detected at the D13S284 locus on chromosome 13q14. 3. A portion of the DNA segment that spans this locus and is flanked by the microsatellite loci D13S153 and D13S163 was lost in 85% of the specimens with allele losses and was designated as a LOH cluster region (LCR). The LCR spans more than 6 Mbp of DNA. The results suggest that a TSG relevant for the development of prostate cancer is located telomeric to the RB locus. There was a significant correlation (P=0.0024) between chromosome 13q LOH and advanced metastatic disease, suggesting that loss of 13q14.3 region is associated with prostate cancer progression. However, further research must be conducted to establish the identity and function of this putative TSG.     

Hypermethylation and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers

A variety of tumor suppressor genes are down-regulated by hypermethylation during carcinogenesis. Using methylated CpG amplification-representation difference analysis, we identified a DNA fragment corresponding to the Tazarotene-induced gene 1 (TIG1) promoter-associated CpG island as one of the genes hypermethylated in the leukemia cell line K562. Because TIG1 has been proposed to act as a tumor suppressor, we tested the hypothesis that cytosine methylation of the TIG1 promoter suppresses its expression and causes a loss of responsiveness to retinoic acid in some neoplastic cells. We examined TIG1 methylation and expression status in 53 human cancer cell lines and 74 primary tumors, including leukemia and head and neck, breast, colon, skin, brain, lung, and prostate cancer. Loss of TIG1 expression was strongly associated with TIG1 promoter hypermethylation (P < 0.001). There was no correlation between TIG1 promoter methylation and that of retinoid acid receptor beta2 (RARbeta2), another retinoic-induced putative tumor suppressor gene (P = 0.78). Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine for 5 days restored TIG1 expression in all eight silenced cell lines tested. TIG1 expression was also inducible by treatment with 1 micro M all-trans-retinoic acid for 3 days except in densely methylated cell lines. Treatment of the K562 leukemia cells with demethylating agent combined with all-trans-retinoic acid induced apoptosis. These findings indicate that silencing of TIG1 promoter by hypermethylation is common in human cancers and may contribute to the loss of retinoic acid responsiveness in some neoplastic cells.     

The RASSF3 candidate tumor suppressor induces apoptosis and G1-S cell-cycle arrest via p53

RASSF3 is the smallest member of the RASSF family of proteins that function as tumor suppressors. Unlike other members of this important family, the mechanisms through which RASSF3 suppresses tumor formation remain unknown. Here, we show that RASSF3 expression induces p53-dependent apoptosis and its depletion attenuates DNA damage-induced apoptosis. We found that RASSF3-induced apoptosis depended upon p53 expression. Exogenous expression of RASSF3 induced G(1)-S arrest, which was also p53 dependent. In contrast, loss of RASSF3 promoted cell-cycle progression, abrogated UVB- and VP-16-induced G(1)-S arrest, decreased p53 protein and target gene expression, and prevented DNA repair. RASSF3 was shown to directly interact with and facilitate the ubiquitination of MDM2, the E3 ligase that targets p53 for degradation, thereby increasing p53 stabilization. Together, our findings show the tumor suppressor activity of RASSF3, which occurs through p53 stabilization and regulation of apoptosis and the cell cycle.     

The von Hippel-Lindau tumor suppressor gene protects cells from UV-mediated apoptosis

The familial cancer syndrome, von Hippel-Lindau (VHL) disease, characterized by a predisposition to renal cell carcinoma and certain other tumor types, is caused by mutational inactivation of the VHL tumor suppressor gene. Loss of VHL gene function is detected also in the vast majority of sporadic renal cell carcinomas. Previous reports have determined a protective role for VHL in response to serum withdrawal and glucose deprivation. In this study, the effect of UV irradiation on VHL-negative and VHL-positive renal carcinoma cells was examined. VHL-negative 786-O renal carcinoma cells underwent apoptosis following UV irradiation. In contrast, reintroduction of wild-type VHL expression protected 786-O cells from UV-mediated cell death. p53 and Bax levels were equivalent in VHL-negative and VHL-positive 786-O cells. Strikingly, cyclin-dependent kinase inhibitors p21 and p27 underwent proteasome-dependent degradation in VHL-negative 786-O cells following UV treatment. However, p21 and p27 protein levels were stable in VHL-positive cells. Also, levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xL were elevated in VHL-positive cells, consistent with the protection from apoptotic stimuli. UV treatment led to increased S phase in VHL-negative, but not VHL-positive cells. Thus, following UV irradiation, diminution of p21 and p27 levels resulted in a hyperproliferative state in VHL-negative cells, leading to apoptosis. These results suggest that loss of VHL function promotes apoptosis and may provide selective pressure toward cells that are able to escape apoptosis, leading to tumorigenesis.     

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), a putative breast tumor suppressor gene

Loss of heterozygosity (LOH) at the mannose 6-phosphate/insulin-like growth factor 2 receptor gene locus (M6P/IGF2R) on 6q26-27 has recently been demonstrated in approximately 30% of both invasive and in situ breast cancers. LOH was coupled with somatic point mutations in the remaining allele in several instances, leading to the proposition that M6P/IGF2R is a tumor suppressor gene [1]. Somatic mutations in M6P/IGF2R have also been described in hepatoma [2] and gastrointestinal cancers with the replication error positive (RER+) phenotype [3]. These data indicate that M6P/IGF2R loss of function mutations may be involved in the pathogenesis of a wide spectrum of malignancies. Extensive data on the normal function of the M6P/IGF2R suggest that loss of M6P/IGF2R activity may contribute to multiple aspects of tumor pathophysiology, including deregulated growth, apoptosis, angiogenesis and invasion.     

Nicotinamide blocks proliferation and induces apoptosis of chronic lymphocytic leukemia cells through activation of the p53/miR-34a/SIRT1 tumor suppressor network

Because of its relatively indolent clinical course, chronic lymphocytic leukemia (CLL) offers a versatile model for testing novel therapeutic regimens and drug combinations. Nicotinamide is the main NAD(+) precursor and a direct inhibitor of four classes of enzymes, including the sirtuins. SIRT1, the main member of the sirtuin family, inactivates p53 by deacetylating a critical lysine residue. In this study, we showed that CLL cells express high levels of functional SIRT1, which is inhibited by exogenous nicotinamide. This agent blocks proliferation and promotes apoptosis selectively in leukemic cells that express wild-type (wt) p53. Nicotinamide modulates the p53-dependent genes p21, NOXA, BAX, and Mcl-1, indicating an activation of the p53 pathway and of caspase-3. DNA-damaging chemotherapeutics, such as etoposide, activate a functional loop linking SIRT1 and p53 through the induction of miR-34a. When leukemic cells are simultaneously exposed to nicotinamide and etoposide, we observe a significant increase in miR-34a levels with a concomitant inhibition of SIRT1. Furthermore, p53 acetylation levels are higher than with either agent used alone. Overall, treatment with both nicotinamde and etoposide shows strongly synergistic effects in the induction of apoptosis. We therefore concluded that nicotinamide has the dual property of inhibiting SIRT1 through a noncompetitive enzymatic block (p53 independent) and at the same time through miR-34a induction (p53 dependent). These observations suggested the therapeutic potential of nicotinamide, a novel, safe, and inexpensive drug, to be used in addition to chemotherapy for CLL patients with wt p53.     

Lats2, a putative tumor suppressor,inhibits G1/S transition

Lats2 is a new member of the Lats tumor suppressor family. The human LATS2 gene is located at chromosome 13q11-12, which has been shown to be a hot spot (67%) for LOH in nonsmall cell lung cancer. In order to understand the function of LATS2 in the control of tumor development, we ectopically expressed mouse Lats2 via retroviral infection in NIH3T3/v-ras cells to examine whether Lats2 plays a role in suppressing tumor development and regulating cell proliferation. We have found that ectopic expression of Lats2 in NIH3T3/v-ras cells suppresses development of tumors in athymic nude mice and inhibits proliferation of NIH3T3/v-ras cells in an in vitro assay. Cell cycle profile analysis demonstrated that ectopic expression of Lats2 inhibited the G1/S transition. Further mechanistic studies revealed that cyclin E/CDK2 kinase activity was downregulated in Lats2-transduced NIH3T3/v-ras cells, while other cell cycle regulators controlling the G1/S transition were not affected. We have also shown that LATS2 kinase activity and two LATS conserved domains (LCDs) are required for Lats2 to suppress tumorigenicity and to inhibit cell growth. In addition, the LATS2 protein is cytoplasmic during interphase in NIH3T3 cells, while it becomes localized to the mitotic apparatus during mitosis. Finally, we propose a model in which a combination of mammalian Lats2 and Lats1 control cell proliferation by negatively regulating different cell cycle check points.     

The von Hippel-Lindau tumor suppressor protein regulates gene expression and tumor growth through histone demethylase JARID1C

In clear-cell renal cell carcinoma (ccRCC), inactivation of the tumor suppressor von Hippel-Lindau (VHL) occurs in the majority of the tumors and is causal for the pathogenesis of ccRCC. Recently, a large-scale genomic sequencing study of ccRCC tumors revealed that enzymes that regulate histone H3 lysine 4 trimethylation (H3K4Me3), such as JARID1C/KDM5C/SMCX and MLL2, were mutated in ccRCC tumors, suggesting that H3K4Me3 might have an important role in regulating gene expression and tumorigenesis. In this study we report that in VHL-deficient ccRCC cells, the overall H3K4Me3 levels were significantly lower than that of VHL+/+ counterparts. Furthermore, this was hypoxia-inducible factor (HIF) dependent, as depletion of HIF subunits by small hairpin RNA in VHL-deficient ccRCC cells restored H3K4Me3 levels. In addition, we demonstrated that only loss of JARID1C, not JARID1A or JARID1B, abolished the difference of H3K4Me3 levels between VHL-/- and VHL+/+ cells, and JARID1C displayed HIF-dependent expression pattern. JARID1C in VHL-/- cells was responsible for the suppression of HIF-responsive genes insulin-like growth factor-binding protein 3 (IGFBP3), DNAJC12, COL6A1, growth and differentiation factor 15 (GDF15) and density-enhanced phosphatase 1. Consistent with these findings, the H3K4Me3 levels at the promoters of IGFBP3, DNAJC12, COL6A1 and GDF15 were lower in VHL-/- cells than in VHL+/+ cells, and the differences disappeared after JARID1C depletion. Although HIF2α is an oncogene in ccRCC, some of its targets might have tumor suppressive activity. Consistent with this, knockdown of JARID1C in 786-O VHL-/- ccRCC cells significantly enhanced tumor growth in a xenograft model, suggesting that JARID1C is tumor suppressive and its mutations are tumor promoting in ccRCC. Thus, VHL inactivation decreases H3K4Me3 levels through JARID1C, which alters gene expression and suppresses tumor growth.     

Wnt antagonist DKK1 acts as a tumor suppressor gene that induces apoptosis and inhibits proliferation in human renal cell carcinoma

The functional significance of Wnt antagonist DKK1 has not been investigated in renal cell carcinoma (RCC). Therefore, we hypothesized that DKK1 may be a tumor suppressor gene and is epigenetically silenced, thus decreased DKK1 may cause progression of RCC. To assess the function of DKK1, we established stable DKK1 transfected cells and monitored them regarding cell viability, colony formation, apoptosis, cell cycle, and invasive capability. RCC cell lines had decreased levels of DKK1, which were increased after treatment with 5‐Aza‐2′‐deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5‐Aza‐2′‐deoxycytidine/trichostatin A treatment in RCC cell lines. Increased methylation was also associated with higher pathological stages in primary RCC tissues. T‐cell factor/lymphoid enhancer factor activity and nuclear beta‐catenin expression were not changed in DKK1 transfectants. Also the expression of cyclinD1 and c‐Myc was not changed in DKK1 transfectants. These results suggest that DKK1 may not be involved in the beta‐catenin dependent pathway. We also evaluated the expression of various related genes. Cleaved caspase3, p53, p21 and puma expression were significantly upregulated in the DKK1 transfected cells. The population of apoptotic cells was increased in stable DKK1 cells and tumor growth suppression was also observed in nude mice with DKK1 transfected cells. In conclusion, this is the first report to show that DKK1 expression is epigenetically silenced in kidney cancer and its reexpression induces apoptosis and cell cycle arrest in RCC.     

LATS1 tumor suppressor regulates G2/M transition and apoptosis

The LATS1 gene is a mammalian member of the novel lats tumor suppressor family. Both lats mosaic flies and LATS1 deficient mice spontaneously develop tumors. Our previous studies have shown that inactivation of Drosophila lats leads to up-regulation of cyclin A in the fly, and the human LATS1 protein associates with CDC2 in early mitosis in HeLa cells, suggesting that the lats gene family may negatively regulate cell proliferation by modulating CDC2/Cyclin A activity. We demonstrate here that transduction of the human breast cancer cell MCF-7 with recombinant LATS1 adenovirus (Ad-LATS1), but not with EGFP adenovirus (Ad-EGFP), inhibits in vitro cell proliferation. Ectopic expression of LATS1 in MCF-7 cells specifically down-regulates Cyclin A and Cyclin B protein levels and dramatically reduces CDC2 kinase activity, leading to a G2/M blockade. Furthermore, Ad-LATS1 suppresses anchorage-independent growth of MCF-7 cells in soft agar and tumor formation in athymic nude mice. We also demonstrate that ectopic expression of LATS1 in MCF-7 cells and human lung cancer cell H460 up-regulates the level of BAX proteins and induces apoptosis. Finally, we show that LATS1 kinase activity is required for its ability to inhibit cell growth and induce apoptosis. The results indicate that the LATS1 tumor suppressor may play an important role in the control of human tumor development and that LATS1 suppresses tumorigenesis by negatively regulating cell proliferation and modulating cell survival.