Wwox suppresses prostate cancer cell growth through modulation of ErbB2-mediated androgen receptor signaling.

The expression of the WWOX tumor suppressor gene is lost or reduced in a large fraction of various cancers, including prostate cancer. We previously reported that Wwox overexpression induced apoptosis and suppressed prostate cancer growth in vitro and in vivo. In this study, pathways through which Wwox contributes to control of prostate cancer cell growth have been investigated. We found that Wwox interacts with Ap2gamma and prevents it from entering the nucleus to bind the ERBB2 promoter region to activate transcription of ERBB2, a mediator of androgen receptor activity and prostate cancer cell growth at limiting androgen concentration. Ectopic expression of Wwox reduced ErbB2 protein expression in vitro and expression of Wwox protein inversely correlated with expression of ErbB2 protein in prostate cancer tissues. Furthermore, Wwox suppressed Ap2gamma/ErbB2-induced prostate cancer cell growth and suppressed prostate-specific antigen secretion through interaction with Ap2gamma and down-modulation of ErbB2, an effect that required functional androgen receptor.

Inhibition of breast cancer cell growth in vitro and in vivo: effect of restoration of Wwox expression.

PURPOSE: The WWOX gene is down-regulated in breast cancer and loss of Wwox expression correlates with important clinical features of breast cancer. Thus, we have examined the effect of restoration of Wwox expression in breast cancer-derived cells. EXPERIMENTAL DESIGN: Wwox protein expression was restored by the following: (a) infection with a recombinant adenovirus carrying WWOX cDNA (Ad-WWOX) or (b) treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, to activate the endogenous WWOX gene, in breast cancer-derived cells in vitro and in vivo. RESULTS: Restoration of Wwox expression led to suppression of growth of Wwox-deficient breast cancer-derived cells, through activation of the intrinsic caspase pathway, but did not affect growth of Wwox-sufficient MCF7 cells. Intratumoral Wwox restoration, through Ad-WWOX infection or endogenous Wwox reactivation by 5-aza-2'-deoxycytidine injection, suppressed tumor growth in nude mice by inducing apoptosis. Alteration of global methylation levels was not observed. CONCLUSIONS: The results confirm that overexpression of exogenous Wwox inhibits breast cancer cell growth in vitro and in vivo and, perhaps more importantly, shows that restoration of endogenous Wwox expression, and likely other proteins, by treatment with a de novo methyltransferase inhibitor, also inhibits breast cancer cell growth and reverses breast cancer xenograft growth.

A role for the WWOX gene in prostate cancer.

Expression of the WWOX gene, encompassing the common chromosome fragile site FRA16D, is altered in a large fraction of cancers of various types, including prostate cancer. We have examined expression and biological functions of WWOX in prostate cancer. WWOX mRNA and protein expression were significantly reduced in prostate cancer-derived cells (LNCaP, DU145, and PC-3) compared with noncancer prostate cells (PWR-1E), and WWOX expression was reduced in 84% of prostate cancers, as assessed by immunohistochemical staining. Down-modulation of WWOX expression in the prostate cancer-derived cells is due to DNA hypermethylation in the WWOX regulatory region. Treatment with 5-aza-2'-deoxycytidine (AZA), a DNA methyltransferase inhibitor, and trichostatin A, a histone deacetylase inhibitor, led to increased WWOX mRNA and protein expression in prostate cancer-derived cells, most strikingly in DU145 cells. Transfection-mediated WWOX overexpression in DU145 cells suppressed colony growth (P = 0.0012), and WWOX overexpression by infection with Ad-WWOX virus induced apoptosis through a caspase-dependent mechanism and suppressed cell growth. Lastly, ectopic expression of WWOX by Ad-WWOX infection suppressed tumorigenicity of xenografts in nude mice, and intratumoral AZA treatment halted tumor growth. The data are consistent with a role for WWOX as a prostate cancer tumor suppressor and suggest that WWOX signal pathways should be further investigated in normal and cancerous prostate cells and tissues.

Alterations of the tumor suppressor gene ARLTS1 in ovarian cancer.

ARLTS1 is a tumor suppressor gene initially described as a low-penetrance cancer gene: a truncated Trp149Stop (MUT) polymorphism is associated with general familial cancer aggregation and, particularly, high-risk familial breast cancer. DNA hypermethylation has been identified as a mechanism of ARLTS1 expression down-regulation in lung carcinomas and B-cell chronic lymphocytic leukemia. We found that, in the majority of ovarian carcinomas (61.5%) and in a significant proportion of ovarian and breast cancer cell lines (45%), ARLTS1 is strongly down-regulated due to DNA methylation in its promoter region. After ARLTS1 restoration by adenoviral transduction, only the negative TOV-112 and the homozygously mutated (MUT) MCF7 cells, but not the OV-90 cells expressing a normal ARLTS1 product, underwent apoptosis and inhibition of cell growth. Furthermore, ARLTS1 reexpression significantly reduced the tumorigenic potential of TOV-112 in nude mice. On the contrary, the ARLTS1-MUT induced significantly lower levels of apoptosis in infected cells and reduced in vivo tumorigenesis only partially, supporting the hypothesis that Trp149Stop polymorphism is retained in the general population and predisposes to cancer because of a reduction, but not full loss, of normal ARLTS1 function.

Activation of signal transducer and activator of transcription 3 through a phosphomimetic serine 727 promotes prostate tumorigenesis independent of tyrosine 705 phosphorylation.

Aberrantly activated signal transducer and activator of transcription 3 (Stat3) is implicated in the development of various human cancers. Y705 phosphorylation is conventionally thought to be required for Stat3 signal-dependent activation and seems to play an essential role in some malignancies. Recently, it was shown that Stat3 is activated through novel and noncanonical mechanisms, including phosphorylation at S727. Here, we investigate S727 phosphorylation of Stat3 and its subsequent effects in prostate cancer development, independent of Y705 phosphorylation, using mutated Stat3 in the human prostate cancer cell line LNCaP. We show mutation of S727 to the phosphomimetic residue Glu, and inactivation of Y705 (Y705F/S727E) resulted in a remarkable growth advantage in low-serum, enhanced anchorage-independent growth in soft agar, and increased tumorigenicity in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, possibly by direct activation of downstream proto-oncogenes c-myc, mcl-1, and survivin. Y705F/S727E mutant cells were more invasive than Y705F/S727A (inactivation of Y705 and S727) mutant cells, and more Y705F/S727E mutant Stat3 was localized in the nuclei relative to Y705F/S727A mutant Stat3 at the steady state. Furthermore, the Y705F/S727E but not the Y705F/S727A mutant induced anchorage-independent growth of noncancerous prostate epithelial cells (RWPE-1). We further show that Stat3 is phosphorylated at S727 in 65% of malignant prostate tissues (n = 20) relative to 25% of normal prostate tissues (n = 4). Moreover, there is a positive correlation between phosphoS727-Stat3 expression and Gleason score in these prostate cancer tissues (P = 0.05). Our data suggest for the first time that S727 phosphorylation is sufficient to activate Stat3, thereby driving prostate tumorigenesis independent of Y705 phosphorylation.

Biological functions of mammalian Nit1, the counterpart of the invertebrate NitFhit Rosetta stone protein, a possible tumor suppressor.

The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies.