The WW domain-containing oxidoreductase (WWOX) gene is located at FRA16D, a common fragile site involved in human cancer. Targeted deletion of Wwox in mice causes increased spontaneous tumor incidence, confirming that WWOX is a bona fide tumor suppressor gene. We show that stable transfection of WWOX into human PEO1 ovarian cancer cells, containing homozygous WWOX deletion, abolishes in vivo tumorigenicity, but this does not correlate with alteration of in vitro growth. Rather, WWOX restoration in PEO1, or WWOX overexpression in SKOV3 ovarian cancer cells, results in reduced attachment and migration on fibronectin, an extracellular matrix component linked to peritoneal metastasis. Conversely, siRNA-mediated knockdown of endogenous WWOX in A2780 ovarian cancer cells increases adhesion to fibronectin. In addition, whereas there is no WWOX-dependent difference in cell death in adherent cells, WWOXtransfected cells in suspension culture display a proapoptotic phenotype. We further show that WWOX expression reduces membranous integrin A 3 protein but not integrin A 3 mRNA levels, and that adhesion of PEO1 cells to fibronectin is predominantly mediated through integrin A 3 . We therefore propose that WWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix and by inducing apoptosis in detached cells. Consistent with this, the suppression of PEO1 tumorigenicity by WWOX can be partially overcome by implanting these tumor cells in Matrigel. These data suggest a possible role for the loss of WWOX in the peritoneal dissemination of human ovarian cancer cells. [Cancer Res 2009;69(11):4835-42]
B Kuske and C Naughton contributed equally to this work. AbstractHormone-dependent estrogen receptor (ER)-positive breast cancer cells may adapt to low estrogen environments such as produced by aromatase inhibitors. In many instances, cells become insensitive to the effects of estrogen but may still retain dependence on ER. We have investigated the expression, function, and activation of ERa in two endocrine-resistant MCF-7 models to identify mechanisms that could contribute to resistance. While MCF-7/LCC1 cells are partially estrogen dependent, MCF-7/LCC9 cells are fully estrogen insensitive and fulvestrant and tamoxifen resistant. In both MCF-7/LCC1 and MCF-7/LCC9 cell lines, high expression of ERa was associated with enhanced binding to the trefoil factor 1 (TFF1) promoter in the absence of estrogen and increased transcription of TFF1 and progesterone receptor. In contrast to the observations derived from hypersensitive and supersensitive models, these cells were truly estrogen independent; nevertheless, removal of ERa by siRNA, or fulvestrant, a specific ER downregulator, inhibited growth indicating dependence on ERa. In the absence of estrogen, neither ERa Ser 118 nor Ser 167 were phosphorylated as frequently found in other ligandindependent cell line models. Addition of estrogen activated ERa Ser 118 in MCF-7 and LCC1 cells but not in LCC9 cells. We suggest that the estrogen-independent growth within these cell lines is accounted for by high levels of ERa expression driving transcription and full estrogen independence explained by lack of ERa activation through Ser 118 .
IntroductionTamoxifen is the most widely prescribed anti-estrogen treatment for patients with estrogen receptor (ER)-positive breast cancer. However, there is still a need for biomarkers that reliably predict endocrine sensitivity in breast cancers and these may well be expressed in a dynamic manner.MethodsIn this study we assessed gene expression changes at multiple time points (days 1, 2, 4, 7, 14) after tamoxifen treatment in the ER-positive ZR-75-1 xenograft model that displays significant changes in apoptosis, proliferation and angiogenesis within 2 days of therapy.ResultsHierarchical clustering identified six time-related gene expression patterns, which separated into three groups: two with early/transient responses, two with continuous/late responses and two with variable response patterns. The early/transient response represented reductions in many genes that are involved in cell cycle and proliferation (e.g. BUB1B, CCNA2, CDKN3, MKI67, UBE2C), whereas the continuous/late changed genes represented the more classical estrogen response genes (e.g. TFF1, TFF3, IGFBP5). Genes and the proteins they encode were confirmed to have similar temporal patterns of expression in vitro and in vivo and correlated with reduction in tumour volume in primary breast cancer. The profiles of genes that were most differentially expressed on days 2, 4 and 7 following treatment were able to predict prognosis, whereas those most changed on days 1 and 14 were not, in four tamoxifen treated datasets representing a total of 404 patients.ConclusionsBoth early/transient/proliferation response genes and continuous/late/estrogen-response genes are able to predict prognosis of primary breast tumours in a dynamic manner. Temporal expression of therapy-response genes is clearly an important factor in characterising the response to endocrine therapy in breast tumours which has significant implications for the timing of biopsies in neoadjuvant biomarker studies.
Differential expression of estrogen receptor-alpha (ERalpha) cofactors has been implicated in endocrine resistance in breast cancer. Using a three-stage MCF-7 cell-based model that emulates the clinical manifestation of acquired endocrine resistant breast cancer we now show, using a combination of chromatin immunoprecipitation and RNA interference, that there is a progressive loss of ERalpha cofactor recruitment to the estrogen-dependent pS2 gene and reduced requirement for cofactor expression. Maximal estrogen induced pS2 induction requires ERalpha and cofactor recruitment in MCF-7 cells, but in the progression to endocrine resistance these requirements are altered and expression has become less dependent on cofactors. Additionally, in estrogen-resistant MCF-7 cells there is a global loss of requirement of individual cofactors for proliferative cell growth indicating that other genes have lost the need for transcriptional cofactors. This loss of the requirement for cofactors may represent an important mechanism for gene misregulation in cancer.
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