BAD-mediated apoptotic pathway is associated with human cancer development.

The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, p<0.001), breast (n=185, p<0.0008; n=61, p=0.04), colon (n=22, p<0.001) and endometrial (n=33, p<0.001) cancers, as well as with ovarian endometriosis (n=20, p<0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, p<0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD.

Gene expression data reveal common pathways that characterize the unifocal nature of ovarian cancer.

OBJECTIVE: The objective of the study was to evaluate the biological validity of ovarian cancer (OVCA) screening and early detection efforts and to characterize signaling pathways associated with human cancer metastasis and patient survival. STUDY DESIGN: Using genome-wide expression profiling and deoxyribonucleic acid sequencing, we compared pelvic and matched extrapelvic implants from 30 patients with advanced-stage OVCA for expression of molecular signaling pathways and p53 gene mutations. Differentially expressed pathways were further evaluated in a series of primary or early-stage vs metastatic or recurrent cancer samples from 389 ovarian, prostate, and oral cancer patients. Metastasis pathways were also evaluated for associations with survival in 9 independent clinicogenomic datasets from 1691 ovarian, breast, colon, brain, and lung cancer and leukemia patients. The inhibitory effects of 1 pathway (transforming growth factor [TGF]-WNT) on in vitro OVCA cell migration were studied. RESULTS: Pelvic and extrapelvic OVCA implants demonstrated similar patterns of signaling pathway expression and identical p53 mutations. However, we identified 3 molecular pathways/cellular processes that were differentially expressed between pelvic and extrapelvic OVCA samples and between primary/early-stage and metastatic/advanced or recurrent ovarian, oral, and prostate cancers. Furthermore, their expression was associated with overall survival from ovarian cancer (P = .006), colon cancer (1 pathway at P = .005), and leukemia (P = .05). Artesunate-induced TGF-WNT pathway inhibition impaired OVCA cell migration. CONCLUSION: Advanced-stage OVCA has a unifocal origin in the pelvis. Molecular pathways associated with extrapelvic OVCA spread are also associated with metastasis from other human cancers and with overall patient survival. Such pathways represent appealing therapeutic targets for patients with metastatic disease.

Characterizing the efficacy of fermented wheat germ extract against ovarian cancer and defining the genomic basis of its activity.

OBJECTIVE: Most women with advanced-stage epithelial ovarian cancer (OVCA) ultimately develop chemoresistant recurrent disease. Therefore, a great need to develop new, more active, and less toxic agents and/or to optimize the efficacy of existing agents exists. METHODS: In this study, we investigated the activity of Avemar, a natural, nontoxic, fermented wheat germ extract (FWGE), against a range of OVCA cell lines, both alone and in combination with cisplatin chemotherapy and delineated the molecular signaling pathways that underlie FWGE activity at a genome-wide level. RESULTS: We found that FWGE exhibited significant antiproliferative effects against 12 human OVCA cell lines and potentiated cisplatin-induced apoptosis. Pearson correlation of FWGE sensitivity and gene expression data identified 2142 genes (false discovery rate < 0.2) representing 27 biologic pathways (P < 0.05) to be significantly associated with FWGE sensitivity. A parallel analysis of genomic data for 59 human cancer cell lines matched to chemosensitivity data for 2,6-dimethoxy-p-benzoquinone, a proposed active component of FWGE, identified representation of 13 pathways common to both FWGE and 2,6-dimethoxy-p-benzoquinone sensitivity. CONCLUSIONS: Our findings confirm the value of FWGE as a natural product with anticancer properties that may also enhance the activity of existing therapeutic agents. Furthermore, our findings provide substantial insights into the molecular basis of FWGE's effect on human cancer cells.

BCL2 antagonist of cell death kinases, phosphatases, and ovarian cancer sensitivity to cisplatin.

OBJECTIVE: The BCL2 family proteins are critical mediators of cellular apoptosis and, as such, have been implicated as determinants of cancer cell chemo-sensitivity. Recently, it has been demonstrated that the phosphorylation status of the BCL2 antagonist of cell death (BAD) protein may influence ovarian cancer (OVCA) cell sensitivity to cisplatin. Here, we sought to evaluate how kinase and phosphatase components of the BAD apoptosis pathway influence OVCA chemo-sensitivity. METHODS: Protein levels of cyclin-dependent kinase 1 (CDK1) and protein phosphatase 2C (PP2C) were measured by immunofluorescence in a series of 64 primary advanced-stage serous OVCA patient samples. In parallel, levels of cAMP-dependent protein kinase (PKA), AKT, and PP2C were quantified by Western blot analysis in paired mother/daughter platinum-sensitive/resistant OVCA cell lines (A2008/C13, A2780S/A2780CP, Chi/ChiR). BAD pathway kinase CDK1 was depleted using siRNA transfection, and the influence on BAD phosphorylation and cisplatin-induced apoptosis was evaluated. RESULTS: OVCA patient samples that demonstrated complete responses to primary platinum-based therapy demonstrated 4-fold higher CDK1 (p<0.0001) and 2-fold lower PP2C (p=0.14) protein levels than samples that demonstrated incomplete responses. Protein levels of PP2C were lower in the platinum-resistant versus that shown in the platinum-sensitive OVCA cell line sub-clones. Levels of PKA were higher in all platinum-resistant than in platinum-sensitive OVCA cell line sub-clones. Selective siRNA depletion of CDK1 increased sensitivity to cisplatin-induced apoptosis (p<0.002). CONCLUSION: BAD pathway kinases and phosphatases, including CDK1 and PP2C, are associated with OVCA sensitivity to platinum and may represent therapeutic opportunities to enhance cytotoxic efficacy.

BAD phosphorylation determines ovarian cancer chemosensitivity and patient survival.

PURPOSE: Despite initial sensitivity to chemotherapy, ovarian cancers (OVCA) often develop drug resistance, which limits patient survival. Using specimens and/or genomic data from 289 patients and a panel of cancer cell lines, we explored genome-wide expression changes that underlie the evolution of OVCA chemoresistance and characterized the BCL2 antagonist of cell death (BAD) apoptosis pathway as a determinant of chemosensitivity and patient survival. EXPERIMENTAL DESIGN: Serial OVCA cell cisplatin treatments were performed in parallel with measurements of genome-wide expression changes. Pathway analysis was carried out on genes associated with increasing cisplatin resistance (EC(50)). BAD-pathway expression and BAD protein phosphorylation were evaluated in patient samples and cell lines as determinants of chemosensitivity and/or clinical outcome and as therapeutic targets. RESULTS: Induced in vitro OVCA cisplatin resistance was associated with BAD-pathway expression (P < 0.001). In OVCA cell lines and primary specimens, BAD protein phosphorylation was associated with platinum resistance (n = 147, P < 0.0001) and also with overall patient survival (n = 134, P = 0.0007). Targeted modulation of BAD-phosphorylation levels influenced cisplatin sensitivity. A 47-gene BAD-pathway score was associated with in vitro phosphorylated BAD levels and with survival in 142 patients with advanced-stage (III/IV) serous OVCA. Integration of BAD-phosphorylation or BAD-pathway score with OVCA surgical cytoreductive status was significantly associated with overall survival by log-rank test (P = 0.004 and P < 0.0001, respectively). CONCLUSION: The BAD apoptosis pathway influences OVCA chemosensitivity and overall survival, likely via modulation of BAD phosphorylation. The pathway has clinical relevance as a biomarker of therapeutic response, patient survival, and as a promising therapeutic target.