Gene expression profiling in R-flurbiprofen-treated prostate cancer: R-Flurbiprofen regulates prostate stem cell antigen through activation of AKT kinase.

We have used gene expression profiling to characterize genes regulated by the anti-tumor non-steroidal anti-inflammatory drug (NSAID)-like agent R-flurbiprofen (RFB) in murine TRAMP prostate cancer. Mice with spontaneous, palpable tumors were treated with RFB 25 mg/(kgd) x 7d orally, or vehicle only. RNA was then extracted from tumor tissue and used for microarray analysis with Affymetrix chips. Fifty-eight genes were reproducibly regulated by RFB treatment. One of the most highly up-regulated genes was prostate stem cell antigen (psca). We used TRAMP C1 murine prostate cancer cells to examine potential mechanisms through which RFB could regulate psca. RFB induced dose-dependent expression of PSCA protein, and activity of the psca promoter, in TRAMP C1 cells in culture. Increased psca promoter activity was also seen following treatment of cells with sulindac sulfone, another NSAID-like agent, but not with celecoxib treatment. RFB activation of the psca promoter could be attenuated by co-transfection of dominant-negative akt and h-ras constructs, but not by dominant-negative mek1 plasmids. Immunoblotting revealed that RFB increased expression of phosphorylated AKT at concentrations that stimulated psca promoter activity, and that increased PSCA protein expression. In addition, RFB-dependent up-regulation of PSCA protein expression could be blocked by AKT inhibitors. These data demonstrate that RFB, and possibly other NSAID-like analogs, can increase expression of the psca gene both in vivo and in culture. They further suggest the utility of combining RFB with AKT inhibitors or with monoclonal antibodies targeting PSCA protein, for treatment or prevention of prostate cancer.

Overexpression of HPV16 E6* Alters ß-Integrin and Mitochondrial Dysfunction Pathways in Cervical Cancer Cells.

BACKGROUND: High-risk human papillomaviruses (HPV) cause nearly all cases of cervical cancer, as well as many types of oral and anogenital cancer. Alternative splicing increases the capacity of the HPV genome to encode the proteins necessary for successful completion of its infectious life cycle. However, the roles of these splice variants, including E6*, the smaller splice isoform of the E6 oncogene, in carcinogenesis are not clear. MATERIALS AND METHODS: SiHa (HPV16(+)) and C33A (HPV(-)) cells were transfected with the E6* plasmid, and tandem mass tag-labeled protein levels were quantified by mass spectrometry. Proteomic analyses identified pathways affected by E6* in both HPV(+) and HPV(-) cells, and pathways were validated using in vitro methods. RESULTS: A total of 4,300 proteins were identified and quantified in lysates of SiHa and C33A cells with and without HPV16 E6* expression. SiHa and C33A cells expressing E6* underwent changes in protein expression affecting integrin signaling and mitochondrial dysfunction pathways, respectively. Subsequent experiments were performed to validate selected E6*-mediated alterations in protein levels. CONCLUSION: E6* modifies the expression of proteins involved in mitochondrial dysfunction and oxidative phosphorylation in C33A cells, and ß-integrin signaling in SiHa cells.

Cellular levels of oxidative stress affect the response of cervical cancer cells to chemotherapeutic agents.

Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT-PCR array. The analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress affect responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy.

The PIM1 kinase is a critical component of a survival pathway activated by docetaxel and promotes survival of docetaxel-treated prostate cancer cells.

A defining characteristic of solid tumors is the capacity to divide aggressively and disseminate under conditions of nutrient deprivation, limited oxygen availability, and exposure to cytotoxic drugs or radiation. Survival pathways are activated within tumor cells to cope with these ambient stresses. We here describe a survival pathway activated by the anti-cancer drug docetaxel in prostate cancer cells. Docetaxel activates STAT3 phosphorylation and transcriptional activity, which in turns induces expression of the PIM1 gene, encoding a serine-threonine kinase activated by many cellular stresses. Expression of PIM1 improves survival of docetaxel-treated prostate cancer cells, and PIM1 knockdown or expression of a dominant-negative PIM1 protein sensitize cells to the cytotoxic effects of docetaxel. PIM1 in turn mediates docetaxel-induced activation of NFkappaB transcriptional activity, and PIM1 depends in part on RELA/p65 proteins for its prosurvival effects. The PIM1 kinase plays a critical role in this STAT3 --> PIM1 --> NFkappaB stress response pathway and serves as a target for intervention to enhance the therapeutic effects of cytotoxic drugs such as docetaxel.