Aurora kinase inhibitors synergize with paclitaxel to induce apoptosis in ovarian cancer cells.

BACKGROUND: A large percentage of patients with recurrent ovarian cancer develop resistance to the taxane class of chemotherapeutics. While mechanisms of resistance are being discovered, novel treatment options and a better understanding of disease resistance are sorely needed. The mitotic kinase Aurora-A directly regulates cellular processes targeted by the taxanes and is overexpressed in several malignancies, including ovarian cancer. Recent data has shown that overexpression of Aurora-A can confer resistance to the taxane paclitaxel. METHODS: We used expression profiling of ovarian tumor samples to determine the most significantly overexpressed genes. In this study we sought to determine if chemical inhibition of the Aurora kinase family using VE-465 could synergize with paclitaxel to induce apoptosis in paclitaxel-resistant and sensitive ovarian cancer cells. RESULTS: Aurora-A kinase and TPX2, an activator of Aurora-A, are two of the most significantly overexpressed genes in ovarian carcinomas. We show that inhibition of the Aurora kinases prevents phosphorylation of a mitotic marker and demonstrate a dose-dependent increase of apoptosis in treated ovarian cancer cells. We demonstrate at low doses that are specific to Aurora-A, VE-465 synergizes with paclitaxel to induce 4.5-fold greater apoptosis than paclitaxel alone in 1A9 cells. Higher doses are needed to induce apoptosis in paclitaxel-resistant PTX10 cells. CONCLUSION: Our results show that VE-465 is a potent killer of taxane resistant ovarian cancer cells and can synergize with paclitaxel at low doses. These data suggest patients whose tumors exhibit high Aurora-A expression may benefit from a combination therapy of taxanes and Aurora-A inhibition.

Sex-determining region Y box 4 is a transforming oncogene in human prostate cancer cells.

Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene.

Loss of HOXC6 expression induces apoptosis in prostate cancer cells.

We have performed whole genome expression profiling of 28 patient prostate tumor samples and 12 normal prostate samples and identified 55 upregulated and 60 downregulated genes significantly changed in prostate tumor samples compared to normal prostate tissues. Among the members of the upregulated gene set was the developmental transcription factor Homeobox C6 (HOXC6). Silencing of HOXC6 expression using small-interfering RNA (siRNA) resulted in decreased proliferation rates for both androgen-dependent LnCaP cells and the LnCaP-derived androgen-independent C4-2 cell line. Flow cytometry and immunoblotting for the caspase-cleaved form of poly-ADP ribose polymerase (PARP) determined that the decrease in cell numbers was due to increased apoptosis. To validate the specificity of the siRNA-induced apoptosis, LnCaP cells were cotransfected with siRNA specific to the HOXC6 3'UTR and a mammalian expression vector containing the HOXC6 open reading frame, but lacking the 3'UTR. Overexpression of HOXC6 rescued the LnCaP cells from HOXC6 siRNA-induced apoptosis, and increased growth of control GFP siRNA-transfected cells. Expression profiling of HOXC6 siRNA transfections and HOXC6 overexpression identified neutral endopeptidase (NEP) and insulin-like growth factor binding protein-3 (IGFBP-3) as potential proapoptotic repression targets of HOXC6. Our data suggest that HOXC6 may be a novel potential therapeutic target for prostate cancer.

Cross-platform expression profiling demonstrates that SV40 small tumor antigen activates Notch, Hedgehog, and Wnt signaling in human cells.

BACKGROUND: We previously analyzed human embryonic kidney (HEK) cell lines for the effects that simian virus 40 (SV40) small tumor antigen (ST) has on gene expression using Affymetrix U133 GeneChips. To cross-validate and extend our initial findings, we sought to compare the expression profiles of these cell lines using an alternative microarray platform. METHODS: We have analyzed matched cell lines with and without expression of SV40 ST using an Applied Biosystems (AB) microarray platform that uses single 60-mer oligonucleotides and single-color quantitative chemiluminescence for detection. RESULTS: While we were able to previously identify only 456 genes affected by ST with the Affymetrix platform, we identified 1927 individual genes with the AB platform. Additional technical replicates increased the number of identified genes to 3478 genes and confirmed the changes in 278 (61%) of our original set of 456 genes. Among the 3200 genes newly identified as affected by SV40 ST, we confirmed 20 by QRTPCR including several components of the Wnt, Notch, and Hedgehog signaling pathways, consistent with SV40 ST activation of these developmental pathways. While inhibitors of Notch activation had no effect on cell survival, cyclopamine had a potent killing effect on cells expressing SV40 ST. CONCLUSIONS: These data show that SV40 ST expression alters cell survival pathways to sensitize cells to the killing effect of Hedgehog pathway inhibitors.

Signaling and transcriptional changes critical for transformation of human cells by simian virus 40 small tumor antigen or protein phosphatase 2A B56gamma knockdown.

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56gamma subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56gamma subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56gamma knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor kappaB activation, while inhibiting cell-cell adhesion pathways.