Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways.

Cdc25C is a cell cycle protein of the dual specificity phosphatase family essential for activating the cdk1/Cyclin B1 complex in cells entering into mitosis. Since altered cell cycle is a hallmark of human cancers, we investigated androgen regulation of Cdc25C protein in human prostate cancer (PCa) cells, including androgen-sensitive (AS) LNCaP C-33 cells and androgen-independent (AI) LNCaP C-81 as well as PC-3 cells. In the regular culture condition containing fetal bovine serum (FBS), Cdc25C protein levels were similar in these PCa cells. In a steroid-reduced condition, Cdc25C protein was greatly decreased in AS C-33 cells but not AI C-81 or PC-3 cells. In androgen-treated C-33 cells, the Cdc25C protein level was greatly elevated, following a dose- and a time-dependent manner, correlating with increased cell proliferation. This androgen effect was blocked by Casodex, an androgen receptor blocker. Nevertheless, epidermal growth factor (EGF), a growth stimulator of PCa cells, could only increase Cdc25C protein level by about 1.5-fold. Altered expression of Cdc25C in C-33 cells and PC-3 cells by cDNA and/or shRNA transfection is associated with the corresponding changes of cell growth and Cyclin B1 protein level. Actinomycin D and cycloheximide could only partially block androgen-induced Cdc25C protein level. Treatments with both proteasomal and lysosomal inhibitors resulted in elevated Cdc25C protein levels. Immunoprecipitation revealed that androgens reduced the ubiquitination of Cdc25C proteins. These results show for the first time that Cdc25C protein plays a role in regulating PCa cell growth, and androgen treatments, but not EGF, greatly increase Cdc25C protein levels in AS PCa cells, which is in part by decreasing its degradation. These results can lead to advanced PCa therapy via up-regulating the degradation pathways of Cdc25C protein.

Steroids up-regulate p66Shc longevity protein in growth regulation by inhibiting its ubiquitination.

BACKGROUND: p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism. METHODS AND FINDINGS: In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins. CONCLUSIONS: The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.

Coexpression of EphB4 and ephrinB2 in tumor advancement of uterine cervical cancers.

OBJECTIVE: Receptor EphB4 and the corresponding ligand ephrinB2 contribute to tumor growth in various human tumors. This prompted us to study the expression and localization of EphB4 and ephrinB2 in uterine cervical cancers to analyze the EphB4/ephrinB2 functions against clinical backgrounds. METHODS: Immunohistochemistry and real-time RT-PCR have been done to determine the histoscores and mRNA levels of EphB4 and ephrinB2, respectively, in sixty-two uterine cervical cancer tissue samples. Patient prognoses were analyzed with a 36-month survival rate. RESULTS: The localization of EphB4 and ephrinB2 was dominantly in the cancer cells of uterine cervical cancers of all cases given. Both the histoscores and mRNA levels of EphB4 and ephrinB2 significantly increased with clinical stages (I

A novel role of Shc adaptor proteins in steroid hormone-regulated cancers.

Tyrosine phosphorylation plays a critical role in growth regulation, and its aberrant regulation can be involved in carcinogenesis. The association of Shc (Src homolog and collagen homolog) adaptor protein family members in tyrosine phosphorylation signaling pathway is well recognized. Shc adaptor proteins transmit activated tyrosine phosphorylation signaling that suggest their plausible role in growth regulation including carcinogenesis and metastasis. In parallel, by sharing a similar mechanism of carcinogenesis, the steroids are involved in the early stage of carcinogenesis as well as the regulation of cancer progression and metastatic processes. Recent evidence indicates a cross-talk between tyrosine phosphorylation signaling and steroid hormone action in epithelial cells, including prostate and breast cancer cells. Therefore, the members of Shc proteins may function as mediators between tyrosine phosphorylation and steroid signaling in steroid-regulated cell proliferation and carcinogenesis. In this communication, we discuss the novel roles of Shc proteins, specifically p52(Shc) and p66(Shc), in steroid hormone-regulated cancers and a novel molecular mechanism by which redox signaling induced by p66(Shc) mediates steroid action via a non-genomic pathway. The p66(Shc) protein may serve as an effective biomarker for predicting cancer prognosis as well as a useful target for treatment.

Role of protease activated receptor-2 in lymph node metastasis of uterine cervical cancers.

BACKGROUND: Protease activated receptor-2 (PAR-2) has been implicated in cellular proliferation, invasion and metastasis in various tumors. Lymph node metastasis is an important patient prognostic factor for uterine cervical cancers. This prompted us to study the role of PAR-2 in lymph node metastasis of uterine cervical cancers. METHODS: Thirty patients underwent surgery for uterine cervical cancers. PAR-2 histoscores and mRNA levels were determined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction, respectively. Patient prognosis was analyzed with a 48-month survival rate. RESULTS: PAR-2 histoscores and mRNA levels significantly (P < 0.05) increased in 12 of 30 metastatic lymph node lesions from the corresponding primary tumor. The 48-month survival rate of the 12 patients with increased PAR-2 levels in metastatic lymph nodes was 42%, while the rate of the other 18 patients with no change in PAR-2 levels was 82%, regardless of histopathological type. CONCLUSION: PAR-2 might work on lymph node metastasis of uterine cervical cancers, and is considered to be a novel prognostic indicator for uterine cervical cancers.

Clinical implication of estrogen-related receptor (ERR) expression in ovarian cancers.

The expression of estrogen receptor (ER)alpha and ERbeta mRNAs did not show any specific manner according to clinical backgrounds in ovarian cancers. On the other hand, the levels of estrogen-related receptor (ERR)alpha mRNA increased with clinical stages regardless of histopathological types in ovarian cancers. However, ERRbeta and ERRgamma mRNA levels were extremely low to determine reliably. ERRalpha can bind to the steroid receptor coactivator family without any ligands, and drive transcription activity of the target genes. The manner of ERR and ER gene expressions might show an independent usage of common cofactors. It is speculated that the up regulation of ERRalpha might be related to advancement of ovarian cancers regardless of plausible interaction via cofactors regulated by ERs. Although ERRalpha is not directly related to growth of ovarian cancer, ERRalpha is a candidate for prognostic factors for ovarian cancer.

Expression of protease activated receptor-2 related to angiogenesis in tumor advancement of uterine endometrial cancers.

Protease activated receptor-2 (PAR-2) is the second member of a novel family of G-protein coupled seven-transmembrane domain receptors. PAR-2 has been reported to be expressed in various tumors and play a vital role in the regulation of cancer cell growth. The purpose of this study was to clarify the roles of PAR-2 in the angiogenic pathway in uterine endometrial cancers. PAR-2 expression was analyzed in 61 uterine endometrial cancer and 15 normal endometrium tissue specimens. PAR-2 histoscores and mRNA levels were determined by immunohistochemistry and real-time RT-PCR, respectively. Microvessel counts were determined by immunohistochemistry for CD31 and factor VIII-related antigen. The localization of PAR-2 was dominant in the cancer cells of endometrial cancer tissues of all cases studied. PAR-2 histoscores highly correlated with PAR-2 mRNA levels in the same tissues (r=0.87, p<0.001). PAR-2 histoscores and mRNA levels both significantly increased in uterine endometrial cancers with clinical stages (I

Expression of cyclooxygenase-2 related to angiogenesis in uterine cervical cancers.

Angiogenesis is essential for development, growth and advancement of solid tumors. Cyclooxygenase (COX)-2 is recognized as an angiogenic factor in various tumors. This prompted us to study the clinical implications of COX-2 expression related to angiogenesis in uterine cervical cancers. There was a significant correlation between microvessel counts and COX-2 levels in uterine cervical cancers. COX-2 localized in the cancer cells, but not in the stromal cells of uterine cervical cancer tissues. COX-2 levels increased with advancement, and the prognosis of the 30 patients with high COX-2 expression in uterine cervical cancers was poor (60%), while the 24-month survival rate of the other 30 patients with low COX-2 expression was 90%. Furthermore, COX-2 levels significantly correlated with VEGF levels in uterine cervical cancers. VEGF associated with COX-2 might work on angiogenesis in advancement. Therefore, long-term administration of COX-2 inhibitors might be effective on the suppression of regrowth or recurrence after intensive treatment for advanced uterine cervical cancers.

Plausible linkage of hypoxia inducible factor-1alpha in uterine cervical cancer.

Angiogenesis is essential for the development, growth and advancement of solid tumors. Angiogenesis is induced by hypoxia with angiogenic transcription factor hypoxia inducible factors (HIF). This prompted us to study the clinical implications of HIF relative to angiogenesis in uterine cervical cancers. Although there was no significant difference in HIF-1alpha histoscores and mRNA levels according to histopathological type or lymph node metastasis, HIF-1alpha histoscores and mRNA levels increased significantly with advancing cancer stages. The prognosis of 30 patients with high HIF-1alpha in uterine cervical cancers was poor (73% survival), whereas the 24-month survival rate of the other 30 patients with low HIF-1alpha was 93%. HIF-1alpha histoscores and mRNA levels were correlated with the levels of the angiogenic factors thymidine phosphorylase and interleukin-8, and HIF-1alpha might be linked with these factors in cervical cancer tissue. HIF-1alpha is a candidate for prognostic indicator as an angiogenic mediator in uterine cervical cancer.

Clinical implications of expression of cyclooxygenase-2 related to angiogenesis in ovarian cancer.

Angiogenesis is essential for the development, growth and advancement of solid tumors. Cyclooxygenase (cox)-2 is recognized as an angiogenic factor in various tumors. This prompted us to study the clinical implications of cox-2 expression and angiogenesis in ovarian cancer. There was a significant correlation between microvessel counts and cox-2 levels. Cox-2 localized in the cancer cells, but not in the stromal cells of ovarian cancer tissue. Cox-2 levels increased with the advancement, and the prognosis of the 30 patients with high cox-2 expression was extremely poor (33%), while the 24-month survival rate of the other 30 patients, those with low cox-2 expression, was 67%. Furthermore, cox-2 levels significantly correlated with VEGF levels. VEGF associated with cox-2 might work on angiogenesis with advancement. Therefore, long-term administration of cox-2 inhibitors might be effective on the suppression of regrowth or recurrence after intensive treatment for advanced ovarian cancer.

Plausible linkage of hypoxia-inducible factor (HIF) in uterine endometrial cancers.

OBJECTIVE: Angiogenesis is essential for the development, growth and advancement of solid tumors. Angiogenesis is induced by hypoxia with the angiogenic transcription factor hypoxia-inducible factor (HIF). This prompted us to study the clinical implications of HIF relative to angiogenesis in uterine endometrial cancers. METHODS: Sixty patients underwent curative resection for uterine endometrial cancers. In the tissue of 60 uterine endometrial cancers, HIF-1alpha, HIF-2alpha and HIF-1beta mRNA levels, and the ratio of angiopoietin (Ang)-2 to Ang-1 (Ang-2/Ang-1) mRNA levels were determined by RT real-time PCR; histochemical scores and localization of HIF-1alpha were determined by immunohistochemistry. Levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP) and interleukin-8 (IL-8) were determined by enzyme immunoassay. RESULTS: In stage I uterine endometrial cancers, HIF-1alpha histochemical scores and mRNA levels significantly increased with myometrial invasion of uterine endometrial cancers. HIF-1alpha histochemical scores and mRNA levels correlated with the levels of Ang-2/Ang-1 and IL-8. CONCLUSION: The angiogenic mediator HIF-1alpha, linked to Angs and IL-8, might work on angiogenesis with myometrial invasion of cancer cells in uterine endometrial cancers.

Sex steroid-dependent angiogenesis in uterine endometrial cancers.

In general, tumors induce angiogenic factors specific to them, which leads to angiogenesis with advancement. However, angiogenesis in uterine endometrial cancers is complicated because hormone dependency in growth also modifies the angiogenic potential. Therefore, anti-angiogenic therapy for tumor dormancy in uterine endometrial cancers must be thoroughly considered. The upstream of vascular endothelial growth factor (VEGF) gene conserves estrogen-responsive elements. Progesterone primed with estrogen induces thymidine phosphorylase (TP) in uterine endometrium. Sex steroid-dependent VEGF and TP are highly expressed in cases of early stage and well-differentiated uterine endometrial cancers, and basic fibroblast growth factor (bFGF) in cases of advanced and poorly differentiated uterine endometrial cancers. A transcriptional factor for angiogenesis, ETS-1, is linked to VEGF in well-differentiated uterine endometrial cancers, and to bFGF in poorly differentiated uterine endometrial cancers. Therefore, even if dedifferentiation and angiogenic switching occur due to advancement and long-term hormone therapy, the inhibition of ETS-1 along with main angiogenic factors might be an effective strategy to suppress uterine endometrial cancers as a novel anti-angiogenic therapy.