[show abstract][hide abstract] ABSTRACT: Cisplatin and paclitaxel are standard chemotherapy for metastatic ovarian cancer, but with limited efficacy. Cancer stem/progenitor cells (or tumor-initiating cells, TICs) are hypothesized to be chemoresistant, and the existence of TICs in ovarian cancer has been previously demonstrated. However, the key signals and molecular events regulating the formation and expansion of ovarian tumor-initiating cells (OTICs) remain elusive. Here, we show that c-Kit is not just a marker of OTICs, but also a critical mediator of the phenotype that can be a viable target for the treatment of ovarian cancer. In contrast to non-OICs, c-Kit was overexpressed in OTICs. Moreover, the use of small interfering RNA to inhibit c-Kit expression markedly attenuated the number and size of OTIC subpopulations, inhibited the expression of stem cell markers and decreased the tumorigenic capabilities of OTICs. Imatinib (Gleevec), a clinical drug that blocks c-Kit kinase activity, also demonstrated its inhibition potency on OTICs. In addition, cisplatin/paclitaxel, which killed non-OTICs, with c-Kit knockdown or imatinib revealed that this was critically required for intervening ovarian cancer progression and recurrence in vitro and in xenograft tumors in vivo. Similar results were obtained with OTICs derived from ovarian carcinoma patients. Studies into the mechanisms suggest an important role for the activation of Wnt/β-catenin and ATP-binding cassette G2 downstream of c-Kit. The tumor-promoting microenvironment, such as hypoxia, could promote OTICs via upregulation of c-Kit expression. These results unravel an integral role for c-Kit in ovarian neoplastic processes and shed light on its mechanisms of action.Oncogene advance online publication, 16 July 2012; doi:10.1038/onc.2012.290.
[show abstract][hide abstract] ABSTRACT: The PFTK1 gene encodes a cdc2-related serine/threonine protein kinase that has been shown to confer cell migratory properties in hepatocellular carcinoma (HCC). However, the prognostic value and biological mechanism by which PFTK1 promotes HCC motility remain largely unknown. Here, we showed from tissue microarray that common upregulations of PFTK1 in primary HCC tumors (n=133/180) correlated significantly with early age onset (40 years), advance tumor grading and presence of microvascular invasion (P0.05). To understand downstream phosphorylated substrate(s) of PFTK1, phospho-proteins in PFTK1 expressing and knockdown Hep3B cells were profiled by two-dimensional-polyacrylamide gel electrophoresis mass spectrometric analysis. Protein identification of differential spots revealed β-actin (ACTB) and transgelin2 (TAGLN2) as the two most profound phosphorylated changes affected by PFTK1. We verified the presence of TAGLN2 serine phosphorylation and ACTB tyrosine phosphorylation. Moreover, reduced TAGLN2 and ACTB phosphorylations in PFTK1-suppressed Hep3B corresponded to distinct actin depolymerizations and marked inhibition on cell invasion and motility. Given that TAGLN2 is a tumor suppressor whose function has been ascribed in cancer metastasis, we examined if TAGLN2 is an intermediate substrate in the biological path of PFTK1. We showed in PFTK1-suppressed cells that knockdown of TAGLN2 over-rode the inhibitory effect on cell invasion and motility, and a recovery on actin polymerization was evident. Interestingly, we also found that unphosphorylated TAGLN2 in PFTK1-suppressed cells elicited strong actin-binding ability, a mechanism that possibly halts the actin cytoskeleton dynamics. Site-directed mutagenesis of TAGLN2 suggested that PFTK1 regulates the actin-binding affinity of TAGLN2 through the S83 and S163 residues, which if mutated can significantly affect HCC cell motility. Taken together, our data propose a novel, oncogene-tumor suppressor interplay, where oncogenic PFTK1 confers HCC cell motility through inactivating the actin-binding motile suppressing function of TAGLN2 via phosphorylation.
[show abstract][hide abstract] ABSTRACT: Gonadotropin-releasing hormone (GnRH) is a potent prometastatic factor in ovarian cancer, but the intracellular signaling events are not well understood. The classical Gα(q)-phospholipase C signal transduction pathway known to operate in the pituitary is not involved in GnRH actions at non-pituitary targets. Here we showed that GnRH treatment of ovarian cancer cells led to a rapid and remarkable tyrosine phosphorylation of p120 catenin (p120(ctn)), which was mediated by P-cadherin. The use of P-cadherin small interfering RNA or neutralizing antibodies to inhibit P-cadherin expression and function resulted in diminished p120(ctn) activation, confirming that the effect was P-cadherin specific. On exploring how P-cadherin, which lacks intrinsic kinase activity, might regulate the activation of p120(ctn), we found that P-cadherin could induce the ligand-independent activation of insulin-like growth factor-1 receptor (IGF-1R). Inhibition of IGF-1R expression or its activity significantly inhibited GnRH-induced p120(ctn) activation, and the subsequent cell migration and invasion. In addition, we showed that IGF-1R regulation by P-cadherin was associated with complex formation between IGF-1R and P-cadherin, and this regulation was also observed to be in vivo correlated with metastasis. Furthermore, using a mouse model of ovarian cancer metastasis, GnRH receptor knockdown was shown to diminish peritoneal dissemination of tumors and ascites formation. These findings suggest for the first time that GnRH can initiate an outside-in p120(ctn) signal transduction through the cross-talk between P-cadherin and IGF-1R, thus providing a novel molecular mechanism by which GnRH may control the high level of aggressiveness and invasion and metastasis potential that are characteristic of ovarian cancer.
[show abstract][hide abstract] ABSTRACT: Ovarian cancer is highly metastatic with a poor prognosis. The serine/threonine kinase, p70 S6 kinase (p70(S6K)), which is a downstream effector of phosphatidylinositol 3-kinase/Akt pathway, is frequently activated in ovarian cancer. Here, we show that p70(S6K) is a critical regulator of the actin cytoskeleton in the acquisition of the metastatic phenotype. This regulation is through two important activities: p70(S6K) acts as an actin filament cross-linking protein and as a Rho family GTPase-activating protein. Ectopic expression of constitutively active p70(S6K) in ovarian cancer cells induced a marked reorganization of the actin cytoskeleton and promoted directional cell migration. Using cosedimentation and differential sedimentation assays, p70(S6K) was found to directly bind to and cross-link actin filaments. Immunofluorescence studies showed p70(S6K) colocalized with cytochalasin D-sensitive actin at the leading edge of motile cells. The p70(S6K) did not affect the kinetics of spontaneous actin polymerization, but could stabilize actin filaments by the inhibition of cofilin-induced actin depolymerization. In addition, we showed that p70(S6K) stimulated the rapid activation of both Rac1 and Cdc42, and their downstream effector p21-activated kinase (PAK1), but not RhoA. Depletion of p70(S6K) expression or inhibition of its activity resulted in significant inhibition of actin cytoskeleton reorganization and reduced migration, with a concomitant reduction in Rac1, Cdc42 and PAK1 activation, confirming that the effect was p70(S6K) specific. Similarly, the actin cytoskeleton reorganization/migratory phenotype could be reversed by expression of dominant negative Rac1 and Cdc42, or inhibition of PAK1. These results reveal a new direction for understanding the oncogenic roles of p70(S6K) in tumor progression.
[show abstract][hide abstract] ABSTRACT: Gonadotropin-releasing hormone (GnRH) receptor expression is often elevated in ovarian cancer, but its potential role in ovarian cancer metastasis has just begun to be revealed. Cadherin switching is a crucial step during tumorigenesis, particularly in metastasis. Here, we showed that GnRH is an inducer of E- to P-cadherin switching, which is reminiscent of that seen during ovarian tumor progression. Overexpression of P-cadherin significantly enhanced, whereas knockdown of P-cadherin reduced migration and invasion regardless of E-cadherin expression, suggesting that inappropriate expression of P-cadherin contributes to the invasive phenotype. These effects of P-cadherin were mediated by activation of the Rho GTPases, Rac1, and Cdc42, through accumulation of p120 catenin (p120(ctn)) in the cytoplasm. The use of p120(ctn) small interfering RNA or chimeric cadherin construct to inhibit p120(ctn) expression and cytoplasmic localization, respectively, resulted in significant inhibition of cell migration and invasion, with a concomitant reduction in Rac1 and Cdc42 activation, confirming that the effect was p120(ctn) specific. Similarly, the migratory/invasive phenotype could be reversed by expression of dominant-negative Rac1 and Cdc42. These results identify for the first time cadherin switching and p120(ctn) signaling as important targets of GnRH function and as novel mediators of invasiveness and tumor progression in ovarian cancer.
[show abstract][hide abstract] ABSTRACT: The tumor suppressor BRCA1 is mutated in a high percentage of familial breast and ovarian cancer, but our understanding of its mechanisms of action remains incomplete. We report here that glucose-regulated protein (GRP)-78, a critical regulator of the unfolded protein response (UPR), is a novel downstream target of BRCA1. We showed that overexpression of wild-type BRCA1 suppressed the expression of GRP78, whereas expression of mutant BRCA1 gene or targeted inhibition of endogenous BRCA1 using small-interfering RNA (siRNA) enhanced GRP78 expression. Knockdown of BRCA1 also led to induction of other components of UPR, such as GRP94 and CHOP. Consistent with a role of BRCA1 knockdown in mediating cell survival, forced expression of GRP78 stimulated cell proliferation and prevented apoptosis, including that induced by endoplasmic reticulum stress and chemotherapy, in ovarian OVCAR-3 and breast MCF-7 cancer cells. Overexpression of wild-type BRCA1 could increase the apoptosis of GRP78-overexpressing cells. Conversely, knockdown GRP78 by siRNA sensitized ovarian and breast cancer cells to apoptosis. This effect was reduced when the expression of BRCA1 was simultaneously knockdown by siRNA, indicating that BRCA1 also negatively regulates GRP78-mediated cell survival and resistance to apoptosis.
[show abstract][hide abstract] ABSTRACT: Chemoresistance and therapeutic selectivity are major obstacles to successful chemotherapy of ovarian cancer. Manganese superoxide disumutase (MnSOD) is an important antioxidant enzyme responsible for the elimination of superoxide radicals. We reported here that MnSOD was significantly elevated in ovarian cancer cells and its overexpression was one of the mechanisms that increased resistance to apoptosis in cancer cells. Knockdown of MnSOD by small-interfering RNA (siRNA) led to an increase in superoxide generation and sensitisation of ovarian cancer cells to the two front-line anti-cancer agents doxorubicin and paclitaxel whose action involved free-radical generation. This synergistic effect was not observed in non-transformed ovarian surface epithelial cells. Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway. Evaluation of signalling pathways showed that MnSOD siRNA enhanced doxorubicin- and paclitaxel-induced phosphorylation of extracellular signal-regulated kinase 1/2. Akt activation was not affected. These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.
British Journal of Cancer 08/2008; 99(2):283-93. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Angiogenesis is a crucial step in tumour growth and metastasis. Ginsenoside-Rb1 (Rb1), the major active constituent of ginseng, potently inhibits angiogenesis in vivo and in vitro. However, the underlying mechanism remains unknown. We hypothesized that the potent anti-angiogenic protein, pigment epithelium-derived factor (PEDF), is involved in regulating the anti-angiogenic effects of Rb1.
Rb1-induced PEDF was determined by real-time PCR and western blot analysis. The anti-angiogenic effects of Rb1 were demonstrated using endothelial cell tube formation assay. Competitive ligand-binding and reporter gene assays were employed to indicate the interaction between Rb1 and the oestrogen receptor (ER).
Rb1 significantly increased the transcription, protein expression and secretion of PEDF. Targeted inhibition of PEDF completely prevented Rb1-induced inhibition of endothelial tube formation, suggesting that the anti-angiogenic effect of Rb1 was PEDF specific. Interestingly, the activation of PEDF occurred via a genomic pathway of ERbeta. Competitive ligand-binding assays indicated that Rb1 is a specific agonist of ERbeta, but not ERalpha. Rb1 effectively recruited transcriptional activators and activated an oestrogen-responsive reporter gene. Furthermore, Rb1-mediated PEDF activation and the subsequent inhibition of tube formation were blocked by the ER antagonist ICI 182,780 or transfection of ERbeta siRNA, indicating ERbeta dependence.
Here we show for the first time that the Rb1 suppressed the formation of endothelial tube-like structures through modulation of PEDF via ERbeta. These findings demonstrate a novel mechanism of the action of this ginsenoside that may have value in anti-cancer and anti-angiogenesis therapy.
British Journal of Pharmacology 10/2007; 152(2):207-15. · 5.07 Impact Factor