Abby Sin-Chi Mak

c-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/β-catenin–ATP-binding cassette G2 signaling

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.

Vitellogenesis in the Sand Shrimp, Metapenaeus ensis: The Contribution from the Hepatopancreas-Specific Vitellogenin Gene (MeVg2)

Abstract An additional vitellogenin gene (MeVg2) that is structurally different from MeVg1 was cloned and characterized from the shrimp Metapenaeus ensis. The MeVg2 gene consists of fewer exons-introns and is most likely evolved from the MeVg1 gene. The cDNA for MeVg2 is 8.0 kilobases (kb) in size, and the deduced MeVg2 precursor shared an overall 54% amino sequence identity to the MeVg1 gene of the same shrimp. As compared to the MeVg1 precursor, MeVg2 precursor consists of more potential subunit cleavage sites, suggesting that the precursor may be processed into many smaller subunits. The MeVg2 is expressed only in the hepatopancreas, and the expression level of MeVg2 in adult female increases from the early vitellogenic stage, reaching a maximum at the middle vitellogenic stage, and remains high toward the end of vitellogenic cycle. In addition to the 8-kb mRNA, smaller transcripts of 1.5–2.5 kb for MeVg2 were identified, and the 8-kb transcript only constitutes less than 10% of the overall MeVg2-derived transcripts. To confirm the presence of the small transcripts, we screened the shrimp hepatopancreas cDNA library and isolated two smaller MeVg2-specific cDNA clones. These clones shared greater than 99% overall identity to the corresponding C-terminal region of the MeVg2 precursor, suggesting that an alternative expression/ splicing of the MeVg2 gene occurred. By immunohistochemical analysis, vitellin-immunopositive signals were localized in the lumen and extracellular fraction of the hepatopancreas. Amino acid sequence determination of the tissue protein and secreted protein from the hepatopancreas revealed that the 76-kDa vitellogenin subunit is most likely processed into smaller-sized subunits. Taken together, these results suggest that the hepatopancreas is an important organ for the synthesis of vitellogenin and may contribute to vitellogenesis by producing a large quantity of smaller MeVg2 subunit for ovarian uptake.

Vitellogenesis in the Red Crab, Charybdis feriatus: Contributions from Small Vitellogenin Transcripts (CfVg) and Farnesoic Acid Stimulation of CfVg Expression

Abstract: During reproductive maturation of the female red crab, Charybdis feriatus, the oocytes rapidly accumulate 110‐ and 78‐kDa major polypeptides. Although the hepatopancreas expresses a high level of vitellogenin (CfVg) mRNA, tissue proteins and secreted proteins of the hepatopancreas consist of only small polypeptides. In addition to the 8.0‐kb transcripts, many smaller mRNAs specific to the CfVg gene can be detected. These results suggest that the hepatopancreas also produces smaller CfVg transcripts for small CfVg subunits. Using an RT‐PCR cloning approach, a population of the small cDNA clones were isolated. Determining the DNA sequence of these clones revealed that these transcripts were most likely the result of alternative splicing and/or alternative expression of the CfVg gene. In vitro treatment of the hepatopancreas fragments with low levels of farnesoic acid stimulated the expression of CfVg.

P-cadherin cooperates with insulin-like growth factor-1 receptor to promote metastatic signaling of gonadotropin-releasing hormone in ovarian cancer via p120 catenin.

Gonadotropin-releasing hormone (GnRH) is a potent prometastatic factor in ovarian cancer, but the intracellular signaling events are not well understood. The classical Gαq-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 (p120ctn), 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 p120ctn activation, confirming that the effect was P-cadherin specific. On exploring how P-cadherin, which lacks intrinsic kinase activity, might regulate the activation of p120ctn, 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 p120ctn 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 p120ctn 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.

β-catenin downregulates Dicer to promote ovarian cancer metastasis.

Ovarian cancer is a nearly uniform lethal disease and its highly aggressive metastatic phenotype portends a poor prognosis. Lack of a well-controlled, relevant experimental model has been a major obstacle to identifying key molecules causing metastasis. Here we describe the creation of a new isogenic model of spontaneous human ovarian cancer metastasis exhibiting opposite phenotypes—highly metastatic (HM) and non-metastatic (NM)—both in vitro and in vivo. HM was unique in its ability to metastasize consistently to the peritoneum, mimicking the major dissemination route of human ovarian cancer. In contrast, NM failed to form detectable metastases, although it was equally tumorigenic. Using comparative label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified β-catenin, which we demonstrated for the first time as having a direct role in the pathogenesis of ovarian cancer metastasis. Our studies also revealed a previously unrecognized role of β-catenin in the downregulation of multiple microRNAs (miRNAs) through attenuating miRNA biogenesis by targeting Dicer, a key component of the miRNA-processing machinery. One such downregulated miRNAs was miR-29s involved in epithelial-to-mesenchymal transition and subsequent stem cell traits. Silencing β-catenin or overexpressing Dicer or miR-29 mimics in HM significantly reduced the ability of these cells to migrate. β-catenin-knockdown cells also failed to metastasize in an orthotopic model of ovarian cancer. Meta-analysis revealed an increase in CTNNB1 and a decrease in DICER1 expression levels in the high-risk group. These results uncover β-catenin as a critical factor in promoting ovarian cancer aggressiveness and a new mechanism linking between β-catenin and miRNA downregulation underlying this process.

A novel p70 S6 kinase-microRNA biogenesis axis mediates multicellular spheroid formation in ovarian cancer progression

Ovarian cancer is the leading cause of death of all gynecologic tumors, associated with widespread peritoneal dissemination and malignant ascites. Key to this is the ability to form multicellular spheroids (MCS); however, the tumor-specific factors that regulate MCS formation are unclear. p70 S6 kinase (p70S6K), which is a downstream effector of phosphatidylinositol 3-kinase/Akt, is frequently constitutively active in ovarian carcinoma. Here we identify p70S6K as a vital regulator of MCS formation. We also uncover a new mechanism of p70S6K function as a component of the microRNA biogenesis machinery in this process. We show that p70S6K phosphorylates, and inhibits the interaction of tristetraprolin (TTP) and Dicer that promotes the expression of a subset of miRNAs, including the maturation of miR-145. Twist and Sox9 are two divergent targets of miR-145, thereby enhancing N-cadherin, but not other cadherin, expression and MCS formation. Activating miR-145 suppresses ovarian tumor growth and metastasis in an orthotopic xenograft mouse model. Meta-analysis in the Oncomine database reveals that high p70S6K and low TTP levels are associated with ovarian tumor progression. These results define a critical link between p70S6K, miRNA maturation, and MCS formation that may underlie poor clinical outcome of ovarian cancer patients for developing novel therapeutic strategies.