Constitutive proteasomal degradation of TWIST-1 in epithelial-ovarian cancer stem cells impacts differentiation and metastatic potential.

Epithelial-mesenchymal transition (EMT) is a critical process for embryogenesis but is abnormally activated during cancer metastasis and recurrence. This process enables epithelial cancer cells to acquire mobility and traits associated with stemness. It is unknown whether epithelial stem cells or epithelial cancer stem cells are able to undergo EMT, and what molecular mechanism regulates this process in these specific cell types. We found that epithelial-ovarian cancer stem cells (EOC stem cells) are the source of metastatic progenitor cells through a differentiation process involving EMT and mesenchymal-epithelial transition (MET). We demonstrate both in vivo and in vitro the differentiation of EOC stem cells into mesenchymal spheroid-forming cells (MSFCs) and their capacity to initiate an active carcinomatosis. Furthermore, we demonstrate that human EOC stem cells injected intraperitoneally in mice are able to form ovarian tumors, suggesting that the EOC stem cells have the ability to 'home' to the ovaries and establish tumors. Most interestingly, we found that TWIST-1 is constitutively degraded in EOC stem cells, and that the acquisition of TWIST-1 requires additional signals that will trigger the differentiation process. These findings are relevant for understanding the differentiation and metastasis process in EOC stem cells.

TWISTing stemness, inflammation and proliferation of epithelial ovarian cancer cells through MIR199A2/214.

Cancer stem cells are responsible for sustaining the tumor and giving rise to proliferating and progressively differentiating cells. However, the molecular mechanisms regulating the process of cancer stem cell (CSC) differentiation is not clearly understood. Recently, we reported the isolation of the epithelial ovarian cancer (EOC) stem cells (type I/CD44+). In this study, we show that type I/CD44+ cells are characterized by low levels of both miR-199a and miR-214, whereas mature EOC cells (type II/CD44-) have higher levels of miR-199a and miR-214. Moreover, these two micro RNAs (miRNAs) are regulated as a cluster on pri-miR-199a2 within the human Dnm3os gene (GenBank FJ623959). This study identify Twist1 as a regulator of this unique miRNA cluster responsible for the regulation of the IKKbeta/NF-kappaB and PTEN/AKT pathways and its association of ovarian CSC differentiation. Our data suggest that Twist1 may be an important regulator of 'stemness' in EOC cells. The regulation of MIR199A2/214 expression may be used as a potential therapeutic approach in EOC patients.

The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71.

Studies using first trimester trophoblast cells may be limited by the inability to obtain patient samples and/or adequate cell numbers. First trimester trophoblast cell lines have been generated by SV40 transformation or similar methods, however, this approach is known to induce phenotypic and karyotypic abnormalities. The introduction of telomerase has been proposed to be a viable alternative for the immortalization of primary human cells. To investigate whether telomerase-induced immortalization might be a more feasible approach for the generation of first trimester trophoblast cell lines, we isolated primary trophoblast cells from a 7-week normal placenta and infected the cells with human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Although this hTERT-infected first trimester trophoblast cell line, which we have named Swan 71, has been propagated for more than 100 passages, it still has attributes that are characteristic of primary first trimester trophoblast cells. The Swan 71 cells are positive for the expression of cytokeratin 7, vimentin and HLA-G, but do not express CD45, CD68 or the Fibroblast Specific Antigen (FSA), CD90/Thy-1. In addition, we also demonstrated that the Swan 71 cells secrete fetal fibronectin (FFN) as well as low levels of human Chorionic Gonadotrophin (hCG). Moreover, the Swan 71 cells exhibit a cytokine and growth factor profile that is similar to primary trophoblast cells and are resistant to Fas, but not TNF-alpha-induced apoptosis. This suggests that the Swan 71 cells may represent a valuable model for future in vitro trophoblast studies.

Regulation of IKKbeta by miR-199a affects NF-kappaB activity in ovarian cancer cells.

Cancer progression is an abnormal form of tissue repair characterized by chronic inflammation. IkappaB kinase-beta (IKKbeta) required for nuclear factor-kappaB (NF-kappaB) activation plays a critical role in this process. Using EOC cells isolated from malignant ovarian cancer ascites and solid tumors, we identified IKKbeta as a major factor promoting a functional TLR-MyD88-NF-kappaB pathway that confers to EOC cell the capacity to constitutively secrete proinflammatory/protumor cytokines and therefore promoting tumor progression and chemoresistance. Furthermore, we describe for the first time the identification of the microRNA hsa-miR-199a as a regulator of IKKbeta expression. Our study describes the property of ovarian cancer cells to enhance the inflammatory microenvironment as a result of the expression of an active IKKbeta pathway. Identification of these markers in patients' tumor samples may facilitate the adequate selection of treatment and open new venues for the development of effective therapy for chemoresistant ovarian cancers.

Cancers take their Toll--the function and regulation of Toll-like receptors in cancer cells.

Cancer could be deemed as an abnormal and uncontrolled tissue repair process. Therefore, it would not be surprising that factors that function in the tissue repair process, such as cytokines, chemokines, growth factors and Toll-like receptor (TLR) ligands, as well as growth signals for compensatory proliferation, would also be key factors in regulating and enhancing cancer progression. The TLR pathways, which play a critical role in tissue repair, are also key regulators in cancer progression as well as chemoresistance. TLRs serve as cell surface sensors that can initiate pathways leading to proliferation and chemoresistance; as well as mediators that are able to regulate the infiltrating immune cells to provide further support for cancer progression.