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 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 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 IKKβ/NFκB and PTEN/AKT pathways and its association of ovarian cancer stem cell 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.

Cancer stem cells are responsible for tumor initiation and chemo-resistance. In ovarian cancer, the CD44+/MyD88+ ovarian cancer stem cells (OCSCs) are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome and patient survival. The previous demonstration that the OCSCs are resistant to apoptotic cell death induced by conventional chemotherapy agents suggests that other forms of targeted therapy should be explored. We show in this study that targeting mitochondrial bioenergetics is a potent stimulus to induce caspase-independent cell death in a panel of OCSCs. Treatment of these cells with the novel isoflavone derivative, NV-128, significantly depressed mitochondrial function exhibited by decrease in ATP, Cox-I, and Cox-IV levels, and increase in mitochondrial superoxide and hydrogen peroxide. This promotes a state of “cellular starvation” that activates two independent pathways: 1) AMPKα1 pathway leading to mTOR inhibition; and 2) mitochondrial MEK/ERK pathway leading to loss of mitochondrial membrane potential. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemo-resistant cell population opens a new venue for treating ovarian cancer patients.

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 i.p 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 TWIST1 is constitutively degraded in EOC stem cells, and that the acquisition of TWIST1 requires additional signals that will trigger the differentiation process. These findings are relevant for understanding the differentiation and metastasis process in EOC stem cells.

Primary ovarian cancer is responsive to treatment, but chemoresistant recurrent disease ensues in majority of patients. Recent compelling evidence demonstrates that a specific population of cancer cells, the cancer stem cells, initiates and sustains tumors. It is therefore possible that this cell population is also responsible for recurrence. We have shown previously that CD44+/MyD88+ epithelial ovarian cancer stem cells (CD44+/MyD88+ EOC stem cells) are responsible for tumor initiation. In this study, we demonstrate that this population drives tumor repair following surgery- and chemotherapy-induced tumor injury. Using in vivo and in vitro models, we also demonstrate that during the process of tumor repair, CD44+/MyD88+ EOC stem cells undergo self-renewal as evidenced by upregulation of stemness-associated genes. More importantly, we show that a pro-inflammatory microenvironment created by the TLR2-MyD88-NFκB pathway supports EOC stem cell-driven repair and self-renewal. Overall, our findings point to a specific cancer cell population, the CD44+/MyD88+ EOC stem cells and a specific pro-inflammatory pathway, the TLR2-MyD88-NFκB pathway, as two of the required players promoting tumor repair, which is associated with enhanced cancer stem cell load. Identification of these key players is the first step in elucidating the steps necessary to prevent recurrence in EOC patients.

Subventricular zone (SVZ) neural stem cells (NSCs) are the cornerstone of the perinatal neurogenic niche. Microglia are immune cells of the nervous system that are enriched in the neonatal SVZ. Although microglia regulate NSCs, the extent to which this interaction is bi-directional is unclear. Extracellular vesicles (EVs) are cell-derived particles that encase miRNA and proteins. Here, we demonstrate that SVZ NSCs generate and release EVs. Neonatal electroporated fluorescent EV fusion proteins were released by NSCs and subsequently cleared from the SVZ. EVs were preferentially targeted to microglia. Small RNA sequencing identified miRNAs within the EVs that regulate microglia physiology and morphology. EVs induced a transition to a CD11b/Iba1 non-stellate microglial morphology. The transition accompanied a microglial transcriptional state characterized by Let-7-regulated cytokine release and a negative feedback loop that controlled NSC proliferation. These findings implicate an NSC-EV-microglia axis and provide insight to normal and pathophysiological brain development.