Vinicius Craveiro

Targeting the Mitochondria Activates Two Independent Cell Death Pathways in Ovarian Cancer Stem Cells

Cancer stem cells are responsible for tumor initiation and chemoresistance. In ovarian cancer, the CD44+/MyD88+ ovarian cancer stem cells 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 ovarian cancer stem cells 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 ovarian cancer stem cells. 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 by increase in mitochondrial superoxide and hydrogen peroxide. This promotes a state of cellular starvation that activates two independent pathways: (i) AMPKα1 pathway leading to mTOR inhibition; and (ii) mitochondrial MAP/ERK kinase/extracellular signal-regulated kinase 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 chemoresistant cell population opens a new venue for treating ovarian cancer patients. Mol Cancer Ther; 10(8); 1385–93. ©2011 AACR.

TLR2 enhances ovarian cancer stem cell self-renewal and promotes tumor repair and recurrence

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.

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.

Distinct Subpopulations of Epithelial Ovarian Cancer Cells Can Differentially Induce Macrophages and T Regulatory Cells Toward a Pro-Tumor Phenotype

Citation Alvero AB, Montagna MK, Craveiro V, Liu L, Mor G. Distinct subpopulations of epithelial ovarian cancer cells can differentially induce macrophages and T regulatory cells toward a pro‐tumor phenotype. Am J Reprod Immunol 2012; 67: 256–265

Effect of Culture Conditions on the Phenotype of THP‐1 Monocyte Cell Line

Macrophage function has many implications in a variety of diseases. Understanding their biology becomes imperative when trying to elucidate immune cell interactions with their environment, and in vitro cell lines allow researchers to manipulate these interactions. A common cell line used is THP‐1, a promyeloid cell line suggestive to outside factors, and therefore sensitive to culture conditions. In this study, we describe how culture conditions can alter THP‐1 morphology and in turn affect their response to differentiation stimuli.

p53 protein aggregation promotes platinum resistance in ovarian cancer

High-grade serous ovarian carcinoma (HGSOC), the most lethal gynecological cancer, often leads to chemoresistant diseases. The p53 protein is a key transcriptional factor regulating cellular homeostasis. A majority of HGSOCs have inactive p53 because of genetic mutations. However, genetic mutation is not the only cause of p53 inactivation. The aggregation of p53 protein has been discovered in different types of cancers and may be responsible for impairing the normal transcriptional activation and pro-apoptotic functions of p53. We demonstrated that in a unique population of HGSOC cancer cells with cancer stem cell properties, p53 protein aggregation is associated with p53 inactivation and platinum resistance. When these cancer stem cells differentiated into their chemosensitive progeny, they lost tumor-initiating capacity and p53 aggregates. In addition to the association of p53 aggregation and chemoresistance in HGSOC cells, we further demonstrated that the overexpression of a p53-positive regulator, p14ARF, inhibited MDM2-mediated p53 degradation and led to the imbalance of p53 turnover that promoted the formation of p53 aggregates. With in vitro and in vivo models, we demonstrated that the inhibition of p14ARF could suppress p53 aggregation and sensitize cancer cells to platinum treatment. Moreover, by two-dimensional gel electrophoresis and mass spectrometry we discovered that the aggregated p53 may function uniquely by interacting with proteins that are critical for cancer cell survival and tumor progression. Our findings help us understand the poor chemoresponse of a subset of HGSOC patients and suggest p53 aggregation as a new marker for chemoresistance. Our findings also suggest that inhibiting p53 aggregation can reactivate p53 pro-apoptotic function. Therefore, p53 aggregation is a potential therapeutic target for reversing chemoresistance. This is paramount for improving ovarian cancer patients’ responses to chemotherapy, and thus increasing their survival rate.

Phenotypic modifications in ovarian cancer stem cells following Paclitaxel treatment

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite initial responsiveness, 80% of EOC patients recur and present with chemoresistant and a more aggressive disease. This suggests an underlying biology that results in a modified recurrent disease, which is distinct from the primary tumor. Unfortunately, the management of recurrent EOC is similar to primary disease and does not parallel the molecular changes that may have occurred during the process of rebuilding the tumor. We describe the characterization of unique in vitro and in vivo ovarian cancer models to study the process of recurrence. The in vitro model consists of GFP+/CD44+/MyD88+ EOC stem cells and mCherry+/CD44−/MyD88− EOC cells. The in vivo model consists of mCherry+/CD44+/MyD88+ EOC cells injected intraperitoneally. Animals received four doses of Paclitaxel and response to treatment was monitored by in vivo imaging. Phenotype of primary and recurrent disease was characterized by quantitative polymerase chain reaction (qPCR) and Western blot analysis. Using the in vivo and in vitro models, we confirmed that chemotherapy enriched for CD44+/MyD88+ EOC stem cells. However, we observed that the surviving CD44+/MyD88+ EOC stem cells acquire a more aggressive phenotype characterized by chemoresistance and migratory potential. Our results highlight the mechanisms that may explain the phenotypic heterogeneity of recurrent EOC and emphasize the significant plasticity of ovarian cancer stem cells. The significance of our findings is the possibility of developing new venues to target the surviving CD44+/MyD88+ EOC stem cells as part of maintenance therapy and therefore preventing recurrence and metastasis, which are the main causes of mortality in patients with ovarian cancer.

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

Abstract 3471: Paclitaxel selects and enriches for CD44+/MyD88+ ovarian cancer stem cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. The current standard of care in the treatment of EOC patients is surgical debulking and combination chemotherapy usually with Carboplatin and Paclitaxel. Although effective in majority of the cases, more than 20% of patients do not respond. Moreover, more than 80% of patients present with recurrent disease within 5 years. Therefore, there is an underlying biology that results in the differential response to treatment as well as the occurrence of recurrent disease. Tumors are heterogeneous and consist of multiple types of cancer cells, which exhibit different chemoresponsiveness. Our group previously described the characterization of CD44+/MyD88+ EOC stem cells and demonstrated their tumor-initiating properties. An important characteristic of these cells, which differentiate them from the CD44-/MyD88- EOC cells, is the presence of a functional TLR4-MyD88-NFkB pathway. This pathway confers Paclitaxel resistance to these cells. We hypothesize that a cause for recurrence following Paclitaxel treatment is the selective survival of cancer stem cells capable to recreate the tumor. In this study, we demonstrate that Paclitaxel enriches for the CD44+/MyD88+ EOC stem cells thus promoting recurrence. Methods: In vitro model: co-culture system consisting of (50%:50%) GFP+ CD44+/MyD88+ and RFP+ CD44-/MyD88- cells treated with 0.2 uM Paclitaxel. In vivo model: Ovarian tumors implanted s.c in Nude mice treated with 10 mg/kg Paclitaxel q3d for 21 days. Levels of GFP+ and CD44+ cells were determined by flow cytometry. Klf-4, Nanog, MyD88, and ALDH1 were determined by qPCR or western blot. Results: Control co-cultures were characterized by overgrowth of CD44-/MyD88- EOC cells while the Paclitaxel treated co-cultures had mainly CD44+/MyD88+ EOC stem cells. Similarly in vivo results show enrichment in CD44+ cells with Paclitaxel (49% CD44+ in control vs 88% CD44+ with treatment). Analysis of genes associated with stemness showed upregulation of Klf-4, Nanog ALDH1, and MyD88 in the cultures treated with Paclitaxel compared to control. Conclusion: We demonstrate that Paclitaxel selectively induce cell death in CD44-/MyD88- EOC cells but has a pro-survival effect and enhances self-renewal in the pleuripotent and chemoresistant CD44+/MyD88+ EOC stem cells. Based on these data, we propose that the mode of management for EOC patients should take into consideration the tumors molecular phenotype. Our results highlight the need to identify patients that should not receive Paclitaxel - not only because they are resistant, but more importantly, because it can enrich for the more aggressive cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3471. doi:1538-7445.AM2012-3471

Abstract LB-286: ME-344 delays tumor kinetics in an ovarian cancer in vivo recurrence model.

Background: Epithelial ovarian cancer (EOC) is the most lethal of all gynecologic malignancies. Despite initial responsiveness to first-line standard of care, consisting of surgical debulking and chemotherapy, 8 out of 10 patients recur. In the recurrence setting, the presentation of widespread micrometastasis, which limits the usefulness of surgery, is complicated by concurrent presentation of chemoresistance. Currently, no adequate therapy is able to prevent or treat recurrence. Consequently, therapies directed against control of tumor burden can improve prognosis in EOC patients. Recently we reported the characterization of CD44+/MyD88+ EOC cells with tumor-initiating properties and inherent chemoresistance. In addition, we have identified ME-344, a novel isoflavone derivate, with potent capacity to induce cell death in these cells. Furthermore, we have developed an intra-peritoneal (i.p.) in vivo model of EOC recurrence based on the capacity of these cells to survive chemotherapy and renew the tumor. Using this model, we show the potential efficacy of ME-344 in delaying carcinomatosis and decreasing tumor burden. Methods: CD44+/MyD88+/mCherry+ EOC stem cells are injected i.p. in nude mice. Tumors are detected and consequently followed by live in vivo imaging using In Vivo FX System. Once tumors are detected, mice received 4 doses of 12 mg/kg i.p. Paclitaxel q3d or until the animals are free of disease. Mice were then randomized to maintenance with Vehicle or ME-344 (100 mg/kg i.p. q3d) and further monitored for recurrence. Recurrence is defined as appearance of tumors with ROI interior area > 2000. Tumor growth delay is defined as the difference in days when treated and control groups reach the maximal tumor burden set at ROI interior area = 10,000. Results: Mice exhibited recurrence with an average time of 6 days in the Vehicle group and 7 days in the ME-344 group. However, a significant delay in tumor kinetics was observed in the group maintained with ME-344. Maximal tumor burden, defined as ROI interior area = 10,000, was reached in the control group within 24 days and in the ME-344 group within 39 days. Thus, tumor growth was delayed for 15 days. Conclusion: Maintenance with ME-344 is able to decrease tumor burden in this very aggressive in vivo model of EOC recurrence. In this study, we show a significant delay in tumor kinetics in mice that were maintained with ME-344 following initial response to Paclitaxel. Decreasing and delaying metastatic load will allow more optimal surgical debulking and may improve survival in EOC patients. These results suggest the potential value of ME-344 therapy after 1st line standard of care in EOC patients. Citation Format: Ayesha B. Alvero, Natalia Sumi, Vinicius Craveiro, Won Duk Joo, Yang Yang-Hartwich, Gil Mor. ME-344 delays tumor kinetics in an ovarian cancer in vivo recurrence model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-286. doi:10.1158/1538-7445.AM2013-LB-286