Yang Yang-Hartwich

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.

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.

Ovulation and extra-ovarian origin of ovarian cancer

The mortality rate of ovarian cancer remains high due to late diagnosis and recurrence. A fundamental step toward improving detection and treatment of this lethal disease is to understand its origin. A growing number of studies have revealed that ovarian cancer can develop from multiple extra-ovarian origins, including fallopian tube, gastrointestinal tract, cervix and endometriosis. However, the mechanism leading to their ovarian localization is not understood. We utilized in vitro, ex vivo, and in vivo models to recapitulate the process of extra-ovarian malignant cells migrating to the ovaries and forming tumors. We provided experimental evidence to support that ovulation, by disrupting the ovarian surface epithelium and releasing chemokines/cytokines, promotes the migration and adhesion of malignant cells to the ovary. We identified the granulosa cell-secreted SDF-1 as a main chemoattractant that recruits malignant cells towards the ovary. Our findings revealed a potential molecular mechanism of how the extra-ovarian cells can be attracted by the ovary, migrate to and form tumors in the ovary. Our data also supports the association between increased ovulation and the risk of ovarian cancer. Understanding this association will lead us to the development of more specific markers for early detection and better prevention strategies.

TRX-E-002-1 Induces c-Jun–Dependent Apoptosis in Ovarian Cancer Stem Cells and Prevents Recurrence In Vivo

Chemoresistance is a major hurdle in the management of patients with epithelial ovarian cancer and is responsible for its high mortality. Studies have shown that chemoresistance is due to the presence of a subgroup of cancer cells with stemness properties and a high capacity for tumor repair. We have developed a library of super-benzopyran analogues to generate potent compounds that can induce cell death in chemoresistant cancer stem cells. TRX-E-002-1 is identified as the most potent analogue and can induce cell death in all chemoresistant CD44+/MyD88+ ovarian cancer stem cells tested (IC50 = 50 nmol/L). TRX-E-002-1 is also potent against spheroid cultures formed from cancer stem cells, chemosensitive CD44−/MyD88− ovarian cancer cells, and heterogeneous cultures of ovarian cancer cells. Cell death was associated with the phosphorylation and increased levels of c-Jun and induction of caspases. In vivo, TRX-E-002-1 given as daily intraperitoneal monotherapy at 100 mg/kg significantly decreased intraperitoneal tumor burden compared with vehicle control. When given in combination with cisplatin, animals receiving the combination of cisplatin and TRX-E-002-1 showed decreased tumor burden compared with each monotherapy. Finally, TRX-E-002-1 given as maintenance treatment after paclitaxel significantly delayed disease recurrence. Our results suggest that TRX-E-002-1 may fill the current need for better therapeutic options in the control and management of recurrent ovarian cancer and may help improve patient survival. Mol Cancer Ther; 15(6); 1279–90. ©2016 AACR.

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.

Detection of p53 protein aggregation in cancer cell lines and tumor samples.

The p53 protein plays a central role in regulating apoptosis. The loss of functional p53 is common in many cancers. In cancer cells, the dysfunctional p53 protein often maintains a misfolded, inactive conformation due to genetic mutations or posttranslational deregulation. The misfolded p53 protein can aggregate and form amyloid-like oligomers and fibrils, which abrogate the pro-apoptotic functions of p53. Therefore, the aggregation of p53 may be a crucial factor in carcinogenesis, tumor progression, and the response of cancer cells to apoptotic signals. In this chapter, we provide details on various methods for detecting p53 aggregation in cancer cell lines and tumor samples.

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

Abstract 5220: The role of brain-derived neurotrophic factor in ovarian cancer initiation and progression

Ovarian cancer is one of the most devastating cancers in women. A fundamental step toward improving detection and treatment of this lethal disease is to understand the mechanism of its initiation and progression. In this study we demonstrated the role of brain-derived neurotrophic factor (BDNF) in ovarian cancer initiation and progression, particularly, the high-grade serous ovarian carcinoma (HGSOC). BDNF was discovered in the brain as a growth factor and chemoattractant inducing the migration, survival, and differentiation of neurons. It is also secreted by the ovary into the follicular fluid and by the adipocytes in the omentum and peritoneum. BDNF suppresses anoikis, the apoptosis induced by lack of proper cell to extra-cellular matrix (ECM) attachment. BDNF has been associated with tumor progression in colon, breast, lung, and gastric cancers. In ovarian tumors both BDNF and its receptor tropomyosin receptor kinase B (TrkB) are expressed. Their overexpression is associated with poor survival. Fallopian tube epithelial cells (FTEs) are hypothesized to be the cell of origin of HGSOC. We used 3 immortalized human normal FTE cell lines to demonstrate the effects of BDNF on the survival, mobility, and adhesion of FTEs in vitro. CellTiter luminescent cell viability assays were conducted to evaluate the effects of BDNF on the short-term and long-term survival of FTEs in 3D culture conditions. Caspase-3 activity assays were used to quantify the extent of which BDNF inhibited anoikis in FTEs. Trans-well and 3D bioprinting migration assays were used to determine whether BDNF could promote the mobility of FTEs. The ECM-coated beads were co-cultured with FTEs in a 3D model and their attachment to ECM was quantified in order to evaluate the effects of BDNF on the cell adhesion. The BDNF-activated intracellular pathways were identified using Western blots, RNA microarray, RT-QPCR analyses. Our data demonstrated that TrkB was expressed by the FTEs in human and mouse fallopian tubes. BDNF significantly enhanced the survival of FTE cell lines in the serum-free 3D culture condition. Their enhanced survival was also evidenced by the decreased caspase-3 activity indicating the inhibition of anoikis. BDNF increased the ability of FTEs to migrate in the migrations assays. The BDNF-treated FTEs attached to the ECM-coated beads faster than the untreated cells. Western blots showed that BDNF activated the phosphorylation of TrkB, AKT, ERK, PLC-gamma1, and CREB. RNA microarray and QPCR data suggest that epithelial-mesenchymal transition (EMT), anti-oxidative stress, and ECM-related pathways were activated by BDNF leading to the enhanced survival, migration, and cell adhesion. These results revealed the potential role of BDNF to promote ovarian cancer initiation. Understanding this molecular pathway will lead to the development of more specific markers for early detection and better prevention and treatment strategies. Citation Format: Min Kang, Kay Y. Chong, Tobias M. Hartwich, Oluwagbemisola O. Madarikan, Jonah Nucci, Sarah L. Cady, Yang Yang-Hartwich. The role of brain-derived neurotrophic factor in ovarian cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5220.

Abstract AP16: THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS

BACKGROUND: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism underlying metastasis is still not clear. Twist1 is a transcriptional factor that promotes epithelial-mesenchymal transition (EMT) and enhances the migration and tumor-initiation of ovarian cancer cells. It also has been associated with cancer stem cells and chemoresistance. The purpose of our study is to better understand the regulation of Twist1 and to identify molecular pathways that can be targeted in order to control metastatic diseases. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We hypothesize that through their interaction, p53 promotes the proteasomedependent degradation of Twist1 and consequently inhibits EMT in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p539s ability to inhibit Twist1, leading to enhanced EMT and metastasis. METHOD: In ubiquitin assay p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Coimmunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Plasmid constructs containing different Twist1 mutants were created to select the degradation resistant mutation. ΔC-Twist1 (C-terminal deletion) was used in in vivo study. RESULT: 1) In EOC cells, proteasome-dependent degradation suppressed Twist1 protein. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In ovarian cancer cells, wild type p53 overexpression reduced Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to enhance Twist1 degradation. E3 ligase, Pirh2, formed complex with p53 and Twist1 to induce Twist1 degradation. 2) ΔC-Twist1 was resistant to p53-regulated degradation. In xenograft mouse model, EOC cells overexpressing ΔC-Twist1 could form more aggressive tumors with increased numbers of metastatic lesions than wild type Twist1 group. 3) In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. Nine samples with high levels of wild type p53 all showed low or no Twist1 expression. CONCLUSION: Our data revealed a mechanism by which Twist1 is regulated through p53-promoted proteasome-dependent degradation. p53 facilitates the formation of a Twist1-p53-Pirh2 complex and the Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP16.