Chunbo He

The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression

The Hippo signaling pathway controls organ size and tumorigenesis through a kinase cascade that inactivates Yes‐associated protein (YAP). Here, we show that YAP plays a central role in controlling the progression of cervical cancer. Our results suggest that YAP expression is associated with a poor prognosis for cervical cancer. TGF‐α and amphiregulin (AREG), via EGFR, inhibit the Hippo signaling pathway and activate YAP to induce cervical cancer cell proliferation and migration. Activated YAP allows for up‐regulation of TGF‐α, AREG, and EGFR, forming a positive signaling loop to drive cervical cancer cell proliferation. HPV E6 protein, a major etiological molecule of cervical cancer, maintains high YAP protein levels in cervical cancer cells by preventing proteasome‐dependent YAP degradation to drive cervical cancer cell proliferation. Results from human cervical cancer genomic databases and an accepted transgenic mouse model strongly support the clinical relevance of the discovered feed‐forward signaling loop. Our study indicates that combined targeting of the Hippo and the ERBB signaling pathways represents a novel therapeutic strategy for prevention and treatment of cervical cancer.

The G-protein-coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian cancer cells by blocking tubulin polymerization.

The G-protein-coupled estrogen receptor 1 (GPER) has recently been reported to mediate the non-genomic action of estrogen in different types of cells and tissues. G-1 (1-[4-(6-bromobenzo[1,3] dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed as a potent and selective agonist for GPER. G-1 has been shown to induce the expression of genes and activate pathways that facilitate cancer cell proliferation by activating GPER. Here we demonstrate that G-1 has an anticancer potential with a mechanism similar to vinca alkaloids, the commonly used chemotherapy drugs. We found that G-1 blocks tubulin polymerization and thereby interrupts microtubule assembly in ovarian cancer cells leading to the arrest of cell cycle in the prophase of mitosis and the suppression of ovarian cancer cell proliferation. G-1 treatment also induces apoptosis of ovarian cancer cells. The ability of G-1 to target microtubules to suppress ovarian cancer cell proliferation makes it a promising candidate drug for treatment of ovarian cancer.

YAP induces high-grade serous carcinoma in fallopian tube secretory epithelial cells.

Accumulating evidence indicates that ovarian high-grade serous carcinoma (HGSC) originates from fallopian tube secretory epithelial cells (FTSECs). However, the molecular mechanisms underlying the initiation and progression of HGSC derived from FTSECs remains unclear. In this study, we found that the Hippo/Yes-associated protein (YAP) signaling pathway has a critical role in the initiation and progression of fallopian tube and ovarian HGSC. Importantly, YAP was overexpressed in inflammatory and cancerous fallopian tube tissues. Further, overexpression of wild-type YAP, or constitutively active YAP in immortalized FTSECs, induced cell proliferation, migration, colony formation and tumorigenesis. Moreover, the Hippo/YAP and the fibroblast growth factor (FGF) signaling pathways formed an autocrine/paracrine-positive feedback loop to drive the progression of the FTSEC-derived HGSC. Evidence in this study strongly suggests that combined therapy with inhibitors of YAP (such as verteporfin) and FGF receptors (such as BGJ398) can provide a novel therapeutic strategy to treat fallopian tube and ovarian HGSC.

G-1 Inhibits breast cancer cell growth via targeting colchicine-binding site of tubulin to interfere with microtubule assembly

G-protein–coupled estrogen receptor 1 (GPER1) has been reported to play a significant role in mediating the rapid estrogen actions in a wide range of normal and cancer cells. G-1 was initially developed as a selective agonist for GPER. However, the molecular mechanisms underlying the actions of G-1 are unknown, and recent studies report inconsistent effects of G-1 on the growth of breast cancer cells. By employing high-resolution laser scanning confocal microscopy and time-lapse imaging technology, as well as biochemical analyses, in the current study, we provide convincing in vitro and in vivo evidence that G-1 is able to suppress the growth of breast cancer cells independent of the expression status of GPERs and classic estrogen receptors. Interestingly, we found that triple-negative breast cancer cells (TNBC) are very sensitive to G-1 treatment. We found that G-1 arrested the cell cycle in the prophase of mitosis, leading to caspase activation and apoptosis of breast cancer cells. Our mechanistic studies indicated that G-1, similar to colchicine and 2-methoxyestradiol, binds to colchicine binding site on tubulin, inhibiting tubulin polymerization and subsequent assembly of normal mitotic spindle apparatus during breast cancer cell mitosis. Therefore, G-1 is a novel microtubule-targeting agent and could be a promising anti-microtubule drug for breast cancer treatment, especially for TNBC treatment. Mol Cancer Ther; 16(6); 1080–91. ©2017 AACR.

Abstract MIP-058: YAP, VIA INTERACTIONS WITH THE FGF SIGNALING PATHWAY, REGULATES TUMORIGENESIS OF FALLOPIAN TUBE SECRETORY EPITHELIAL CELLS

INTRODUCTION: Accumulating evidence indicates that ovarian high-grade serous carcinoma (HGSC) originates from Fallopian tube secretory epithelial cells (FTSECs). Our recent studies indicated that the Hippo/YAP signaling pathway contributes to transformation of ovarian surface epithelial cells. However, whether this pathway is also involved in the initiation and progression of FTSEC-derived ovarian HGSC has not been examined. AIMS: The present study aims to determine whether the Hippo/YAP signaling pathway plays a role in initiation and progression of Fallopian tube-derived HGSC and the potential mechanism(s) by which YAP regulates FTSEC tumorigenesis. RESULTS: Immunohistochemical analysis showed that YAP expression was very low in normal human fallopian tube and ovarian tissues. However, YAP immunosignals significantly increased in ovarian HGSCs, cancerous and inflammatory fallopian tube tissues. Data extracted from multidimensional human cancer genomics datasets revealed that the YAP and TEADs (the major target transcription factors of YAP) genes are frequently amplified and/or up-regulated, while the upstream tumor suppressor genes such as NF1 and LATS1, are frequently deleted and/or mutated in ovarian HGSC tissues. Overexpression of YAP in immortalized non-tumorigenic FTSECs promoted cell proliferation in both 2D and 3D culture systems, induced colony formation in soft agar, and drove tumorigenesis in athymic nude mice. Pathohistological and biochemical analyses clearly indicated that tumors derived from the FTSECs resembled ovarian HGSC. In addition, our mechanistic studies showed that activation of YAP in human FTSECs upregulated the expression of fibroblast growth factors (FGF1, FGF2) and their receptors (FGFR1, FGFR2, FGFR3 and FGFR4). Intriguingly, we found that the activated FGF signaling in turn suppressed the Hippo pathway and activated YAP protein. Consistent with this result, treatment of FTSECs with YAP antagonist (Verteporfin) and/or FGFR inhibitor (BGI398) not only blocked YAP-induced FTSECs transformation, but also suppressed tumor cell growth and induced tumor cell death in a human HGSC xenograft model. CONCLUSION: Our results demonstrate that the disruption of the Hippo/YAP signaling pathway contributes to the carcinogenesis of the fallopian tube secretory epithelial cells. The Hippo/YAP and FGF signaling pathways formed an autocrine/paracrine positive feedback loop to drive the progression of the FTSECs-derived HGSC. Evidence in this study strongly suggests that combined targeting of YAP and FGFRs represents a novel therapeutic strategy for ovarian HGSCs. Citation Format: Chunbo He, Guohua Hua, Xiangmin Lv, Zhengfeng Wang, Ronny I. Drapkin, John S. Davis, Cheng Wang. YAP, VIA INTERACTIONS WITH THE FGF SIGNALING PATHWAY, REGULATES TUMORIGENESIS OF FALLOPIAN TUBE SECRETORY EPITHELIAL CELLS [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 MIP-058.

Abstract A37: Yes-associated protein 1 (YAP) in the growth and tumorigenesis of ovarian granulosa cells.

Ovarian granulosa cells are essential for successful follicle development. Dysregulation of granulosa cell proliferation and differentiation may lead to impairment of female fertility or development of ovarian tumors. Although studies indicate that the Hippo signaling pathway plays critical roles in both development and tumorigenesis of several organs, its role in ovarian physiology and pathology remains largely unknown. The present study aims to investigate the role of YAP in ovarian granulosa cell proliferation, differentiation and transformation. Immunohistochemical analyses showed that the active form of YAP (nuclear YAP) was highly expressed in proliferative granulosa cells, whereas the inactive form of YAP (cytoplasmic YAP) was detected mainly in terminally-differentiated luteal cells, indicating that YAP may be involved in proliferation and differentiation of ovarian granulosa cells. Knockdown of YAP or pharmacological inhibition of YAP activity in human granulosa cells suppressed cell growth and reduced cell survival in both traditional 2D cell culture system and a novel 3D hanging-drop culture system, suggesting that YAP is essential for the growth and survival of human granulosa cells. In addition, ectopic expression of wild-type or constitutively active YAP impaired gonadotropin-induced differentiation of granulosa cells and stimulated the rapid growth and transformation of granulosa cells, findings which were reflected by increased colony formation in soft agar assay and growth of tumors in a xenograft mouse model. Injection of verteporfin (a selective antagonist of YAP) into female CD1 mice inhibited granulosa cell growth, induced GC apoptosis, and disrupted mouse ovarian follicle development, leading to severe subfertility. Consistently, mice with ovarian granulosa cell-specific expression of constitutively active YAP demonstrated disruption of granulosa cell differentiation and failure of ovarian follicle development. In conclusion, our studies demonstrate that homeostatic YAP expression and activation are essential for ovarian granulosa cell proliferation, differentiation and survival. YAP is a novel therapeutic target for treatment of ovarian diseases associated with granulosa cell dysregulation. Citation Format: Xiangmin Lv, Chunbo He, Guohua Hua, Jixin Dong, John S. Davis, Cheng Wang. Yes-associated protein 1 (YAP) in the growth and tumorigenesis of ovarian granulosa cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A37.

Abstract A29: YAP interacts with the ERBB signaling pathway to control ovarian surface epithelial cell tumorigenesis.

Introduction: The Hippo signaling pathway controls organ size and inhibits tumorigenesis through a kinase cascade that leads to the phosphorylation of the transcriptional co-activator Yes-associated protein 1 (YAP1, more commonly referred to as YAP). Recent studies have shown that YAP may play a role in the progression of ovarian cancer. However, current reports considering the relationships between YAP expression and clinicopathological outcomes in ovarian cancer are inconsistent. Additionally, the role and functional mechanism of YAP on ovarian tumorigenesis and ovarian cancer progression are not fully understood. Aims: The aims of the present study are to examine the role of YAP in the tumorigenesis of ovarian surface epithelial cells, and to uncover the molecular mechanisms underlying YAP regulation of OSE malignant transformation. Methods: We used a large cohort of ovarian cancer patient samples, an ovarian tissue-specific YAP-overexpression mouse model, and a series of unique cellular models to address above questions. Results: Immunohistochemical analysis using a large cohort of patient samples indicated that YAP expression is associated with poor clinical outcomes in ovarian cancer patients. Overexpression of wild-type YAP or constitutively active YAP in immortalized human ovarian surface epithelial cells (HOSE) induced anchorage-independent growth of these cells in a soft agar assay and tumorigenesis in a xenograft mouse model, suggesting that YAP is sufficient to induce transformation of immortalized human OSEs. To confirm the pathological relevance of our findings, we created an ovarian tissue-specific YAP overexpression mouse model by breeding AMHR2-cre mice to ROSA26-rtTA mice and tetO-YAPS127A mice. The created AMHR2-Cre-rtTA-tetO-YAPS127A mice specifically express YAPS127A (constitutively active YAP) in ovarian tissues once these mice are treated with doxycycline (Dox). We found that AMHR2-induced ovarian tissue specific expression of YAPS127A, which led to drastic neoplasia of ovarian surface epithelial (OSE) cells in AMHR2-Cre-rtTA-tetO-YAPS127A mice after induction with Dox for 3 weeks. In addition, our mechanistic studies indicated that activation of YAP in human OSE cells induced expression of EGFR, ERBB3, HBEGF, NRG1 and NRG2 both in vitro and in vivo. Intriguingly, the activated ERBB signaling in turn suppressed the Hippo pathway and activated YAP protein. Conclusion: Our results demonstrate the existence of a NRGs & HBEGF/ERBBs/YAP/NRGsH Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A29.

Abstract POSTER-BIOL-1315: The hippo signaling pathway regulates ovarian cancer initiation and progression via an autocrine loop

Ovarian cancers are the most lethal gynecological cancers. Mechanisms underlying their initiation and progression are unclear. The Hippo signaling pathway plays a critical role in tissue homeostasis and organ size control by regulating cell proliferation. Recent studies have indicated that dysregulation of the Hippo pathway is associated with cancer in various tissues and organs. However, the exact role and mechanisms used by the Hippo pathway in the initiation and progression of ovarian cancer are unclear. The aim of this study is to determine the role and the regulatory mechanism of the Hippo pathway in the initiation and progression of ovarian cancer. Immunohistochemistry was used to analyze the expression of YAP, a major downstream effector of the Hippo signaling pathway, in paraffin-embedded human normal and cancerous ovarian tissues. HOSE (an immortalized normal human ovarian surface epithelial cell line), TOV21G (an epithelial ovarian cancer cell line) and KGN (an ovarian granulosa cell tumor cell line) cells were used as cellular models to analyze cell proliferation and tumorigenesis. Cell proliferation and viability were examined by cell number counting and MTT assays, respectively. Cell cycle was monitored by flow cytometry. Key molecules associated with cell proliferation were detected with RT-PCR, Western blot, or fluorescent immunohistochemistry. Cell transformation was determined by soft agar assay. Tumorigenesis and tumor growth were determined using human tumor xenografts in an athymic nude mouse model. Immunohistochemistry using large cohort of patient samples (384 samples) indicate that YAP expression is associated with poor clinical outcomes in patients. Overexpression of YAP or a constitutively active YAP mutant lead to transformation and tumorigenesis in immortalized, non-tumorigenic human ovarian surface epithelial cells, and promoted growth of cancer cells in vivo and in vitro. Expression of YAP also induced expression of ERBB receptors (EGFR, ERBB3) and ligands (HBEGF, NRG1 and NRG2). Treatment of cancer cells with HBEGF or NRG1, in turn, activated YAP and stimulated anchorage-dependent and -independent cell growth and increased ERBB3 mRNA expression. Knockdown of ERBB3 or HBEGF eliminated the stimulatory effects of YAP on cell growth and transformation, while knockdown of YAP abrogated NRG1- and HBEGF-stimulated cell proliferation. Our results demonstrate that YAP has the potential to transform ovarian surface epithelial cells, leading to initiation, early expansion, and progression of ovarian cancer. Our study identified an autocrine loop (NRG1/ERBB3/YAP/NRG1) in ovarian cells, which may control tumorigenesis and promote cancer progression. Combined targeting of the Hippo/YAP and ERBB pathways, especially the NRG1/ERBB3 pathway, has potential to provide a novel strategy for ovarian cancer therapy. Citation Format: Chunbo He, Xiangmin Lv, Guohua Hua, John S Davis, Cheng Wang. The hippo signaling pathway regulates ovarian cancer initiation and progression via an autocrine loop [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1315.

Abstract B82: G-protein coupled estrogen receptor (GPER) agonist G-1 inhibits growth of human granulosa cell tumor cells via blocking microtubule assembly

Accumulating evidence indicates that the G-protein coupled estrogen receptor (GPER) mediates the non-genomic actions of estrogen in many different cells and tissues. The selective GPER agonist G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro- 3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed to distinguish estrogen actions mediated by GPER from those mediated by the traditional nuclear estrogen receptors. Our recent data showed that G-1 suppresses the growth of breast and ovarian cancer cells in a GPER-independent manner. However, the mechanisms underlying the suppressive actions of G-1 on cancer cell growth are still unclear. The aim of the present study was to explore potential mechanisms of G-1 action in human ovarian granulosa tumor cells. The KGN cell line, a cell line derived from a metastatic adult ovarian granulosa cell tumor (aGCT), and the COV434 cell line, a cell line derived from a juvenile granulosa cell tumor (jGCT), were used as cellular models. Cell proliferation and viability were determined by cell number counting and MTT assay, respectively. Cell cycle analysis was monitored by flow cytometry. Key proteins associated with cell cycle progression were analyzed with Western blot. Cell apoptosis was detected by analysis of caspase activity and DNA integrity. Cellular cytoskeleton changes were monitored by fluorescent immunocytochemistry, confocal microscopy and in vitro microtubule assembly assays. Consistent with our previous results, the putative GPER agonist G-1 suppressed proliferation in a concentration- and time-dependent manner in both KGN and COV434 cells. G-1 inhibited GCT cell growth via suppressing cell viability, which was indicated by a significant decrease in the MTT metabolism, an increase in DNA fragmentation, and an increase in PARP cleavage. G-1 treatment did not affect the expression of cyclin D1, cyclin D2 and cyclin E, suggesting that G-1 treatment had no effect on the early stage cell cycle progression, a result further confirmed by flow cytometry. However, flow cytometry showed that G-1 treatment arrested the cell cycle in the G2/M phase. G2/M phase arrest of KGN cells was confirmed by a significant increase in the phosphorylation of histone H3 after G-1 treatment. Surprisingly, the Western blot analysis showed that G-1 treatment did not affect the activation of mitotic promoting factors (MPF) and had no effect on G2/M entry checkpoint proteins and kinases. Fluorescent immunocytochemistry and confocal microscopy showed that G-1 treatment disrupted the segregation of chromosomes during mitosis, an effect that may be attributed to the abnormal assembly of microtubules. In vitro microtubule assembly assays demonstrated that G-1 treatment indeed suppressed tubulin polymerization and thereby inhibited the assembly of the microtubules. In conclusion, our results from the present study demonstrate that G-1, the putative GPER agonist, suppresses GCT cell proliferation and induces tumor cell death via blocking microtubule assembly. The ability of G-1 to suppress tumor cell proliferation and induce tumor cell apoptosis by targeting microtubules makes it a promising candidate drug for the treatment of GCT. Citation Format: Xiangmin Lv, Guohua Hua, Chunbo He, John S Davis, Cheng Wang. G-protein coupled estrogen receptor (GPER) agonist G-1 inhibits growth of human granulosa cell tumor cells via blocking microtubule assembly. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B82.