EGFL6 promotes endometrial cancer cell migration and proliferation.

OBJECTIVE: EGFL6, a growth factor produced by adipocytes, is upregulated in and implicated in the tumorigenesis of multiple tumor types. Given the strong link between obesity and endometrial cancer, we sought to determine the impact of EGFL6 on endometrial cancer. METHODS: EGFL6 expression in endometrial cancer and correlation with patient outcomes was evaluated in the human protein atlas and TCGA. EGFL6 treatment, expression upregulation, and shRNA knockdown were used to evaluate the impact of EGFL6 on the proliferation and migration of 3 endometrial cancer cell lines in vitro. Similarly, the impact of EGFL6 expression and knockdown on tumor growth was evaluated. Western blotting was used to evaluate the impact of EGFL6 on MAPK phosphorylation. RESULTS: EGFL6 is upregulated in endometrial cancer, primarily in cony-number high tumors. High tumor endometrial cancer expression of EGFL6 predicts poor patient prognosis. We find that EGFL6 acts to activate the MAPK pathway increasing cellular proliferation and migration. In xenograft models, EGFL6 overexpression increases endometrial cancer tumor growth while EGFL6 knockdown decreases endometrial cancer tumor growth. CONCLUSIONS: EGFL6 is a marker of poor prognosis endometrial cancers, driving cancer cell proliferation and growth. As such EGFL6 represents a potential therapeutic target in endometrial cancer.

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies.

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of Significance: We developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

Generation and characterization of humanized affinity-matured EGFL6 antibodies for ovarian cancer therapy.

OBJECTIVES: Epidermal growth factor EGF-like domain multiple-6 (EGFL6) is highly expressed in high grade serous ovarian cancer and promotes both endothelial cell proliferation/angiogenesis and cancer cell proliferation/metastasis. As such it has been implicated as a therapeutic target. As a secreted factor, EGFL6 is a candidate for antibody therapy. The objectives of this study were to create and validate humanized affinity-matured EGFL6 neutralizing antibodies for clinical development. METHODS: A selected murine EGFL6 antibody was humanized using CDR grafting to create 26 variant humanized antibodies. These were screened and the lead candidate was affinity matured. Seven humanized affinity-matured EGFL6 antibodies were screened for their ability to block EGFL6 activity on cancer cells in vitro, two of which were selected and tested their therapeutic activity in vivo. RESULTS: Humanized affinity matured antibodies demonstrated high affinity for EGFL6 (150 pM to 2.67 nM). We found that several humanized affinity-matured EGFL6 antibodies specifically bound to recombinant, and native human EGFL6. Two lead antibodies were able to inhibit EGFL6-mediated (i) cancer cell migration, (ii) proliferation, and (iii) increase in ERK phosphorylation in cancer cells in vitro. Both lead antibodies restricted growth of an EGFL6 expressing ovarian cancer patient derived xenograft. Analysis of treated human tumor xenografts indicated that anti-EGFL6 therapy suppressed angiogenesis, inhibited tumor cell proliferation, and promoted tumor cell apoptosis. CONCLUSIONS: Our studies confirm the ability of these humanized affinity-matured antibodies to neutralize EGFL6 and acting as a therapeutic to restrict cancer growth. This work supports the development of these antibody for first-in-human clinical trials.

Targeting Therapeutic Resistance and Multinucleate Giant Cells in CCNE1-Amplified HR-Proficient Ovarian Cancer.

Approximately 20% of high-grade serous ovarian cancers (HGSOC) have CCNE1 amplification. CCNE1-amplified tumors are homologous recombination (HR) proficient and resistant to standard therapies. Therapy resistance is associated with increased numbers of polyploid giant cancer cells (PGCC). We sought to identify new therapeutic approaches for patients with CCNE1-amplified tumors. Using TCGA data, we find that the mTOR, HR, and DNA checkpoint pathways are enriched in CCNE1-amplified ovarian cancers. Furthermore, Interactome Mapping Analysis linked the mTOR activity with upregulation of HR and DNA checkpoint pathways. Indeed, we find that mTOR inhibitors (mTORi) downregulate HR/checkpoint genes in CCNE1-amplified tumors. As CCNE1-amplified tumors are dependent on the HR pathway for viability, mTORi proved selectively effective in CCNE1-amplified tumors. Similarly, via downregulation of HR genes, mTORi increased CCNE1-amplifed HGSOC response to PARPi. In contrast, overexpression of HR/checkpoint proteins (RAD51 or ATR), induced resistance to mTORi. In vivo, mTORi alone potently reduced CCNE1-amplified tumor growth and the combination of mTORi and PARPi increased response and tumor eradication. Tumors treated with mTORi demonstrated a significant reduction in ALDH+ PGCCs. Finally, as a proof of principle, we identified three patients with CCNE1 amplified tumors who were treated with an mTORi. All three obtained clinical benefits from the therapy. Our studies and clinical experience indicate mTORi are a potential therapeutic approach for patients with CCNE1-amplified tumors.

An evolving paradigm of cancer stem cell hierarchies: therapeutic implications.

Over a decade of research has confirmed the critical role of cancer stem-like cells (CSCs) in tumor initiation, chemoresistance, and metastasis. Increasingly, CSC hierarchies have begun to be defined with some recurring themes. This includes evidence that these hierarchies are 'flexible,' with both cell state transitions and dedifferentiation events possible. These findings pose therapeutic hurdles and opportunities. Here, we review cancer stem cell hierarchies and their interactions with the tumor microenvironment. We also discuss the current therapeutic approaches designed to target CSC hierarchies and initial clinical trial results for CSC targeting agents. While cancer stem cell targeted therapies are still in their infancy, we are beginning to see encouraging results that suggest a positive outlook for CSC-targeting approaches.

NFATC4 promotes quiescence and chemotherapy resistance in ovarian cancer.

Development of chemotherapy resistance is a major problem in ovarian cancer. One understudied mechanism of chemoresistance is the induction of quiescence, a reversible nonproliferative state. Unfortunately, little is known about regulators of quiescence. Here, we identify the master transcription factor nuclear factor of activated T cells cytoplasmic 4 (NFATC4) as a regulator of quiescence in ovarian cancer. NFATC4 is enriched in ovarian cancer stem-like cells and correlates with decreased proliferation and poor prognosis. Treatment of cancer cells with cisplatin resulted in NFATC4 nuclear translocation and activation of the NFATC4 pathway, while inhibition of the pathway increased chemotherapy response. Induction of NFATC4 activity resulted in a marked decrease in proliferation, G0 cell cycle arrest, and chemotherapy resistance, both in vitro and in vivo. Finally, NFATC4 drove a quiescent phenotype in part via downregulation of MYC. Together, these data identify NFATC4 as a driver of quiescence and a potential new target to combat chemoresistance in ovarian cancer.

CD105 Is Expressed in Ovarian Cancer Precursor Lesions and Is Required for Metastasis to the Ovary.

: Most high-grade serous ovarian cancers (HGSCs) initiate from the fallopian tube epithelium and then metastasize to the ovary and throughout the abdomen. Genomic analyses suggest that most HGSCs seed the ovary prior to abdominal dissemination. Similarly, animal models support a critical role for the ovary in driving abdominal dissemination. Thus, HGSC cell recruitment to the ovary appears to be a critical component of HGSC cell metastasis. We sought to identify factors driving HGSC recruitment to the ovary. We identified CD105 (endoglin, or ENG, a TGF- receptor family member) as a mediator of HGSC cell ovarian recruitment. We found that CD105 was expressed on both serous tubal intraepithelial carcinoma (STIC) cells (STICs-HGSC precursors in the fallopian tube epithelium) and HGSC cells. Using data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE), we showed that high CD105 expression by HGSC cells correlated with a metastatic signature. Furthermore, intravenous injection of CD105(+) HGSC tumor cells, but not CD105(-), resulted in ovarian-specific metastasis and abdominal dissemination of disease. CD105 knockdown or blockade with a clinically relevant CD105-neutralizing mAb (TRC105), inhibited HGSC metastasis, reduced ascites, and impeded growth of abdominal tumor nodules, thereby improving overall survival in animal models of ovarian cancer. CD105 knockdown was associated with a reduction in TGF-signaling. Together, our data support CD105 as a critical mediator of ovarian cancer spread to the ovary and implicate it as a potential therapeutic target.

Sampling from single-cell observations to predict tumor cell growth in-vitro and in-vivo.

Cancer stem-like cells (CSCs) are a topic of increasing importance in cancer research, but are difficult to study due to their rarity and ability to rapidly divide to produce non-self-cells. We developed a simple model to describe transitions between aldehyde dehydrogenase (ALDH) positive CSCs and ALDH(-) bulk ovarian cancer cells. Microfluidics device-isolated single cell experiments demonstrated that ALDH+ cells were more proliferative than ALDH(-) cells. Based on our model we used ALDH+ and ALDH(-) cell division and proliferation properties to develop an empiric sampling algorithm and predict growth rate and CSC proportion for both ovarian cancer cell line and primary ovarian cancer cells, in-vitro and in-vivo. In both cell line and primary ovarian cancer cells, the algorithm predictions demonstrated a high correlation with observed ovarian cancer cell proliferation and CSC proportion. High correlation was maintained even in the presence of the EGF-like domain multiple 6 (EGFL6), a growth factor which changes ALDH+ cell asymmetric division rates and thereby tumor growth rates. Thus, based on sampling from the heterogeneity of in-vitro cell growth and division characteristics of a few hundred single cells, the simple algorithm described here provides rapid and inexpensive means to generate predictions that correlate with in-vivo tumor growth.

EGFL6 Regulates the Asymmetric Division, Maintenance, and Metastasis of ALDH+ Ovarian Cancer Cells.

Little is known about the factors that regulate the asymmetric division of cancer stem-like cells (CSC). Here, we demonstrate that EGFL6, a stem cell regulatory factor expressed in ovarian tumor cells and vasculature, regulates ALDH+ ovarian CSC. EGFL6 signaled at least in part via the oncoprotein SHP2 with concomitant activation of ERK. EGFL6 signaling promoted the migration and asymmetric division of ALDH+ ovarian CSC. As such, EGFL6 increased not only tumor growth but also metastasis. Silencing of EGFL6 or SHP2 limited numbers of ALDH+ cells and reduced tumor growth, supporting a critical role for EGFL6/SHP2 in ALDH+ cell maintenance. Notably, systemic administration of an EGFL6-neutralizing antibody we generated restricted tumor growth and metastasis, specifically blocking ovarian cancer cell recruitment to the ovary. Together, our results offer a preclinical proof of concept for EGFL6 as a novel therapeutic target for the treatment of ovarian cancer. Cancer Res; 76(21); 6396-409. ©2016 AACR.

New models of hematogenous ovarian cancer metastasis demonstrate preferential spread to the ovary and a requirement for the ovary for abdominal dissemination.

Emerging evidence suggest that many high-grade serous "ovarian" cancers (HGSOC) start in the fallopian tube. Cancer cells are then recruited to the ovary and then spread diffusely through the abdomen. The mechanism of ovarian cancer spread was thought to be largely due to direct shedding of tumor cells into the peritoneal cavity with vascular spread being of limited importance. Recent work challenges this dogma, suggesting hematogenous spread of ovarian cancer may play a larger role in ovarian cancer cell metastasis than previously thought. One reason the role of vascular spread of ovarian cancer has not been fully elucidated is the lack of easily accessible models of vascular ovarian cancer metastasis. Here, we present 3 metastatic models of ovarian cancer which confirm the ability of ovarian cancer to hematogenously spread. Strikingly, we observe a high rate of metastasis to the ovary with the development of ascites in these models. Interestingly, oophorectomy resulted in a complete loss of peritoneal metastases and ascites. Taken together, our data indicate that hematogenously disseminated HGSOC cells have a unique tropism for the ovary and that hematogenous spread in ovarian cancer may be more common than appreciated. Furthermore, our studies support a critical role for the ovary in promoting HGSOC cell metastasis to the abdomen. The models developed here represent important new tools to evaluate both the mechanism of cancer cell recruitment to the ovary and understand and target key steps in ovarian cancer metastasis.

Identifying an ovarian cancer cell hierarchy regulated by bone morphogenetic protein 2.

Whether human cancer follows a hierarchical or stochastic model of differentiation is controversial. Furthermore, the factors that regulate cancer stem-like cell (CSC) differentiation potential are largely unknown. We used a novel microfluidic single-cell culture method to directly observe the differentiation capacity of four heterogeneous ovarian cancer cell populations defined by the expression of the CSC markers aldehyde dehydrogenase (ALDH) and CD133. We evaluated 3,692 progeny from 2,833 cells. We found that only ALDH(+)CD133(+) cells could generate all four ALDH(+/-)CD133(+/-) cell populations and identified a clear branched differentiation hierarchy. We also observed a single putative stochastic event. Within the hierarchy of cells, bone morphologenetic protein 2 (BMP2) is preferentially expressed in ALDH(-)CD133(-) cells. BMP2 promotes ALDH(+)CD133(+) cell expansion while suppressing the proliferation of ALDH(-)CD133(-) cells. As such, BMP2 suppressed bulk cancer cell growth in vitro but increased tumor initiation rates, tumor growth, and chemotherapy resistance in vivo whereas BMP2 knockdown reduced CSC numbers, in vivo growth, and chemoresistance. These data suggest a hierarchical differentiation pattern in which BMP2 acts as a feedback mechanism promoting ovarian CSC expansion and suppressing progenitor proliferation. These results explain why BMP2 suppresses growth in vitro and promotes growth in vivo. Together, our results support BMP2 as a therapeutic target in ovarian cancer.

A novel model for evaluating therapies targeting human tumor vasculature and human cancer stem-like cells.

Human tumor vessels express tumor vascular markers (TVM), proteins that are not expressed in normal blood vessels. Antibodies targeting TVMs could act as potent therapeutics. Unfortunately, preclinical in vivo studies testing anti-human TVM therapies have been difficult to do due to a lack of in vivo models with confirmed expression of human TVMs. We therefore evaluated TVM expression in a human embryonic stem cell-derived teratoma (hESCT) tumor model previously shown to have human vessels. We now report that in the presence of tumor cells, hESCT tumor vessels express human TVMs. The addition of mouse embryonic fibroblasts and human tumor endothelial cells significantly increases the number of human tumor vessels. TVM induction is mostly tumor-type-specific with ovarian cancer cells inducing primarily ovarian TVMs, whereas breast cancer cells induce breast cancer specific TVMs. We show the use of this model to test an anti-human specific TVM immunotherapeutics; anti-human Thy1 TVM immunotherapy results in central tumor necrosis and a three-fold reduction in human tumor vascular density. Finally, we tested the ability of the hESCT model, with human tumor vascular niche, to enhance the engraftment rate of primary human ovarian cancer stem-like cells (CSC). ALDH(+) CSC from patients (n = 6) engrafted in hESCT within 4 to 12 weeks whereas none engrafted in the flank. ALDH(-) ovarian cancer cells showed no engraftment in the hESCT or flank (n = 3). Thus, this model represents a useful tool to test anti-human TVM therapy and evaluate in vivo human CSC tumor biology.

Endothelin receptor-A is required for the recruitment of antitumor T cells and modulates chemotherapy induction of cancer stem cells.

BACKGROUND: The endothelin receptor-A (ETRA) plays an important role in tumor cell migration, metastasis, and proliferation. The endothelin receptor B (ETRB) plays a critical role in angiogenesis and the inhibition of anti-tumor immune cell recruitment. Thus dual blockade of ETRA and ETRB could have significant anti-tumor effects. RESULTS: Dual ETRA/ETRB blockade with macitentan (or the combination of the ETRA and ETRB antagonists BQ123 and BQ788) did not enhance antitumor immune cell recruitment. In vitro studies demonstrate that ETRA inhibition prevents the induction of ICAM1 necessary for immune cell recruitment. When used as a single agent against human tumor xenografts, macitentan demonstrated non-significant anti-tumor activity. However, when used in combination with chemotherapy, macitentan specifically reduced tumor growth in cell lines with CD133+ cancer stem cells. We found that ETRA is primarily expressed on CD133+ CSC in both cell lines and primary human tumor cells. ETRA inhibition of CSC prevented chemotherapy induced increases in tumor stem cells. Furthermore, ETRA inhibition in combination with chemotherapy reduced the formation of tumor spheres. METHODS: We tested the dual ETRA/ETRB antagonist macitentan in conjunction with (1) an anti-tumor vaccine and (2) chemotherapy, in order to assess the impact of dual ETRA/ETRB blockade on anti-tumor immune cell infiltration and ovarian tumor growth. In vitro murine and human cell line, tumor sphere assays and tumor xenograft models were utilized to evaluate the effect of ETRA/ETRB blockade on cell proliferation, immune cell infiltration and cancer stem cell populations. CONCLUSIONS: These studies indicate a critical role for ETRA in the regulation of immune cell recruitment and in the CSC resistance to chemotherapy.