NLRP7 Enhances Choriocarcinoma Cell Survival and Camouflage in an Inflammasome Independent Pathway.

BACKGROUND: Gestational choriocarcinoma (GC) is a highly malignant trophoblastic tumor that often develops from a complete hydatidiform mole (HM). NLRP7 is the major gene responsible for recurrent HM and is involved in the innate immune response, inflammation and apoptosis. NLRP7 can function in an inflammasome-dependent or -independent pathway. Recently, we have demonstrated that NLRP7 is highly expressed in GC tumor cells and contributes to their tumorigenesis. However, the underlying mechanisms are still unknown. Here, we investigated the mechanism by which NLRP7 controls these processes in malignant (JEG-3) and non-tumor (HTR8/SVneo) trophoblastic cells. Cell survival, dedifferentiation, camouflage, and aggressiveness were compared between normal JEG-3 cells or knockdown for NLRP7, JEG-3 Sh NLRP7. In addition, HTR8/SVneo cells overexpressing NLRP7 were used to determine the impact of NLRP7 overexpression on non-tumor cells. NLRP7 involvement in tumor cell growth and tolerance was further characterized in vivo using the metastatic mouse model of GC. RESULTS: We demonstrate that NLRP7 (i) functions in an inflammasome-dependent and -independent manners in HTR8/SVneo and JEG-3 cells, respectively; (ii) differentially regulates the activity of NF-κB in tumor and non-tumor cells; (iii) increases malignant cell survival, dedifferentiation, and camouflage; and (iv) facilitates tumor cells colonization of the lungs in the preclinical model of GC. CONCLUSIONS: This study demonstrates for the first time the mechanism by which NLRP7, independently of its inflammasome machinery, contributes to GC growth and tumorigenesis. The clinical relevance of NLRP7 in this rare cancer highlights its potential therapeutic promise as a molecular target to treat resistant GC patients.

Role of NLRP7 in Normal and Malignant Trophoblast Cells.

Gestational choriocarcinoma (CC) is an aggressive cancer that develops upon the occurrence of abnormal pregnancies such as Hydatidiform moles (HMs) or upon non-molar pregnancies. CC cells often metastasize in multiple organs and can cause maternal death. Recent studies have established an association between recurrent HMs and mutations in the Nlrp7 gene. NLRP7 is a member of a new family of proteins that contributes to innate immune processes. Depending on its level of expression, NLRP7 can function in an inflammasome-dependent or independent pathway. To date, the role of NLRP7 in normal and in malignant human placentation remains to be elucidated. We have recently demonstrated that NLRP7 is overexpressed in CC trophoblast cells and may contribute to their acquisition of immune tolerance via the regulation of key immune tolerance-associated factors, namely HLA family, ßCG and PD-L1. We have also demonstrated that NLRP7 increases trophoblast proliferation and decreases their differentiation, both in normal and tumor conditions. Actual findings suggest that NLRP7 expression may ensure a strong tolerance of the trophoblast by the maternal immune system during normal pregnancy and may directly affect the behavior and aggressiveness of malignant trophoblast cells. The proposed review summarizes recent advances in the understanding of the significance of NLRP7 overexpression in CC and discusses its multifaceted roles, including its function in an inflammasome-dependent or independent pathways.

NLRP7 Promotes Choriocarcinoma Growth and Progression through the Establishment of an Immunosuppressive Microenvironment.

The inflammatory gene NLRP7 is the major gene responsible for recurrent complete hydatidiform moles (CHM), an abnormal pregnancy that can develop into gestational choriocarcinoma (CC). However, the role of NLRP7 in the development and immune tolerance of CC has not been investigated. Three approaches were employed to define the role of NLRP7 in CC development: (i) a clinical study that analyzed human placenta and sera collected from women with normal pregnancies, CHM or CC; (ii) an in vitro study that investigated the impact of NLRP7 knockdown on tumor growth and organization; and (iii) an in vivo study that used two CC mouse models, including an orthotopic model. NLRP7 and circulating inflammatory cytokines were upregulated in tumor cells and in CHM and CC. In tumor cells, NLRP7 functions in an inflammasome-independent manner and promoted their proliferation and 3D organization. Gravid mice placentas injected with CC cells invalidated for NLRP7, exhibited higher maternal immune response, developed smaller tumors, and displayed less metastases. Our data characterized the critical role of NLRP7 in CC and provided evidence of its contribution to the development of an immunosuppressive maternal microenvironment that not only downregulates the maternal immune response but also fosters the growth and progression of CC.

Evidence-Based View of Safety and Effectiveness of Prokineticin Receptors Antagonists during Pregnancy.

Endocrine gland derived vascular endothelial growth factor (EG-VEGF) is a canonical member of the prokineticin (PROKs) family. It acts via the two G-protein coupled receptors, namely PROKR1 and PROKR2. We have recently demonstrated that EG-VEGF is highly expressed in the human placenta; contributes to placental vascularization and growth and that its aberrant expression is associated with pregnancy pathologies including preeclampsia and fetal growth restriction. These findings strongly suggested that antagonization of its receptors may constitute a potential therapy for the pregnancy pathologies. Two specific antagonists of PROKR1 (PC7) and for PROKR2 (PKRA) were reported to reverse PROKs adverse effects in other systems. In the view of using these antagonists to treat pregnancy pathologies, a proof of concept study was designed to determine the biological significances of PC7 and PKRA in normal pregnancy outcome. PC7 and PKRA were tested independently or in combination in trophoblast cells and during early gestation in the gravid mouse. Both independent and combined treatments uncovered endogenous functions of EG-VEGF. The independent use of antagonists distinctively identified PROKR1 and PROKR2-mediated EG-VEGF signaling on trophoblast differentiation and invasion; thereby enhancing feto-placental growth and pregnancy outcome. Thus, our study provides evidence for the potential safe use of PC7 or PKRA to improve pregnancy outcome.

The Deletion of TRPC6 Channels Perturbs Iron and Zinc Homeostasis and Pregnancy Outcome in Mice.

BACKGROUND/AIMS: Transient receptor potential canonical 6 (TRPC6) protein is a nonselective cation channel permitting the uptake of essential elements such as iron (Fe) and zinc (Zn). TRPC6 is found throughout the body with high expression levels in the placenta. However, its role in this organ is still to be determined. To further advance our understanding of the physiological relevance of TRPC6, we have studied the placental histology, pregnancy outcome and the Fe and Zn status of organs (placenta, brain, kidney, liver and lung) collected from TRPC6 deficient (TRPC6-/-) mice and sex and age-matched C57Bl6/J and B6129SF2/J mice. METHODS: Metal content was quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Quantitative reverse transcriptase PCR (qRT-PCR) and Western Blottings (WB) were performed to analyze the expression of placental markers and TRPC6. RESULTS: Our data show that TRPC6-/- mice displayed reduced litter sizes, structural changes of the placenta, along with altered mRNA levels of CD31 and Gcm1, two markers of placental development. Furthermore, immunoblots revealed elevated amounts of TRPC6 proteins in placentas from women diagnosed with preeclampsia, a common gestational disease. When compared to C57Bl6/J and B6129SF2/J, TRPC6-/- mice had elevated Zn levels in placenta, liver and kidney during embryonic development and postnatally, but not at adulthood. High amounts of Fe were found in the adult brain and liver of TRPC6-/- mice. The lung was however not affected by the deletion of TRPC6, indicating that this mouse strain developed organ and age-dependent perturbations in their Zn and Fe status. CONCLUSION: This work indicates that TRPC6 exerts critical pathophysiological functions in placenta, and provides further evidence for a role of this channel in the homeostasis of cations like Zn and Fe.

EG-VEGF Maintenance Over Early Gestation to Develop a Pregnancy-Induced Hypertensive Animal Model.

During the last decade, multiple animal models have been developed to mimic hallmarks of pregnancy-induced hypertension (PIH) diseases, which include gestational hypertension, preeclampsia (PE), or eclampsia. Converging in vitro, ex vivo, and clinical studies from our group strongly suggested the potential involvement of the new angiogenic factor EG-VEGF (endocrine gland-derived-VEGF) in the development of PIH. Here, we described the protocol that served to demonstrate that maintenance of EG-VEGF production over 11.5 days post coitus (dpc) in the gravid mice caused the development of PIH. The developed model exhibited most hallmarks of preeclampsia.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and its receptor PROKR2 are associated to human colorectal cancer progression and peritoneal carcinomatosis.

BACKGROUND: The highest risk factor for mortality among malignant tumors is metastasis. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an angiogenic factor which biological activity is mediated via two G protein-coupled receptors, prokineticin receptor1 (PROKR1) and PROKR2. Recent studies suggested that EG-VEGF expression is deregulated in multiple cancers including colorectal cancer (CRC). METHODS: Using distinctive CRC and peritoneal carcinomatosis (PC) cohorts and a corresponding control cohort, we determined the circulating levels of EG-VEGF and its in situ expression, and that of its related receptors. RESULTS: Circulating EG-VEGF levels were significantly increased in patients with metastatic PC compared to CRC and control patients (p< 0.05). Furthermore, according to clinicopathologic examinations, local EG-VEGF expression correlated with higher tumor and nodal stages (p< 0.001) of CRC. EG-VEGF and PROKR2 were highly expressed in colorectal primary lesions compared to positive controls. PROKR1 expression was lower and did not change in tumor specimens. Also, EG-VEGF and its receptor PROKR2 were differentially expressed in the colorectal primary lesions and in the control groups. CONCLUSION: Altogether these findings suggest that EG-VEGF/receptors system might be an important actor in the CRC progression into PC and might be involved in the ability of tumor cells to invade other organs. Circulating EG-VEGF could be proposed as a prognostic marker in human CRC and its progression into PC.

Antagonism of EG-VEGF Receptors as Targeted Therapy for Choriocarcinoma Progression In Vitro and In Vivo.

Purpose: Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland-derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy.Experimental Design: Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control (n = 20) and in distinctive CHM (n = 38) and CC (n = 9) cohorts, (ii) an in vitro study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an in vivo study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice.Results: Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions.Conclusions: Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. Clin Cancer Res; 23(22); 7130-40. ©2017 AACR.

Sustained Endocrine Gland-Derived Vascular Endothelial Growth Factor Levels Beyond the First Trimester of Pregnancy Display Phenotypic and Functional Changes Associated With the Pathogenesis of Pregnancy-Induced Hypertension.

Pregnancy-induced hypertension diseases are classified as gestational hypertension, preeclampsia, or eclampsia. The mechanisms of their development and prediction are still to be discovered. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an angiogenic factor secreted by the placenta during the first trimester of human pregnancy that was shown to control trophoblast invasion, to be upregulated by hypoxia, and to be abnormally elevated in pathological pregnancies complicated with preeclampsia and intrauterine growth restriction. These findings suggested that sustaining EG-VEGF levels beyond the first trimester of pregnancy may contribute to pregnancy-induced hypertension. To test this hypothesis, osmotic minipumps delivering EG-VEGF were implanted subcutaneously into gravid OF1 (Oncins France 1) mice on day 11.5 post coitus, which is equivalent to the end of the first trimester of human pregnancy. Mice were euthanized at 15.5 and 18.5 days post coitus to assess (1) litter size, placental, and fetal weights; (2) placental histology and function; (3) maternal blood pressure; (4) renal histology and function; and (5) circulating soluble fms-like tyrosine kinase 1 and soluble endoglin. Increased EG-VEGF levels caused significant defects in placental organization and function. Both increased hypoxia and decreased trophoblast invasion were observed. Treated mice had elevated circulating soluble fms-like tyrosine kinase 1 and soluble endoglin and developed gestational hypertension with dysregulated maternal kidney function. EG-VEGF effect on the kidney function was secondary to its effects on the placenta as similarly treated male mice had normal kidney functions. Altogether, these data provide a strong evidence to confirm that sustained EG-VEGF beyond the first trimester of pregnancy contributes to the development of pregnancy-induced hypertension.

Prokineticins in central and peripheral control of human reproduction.

Prokineticin 1 (PROK1) and (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. PROKs activate two G-protein linked receptors (prokineticin receptor 1 and 2, PROKR1 and PROKR2). Both PROK1 and PROK2 have been found to regulate a stunning array of biological functions. In particular, PROKs stimulate gastrointestinal motility, thus accounting for their family name "prokineticins". PROK1 acts as a potent angiogenic mitogen, thus earning its other name, endocrine gland-derived vascular endothelial factor. In contrast, PROK2 signaling pathway has been shown to be a critical regulator of olfactory bulb morphogenesis and sexual maturation. During the last decade, strong evidences established the key roles of prokineticins in the control of human central and peripheral reproductive processes. PROKs act as main regulators of the physiological functions of the ovary, uterus, placenta, and testis, with marked dysfunctions in various pathological conditions such as recurrent pregnancy loss, and preeclampsia. PROKs have also been associated to the tumor development of some of these organs. In the central system, prokineticins control the migration of GnRH neurons, a key process that controls reproductive functions. Importantly, mutations in PROK2 and PROKR2 are associated to the development of Kallmann syndrome, with direct consequences on the reproductive system. This review describes the finely tuned actions of prokineticins in the control of the central and peripheral reproductive processes. Also, it discusses future research directions for the use of these cytokines as diagnostic markers for several reproductive diseases.