Extragonadal FSHR Expression and Function—Is It Real?

Expression of the follicle-stimulating hormone receptor (FSHR), besides gonadal tissues, has recently been detected in several extragonadal normal and tumorous tissues, including different types of primary and metastatic cancer and tumor vessel endothelial cells (TVEC). The suggested FSH actions in extragonadal tissues include promotion of angiogenesis, myometrial contractility, skeletal integrity, and adipose tissue accumulation. Non-malignant cells within cancer tissue have been shown to be devoid of FSHR expression, which implies a potential role of FSHR as a diagnostic, prognostic, or even a therapeutic tool. There are shared issues between several of the published reports questioning the validity of some of the conclusion. Firstly, protein expression of FSHR was performed solely with immunohistochemistry (IHC) using either an unavailable "in house" FSHR323 monoclonal antibody or poorly validated polyclonal antibodies, usually without additional methodological quality control and confirmations. Secondly, there is discrepancy between the hardly traceable or absent FSHR gene amplification/transcript data and non-reciprocal strong FSHR protein immunoreactivity. Thirdly, the pharmacological high doses of recombinant FSH used in in vitro studies also jeopardizes the physiological or pathophysiological meaning of the findings. We performed in this review a critical analysis of the results presenting extragonadal expression of FSHR and FSH action, and provide a rationale for the validation of the reported results using additional more accurate and sensitive supplemental methods, including in vivo models and proper positive and negative controls

Revisiting the expression and function of follicle-stimulation hormone receptor in human umbilical vein endothelial cells

Expression of follicle-stimulation hormone receptor (FSHR) is confined to gonads and at low levels to some extragonadal tissues like human umbilical vein endothelial cells (HUVEC). FSH-FSHR signaling was shown to promote HUVEC angiogenesis and thereafter suggested to have an influential role in pregnancy. We revisited hereby the expression and functionality of FSHR in HUVECs angiogenesis, and were unable to reproduce the FSHR expression in human umbilical cord, HUVECs or immortalized HUVECs (HUV-ST). Positive controls as granulosa cells and HEK293 cells stably transfected with human FSHR cDNA expressed FSHR signal. In contrast to positive control VEGF, FSH treatment showed no effects on tube formation, nitric oxide production, wound healing or cell proliferation in HUVEC/HUV-ST. Thus, it remains open whether the FSH-FSHR activation has a direct regulatory role in the angiogenesis of HUVECs

Extragonadal FSHR Expression and Function—Is It Real?

Expression of the follicle-stimulating hormone receptor (FSHR), besides gonadal tissues, has recently been detected in several extragonadal normal and tumorous tissues, including different types of primary and metastatic cancer and tumor vessel endothelial cells (TVEC). The suggested FSH actions in extragonadal tissues include promotion of angiogenesis, myometrial contractility, skeletal integrity, and adipose tissue accumulation. Non-malignant cells within cancer tissue have been shown to be devoid of FSHR expression, which implies a potential role of FSHR as a diagnostic, prognostic, or even a therapeutic tool. There are shared issues between several of the published reports questioning the validity of some of the conclusion. Firstly, protein expression of FSHR was performed solely with immunohistochemistry (IHC) using either an unavailable “in house” FSHR323 monoclonal antibody or poorly validated polyclonal antibodies, usually without additional methodological quality control and confirmations. Secondly, there is discrepancy between the hardly traceable or absent FSHR gene amplification/transcript data and non-reciprocal strong FSHR protein immunoreactivity. Thirdly, the pharmacological high doses of recombinant FSH used in in vitro studies also jeopardizes the physiological or pathophysiological meaning of the findings. We performed in this review a critical analysis of the results presenting extragonadal expression of FSHR and FSH action, and provide a rationale for the validation of the reported results using additional more accurate and sensitive supplemental methods, including in vivo models and proper positive and negative controls

Functional Implications of LH/hCG Receptors in Pregnancy-Induced Cushing Syndrome

Context: Elevated human choriogonadotropin (hCG) may stimulate aberrantly expressed luteinizing hormone (LH)/hCG receptor (LHCGR) in adrenal glands, resulting in pregnancy-induced bilateral macronodular adrenal hyperplasia and transient Cushing syndrome (CS). Objective: To determine the role of LHCGR in transient, pregnancy-induced CS. Design, Setting, Patient, and Intervention: We investigated the functional implications of LHCGRs in a patient presenting, at a tertiary referral center, with repeated pregnancy-induced CS with bilateral adrenal hyperplasia, resolving after parturition. Main Outcome Measures and Results: Acute testing for aberrant hormone receptors was negative except for arginine vasopressin (AVP)–increased cortisol secretion. Long-term hCG stimulation induced hypercortisolism, which was unsuppressed by dexamethasone. Postadrenalectomy histopathology demonstrated steroidogenically active adrenocortical hyperplasia and ectopic cortical cell clusters in the medulla. Quantitative polymerase chain reaction showed upregulated expression of LHCGR, transcription factors GATA4, ZFPM2, and proopiomelanocortin (POMC), AVP receptors (AVPRs) AVPR1A and AVPR2, and downregulated melanocortin 2 receptor (MC2R) vs control adrenals. LHCGR was localized in subcapsular, zona glomerulosa, and hyperplastic cells. Single adrenocorticotropic hormone–positive medullary cells were demonstrated in the zona reticularis. The role of adrenal adrenocorticotropic hormone was considered negligible due to downregulated MC2R. Coexpression of CYP11B1/CYP11B2 and AVPR1A/AVPR2 was observed in ectopic cortical cells in the medulla. hCG stimulation of the patient’s adrenal cell cultures significantly increased cyclic adenosine monophosphate, corticosterone, 11-deoxycortisol, cortisol, and androstenedione production. CTNNB1, PRKAR1A, ARMC5, and PRKACA gene mutational analyses were negative. Conclusion: Nongenetic, transient, somatic mutation-independent, pregnancy-induced CS was due to hCG-stimulated transformation of LHCGR- positive undifferentiated subcapsular cells (presumably adrenocortical progenitors) into LHCGR-positive hyperplastic cortical cells. These cells respond to hCG stimulation with cortisol secretion. Without the ligand, they persist with aberrant LHCGR expression and the ability to respond to the same stimulus

A tumor cell specific Zona Pellucida glycoprotein 3 RNA transcript encodes an intracellular cancer antigen

BackgroundExpression of Zona Pellucida glycoprotein 3 (ZP3) in healthy tissue is restricted to the extracellular Zona Pellucida layer surrounding oocytes of ovarian follicles and to specific cells of the spermatogenic lineage. Ectopic expression of ZP3 has been observed in various types of cancer, rendering it a possible therapeutic target.MethodsTo support its validity as therapeutic target, we extended the cancer related data by investigating ZP3 expression using immunohistochemistry (IHC) of tumor biopsies. We performed a ZP3 transcript specific analysis of publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, and validated expression data by independent computational analysis and real-time quantitative PCR (qPCR). A correlation between the ZP3 expression level and pathological and clinical parameters was also investigated.ResultsIHC data for several cancer types showed abundant ZP3 protein staining, which was confined to the cytoplasm, contradicting the extracellular protein localization in oocytes. We noticed that an alternative ZP3 RNA transcript, which we term ‘ZP3-Cancer’, was annotated in gene databases that lacks the genetic information encoding the N-terminal signal peptide that governs entry into the secretory pathway. This explains the intracellular localization of ZP3 in tumor cells. Analysis of publicly available RNA-seq data of 1339 cancer cell lines (CCLs), 10386 tumor tissues (The Cancer Genome Atlas) and 7481 healthy tissues (Genotype-Tissue Expression) indicated that ZP3-Cancer is the dominant ZP3 RNA transcript in tumor cells and is highly enriched in many cancer types, particularly in rectal, ovarian, colorectal, prostate, lung and breast cancer. Expression of ZP3-Cancer in tumor cells was confirmed by qPCR. Higher levels of the ZP3-Cancer transcript were associated with more aggressive tumors and worse survival of patients with various types of cancer.ConclusionThe cancer-restricted expression of ZP3-Cancer renders it an attractive tumor antigen for the development of a therapeutic cancer vaccine, particularly using mRNA expression technologies

Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3 weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist

Functional Characterization of MicroRNA-27a-3p Expression in Human Polycystic Ovary Syndrome

The goal of this study was to characterize the function of microRNA-27a-3p (miR-27a-3p) in polycystic ovary syndrome (PCOS). miR-27a-3p expression was analyzed in excised granulosa cells (GCs) from 21 patients with PCOS and 12 normal patients undergoing in vitro fertilization cycle treatments and in 17 nontreated cuneiform ovarian resection PCOS samples and 13 control ovarian samples from patients without PCOS. We found that the expression of miR-27a-3p was significantly increased in both excised GCs and the ovaries of patients with PCOS compared with the controls. Insulin treatment of the human granulosa-like tumor cell line (KGN) resulted in decreased downregulated expression of miR-27a-3p, and this effect appeared to be mediated by signal transducer and activator of transcription STAT1 and STAT3. The overexpression of miR-27a-3p in KGN cells inhibited SMAD5, which in turn decreased cell proliferation and promoted cell apoptosis. After KGN cells were stimulated with insulin for 48 hours, there was increased expression of SMAD5 protein and decreased apoptosis. Additionally, knockdown/overexpression of SMAD5 in KGN cells reduced/increased cell number and promoted/inhibited cell apoptosis. Insulin-stimulated primary GCs isolated from patients with PCOS, in contrast to normal GCs or KGN cells, did not exhibit decreased miR-27a-3p expression. The differences in the expression levels in KGN cells and human PCOS GCs are likely explained by increased miR-27a-3p expression in the GCs caused by insulin resistance in PCOS. Taken together, our data provided evidence for a functional role of miR-27a-3p in the GCs' dysfunction that occurs in patients with PCOS

Novel Expression of Zona Pellucida 3 Protein in Normal Testis; Potential Functional Implications

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.</p