Activation of G-Protein-Coupled Estrogen Receptor 1 (GPER1) Reduces Progression of Vulvar Carcinoma Cells.

Whether G protein-coupled estrogen receptor 1 (GPER1) is tumor-promoting or tumor-suppressive depends in part on tumor entity. Little is known about the function of GPER1 in vulvar carcinoma. In this work, we aim to clarify what role GPER1 plays in vulvar cancer, tumor-promoting or tumor-suppressive. Localization of GPER1 in A431 and CAL-39 vulvar carcinoma cells was examined by immunofluorescence. Using a tissue microarray of vulvar neoplasias, the correlation between GPER1 expression and grade of malignancy was investigated. A431 and CAL-39 cells were treated either with GPER1 agonist G1 or antagonist G36. Proliferation was quantified by BrdU assay and viability examined using Resazurin assay. Morphological changes were analyzed by microscopy and measured using ImageJ. Cell migration was analyzed by gap closure assay. Clonogenic potential was tested by colony and sphere formation. Expression of estrogen receptors was examined by Western blot. GPER1 was found consistently expressed in vulvar neoplasia tissues. The immune-reactive score was found to be significantly higher in tissue samples of lymph node metastases and neoplasias with grade 3. In A431 and CAL-39 vulvar carcinoma cells, GPER1 expression was mainly found in the cytoplasm and nuclei. Treatment of A431 and CAL-39 cells with GPER1 agonist G1 resulted in a decrease in proliferation and migration. In addition, colony formation and tumor sphere formation were reduced. Furthermore, morphological signs of necrosis and reduction in cell viability after G1 treatment were observed. The GPER1 antagonist G36 did not have significant effects on vulvar carcinoma cells. Neither agonist G1 nor antagonist G36 treatment resulted in altered expression of estrogen receptors. Activation of GPER1 with GPER1 agonist G1 reduces the tumorigenic potential of the vulvar carcinoma cells. It can be deduced from this that GPER1 appears to have a tumor-suppressive effect in vulvar carcinoma.

Like Brothers in Arms: How Hormonal Stimuli and Changes in the Metabolism Signaling Cooperate, Leading HPV Infection to Drive the Onset of Cervical Cancer.

Despite all precautionary actions and the possibility of using vaccinations to counteract infections caused by human papillomaviruses (HPVs), HPV-related cancers still account for approximately 5% of all carcinomas. Worldwide, many women are still excluded from adequate health care due to their social position and origin. Therefore, immense efforts in research and therapy are still required to counteract the challenges that this disease entails. The special thing about an HPV infection is that it is not only able to trick the immune system in a sophisticated way, but also, through genetic integration into the host genome, to use all the resources available to the host cells to complete the replication cycle of the virus without activating the alarm mechanisms of immune recognition and elimination. The mechanisms utilized by the virus are the metabolic, immune, and hormonal signaling pathways that it manipulates. Since the virus is dependent on replication enzymes of the host cells, it also intervenes in the cell cycle of the differentiating keratinocytes and shifts their terminal differentiation to the uppermost layers of the squamocolumnar transformation zone (TZ) of the cervix. The individual signaling pathways are closely related and equally important not only for the successful replication of the virus but also for the onset of cervical cancer. We will therefore analyze the effects of HPV infection on metabolic signaling, as well as changes in hormonal and immune signaling in the tumor and its microenvironment to understand how each level of signaling interacts to promote tumorigenesis of cervical cancer.

Inhibition of aerobic glycolysis enhances the anti-tumor efficacy of Zoptarelin Doxorubicin in triple-negative breast cancer cells.

AIM: A characteristic of cancer cells including triple-negative breast cancers (TNBC) is an increased aerobic glycolysis for ATP production representing a selective therapeutic target. More than 70% of TNBC express gonadotropin-releasing hormone receptors (GnRH-R). These receptors can be used for targeted chemotherapy with cytotoxic GnRH agonists such as Zoptarelin Doxorubicin, in which doxorubicin is covalently linked to [D-Lys6 ]GnRH. In this study, we have analyzed whether inhibition of aerobic glycolysis can enhance the antitumor efficacy of GnRH-R-targeted chemotherapy using Zoptarelin Doxorubicin. METHODS: Triple-negative breast cancers cell lines MDA-MB-231 and HCC1806 were treated with Zoptarelin Doxorubicin, glycolysis inhibitor 2-deoxy-D-glucose (2DG) or the combination of both agents. Cell viability was measured using Alamar blue. Induction of apoptosis was quantified by measurement of loss of mitochondrial membrane potential. In vivo experiments were performed using nude mice bearing xenografted MDA-MB-231 tumors. RESULTS: Treatment of TNBC cells with Zoptarelin Doxorubicin or with 2DG resulted in a significant decrease of cell viability and a significant increase of apoptosis. Treatment with Zoptarelin Doxorubicin in combination with 2DG resulted in significantly reduced viability and enhanced apoptosis compared with single-agent treatments. Combinational index (CI) analysis revealed the co-treatment effect as a synergistic. The antitumor effects of Zoptarelin Doxorubicin or 2DG were confirmed in nude mice. The tumor reducing effects of Zoptarelin Doxorubicin were enhanced by combination with 2DG. CONCLUSION: The glycolytic phenotype of TNBC can be used to improve antitumor therapies. Co-treatment of Zoptarelin Doxorubicin with glycolysis inhibitor 2DG might be a suitable therapeutic option for GnRH receptor-positive TNBC.

Inhibition of SDF-1/CXCR4-induced epithelial-mesenchymal transition by kisspeptin-10.

Recently we have shown that breast cancer cell invasion was dramatically increased when co-cultured with MG63 cells. In addition we have generated mesenchymal transformed MCF-7 breast cancer cells (MCF-7-EMT), showing significantly increased invasion in contrast to wild type MCF-7 cells (MCF-7 WT). In this study we have analyzed whether stromal derived factor-1 (SDF-1) is responsible for MCF-7 and T-47-D breast cancer cell invasion and epithelial-mesenchymal-transition (EMT). In addition we have analyzed whether kisspeptin-10 (KP-10) treatment affects SDF-1-induced invasion and EMT. Invasion was quantified by assessment of MCF-7 and T-47-D breast cancer cell migration rate through an artificial basement membrane in a modified Boyden chamber during co-culture with MG63 cells or after treatment with SDF-1α, SDF-1ß or the combination of both isoforms. Induction of EMT was verified by analysis of protein expression of epithelial marker E-cadherin (CDH1) and mesenchymal markers N-cadherin (CDH2) and Vimentin (VIM). The role of SDF-1 for invasion and induction of EMT in breast cancer cells was analyzed by blocking SDF-1 secretion during co-culture with MG63 cells. In addition effects of KP-10 treatment on SDF-1-induced invasion and EMT were analyzed. Breast cancer cell invasion was significantly increased when co-cultured with MG63 cells. During co-culture SDF-1 protein expression of MG63 cells was significantly induced. The increased breast cancer cell invasion could be blocked by anti-SDF-1 antibodies. Treatment of breast cancer cells in monoculture (without MG63) with SDF-1α, SDF-1ß or the combination of both isoforms resulted in a significant escalation of breast cancer cell invasion and induction of EMT. Protein expression of mesenchymal markers CDH2 and VIM was clearly elevated, whereas protein expression of epithelial marker CDH1 was clearly decreased. The SDF-1-induced increase of cell invasion was significantly reduced after treatment with KP-10. In addition, induction of EMT was inhibited. Furthermore, protein expression of the binding site of SDF-1, CXC-motive-chemokine receptor 4 (CXCR-4), was reduced by KP-10. Treatment of MCF-7-EMT cells with KP-10 resulted in a significant drop of cell invasion and CXCR-4 protein expression. Our findings suggest that SDF-1 plays a major role in breast cancer invasion and EMT. SDF-1-induced invasion and EMT can be inhibited by KP-10 treatment by down-regulating CXCR-4 expression.

Generation of MCF-7 cells with aggressive metastatic potential in vitro and in vivo.

Epithelial-mesenchymal transition (EMT) is a cellular development program characterized by loss of cell adhesion and increased cell mobility. It is essential for numerous processes including metastasis. In this study we have generated "aggressive" MCF-7 breast cancer cells (MCF-7-EMT), which show significantly increased invasion in contrast to wild type MCF-7 (MCF-7 WT) cells. In addition, we have analyzed, whether these cell lines differ in their metastatic behavior in vivo and in expression of invasion and/or EMT-relevant genes. Invasive behavior of different human breast cancer cell lines was tested. "Aggressive" MCF-7 cells (MCF-7-EMT) were generated using coculture and mammosphere culture techniques. To analyze whether or not MCF-7-EMT cells in contrast to MCF-7 WT cells form metastases in vivo, we assessed metastases in a nude mouse model. mRNA expression profiles of MCF-7 WT cells and MCF-7-EMT cells were compared using the Affymetrix micro array technique. Expression of selected genes was validated using real-time PCR. In addition, protein expression of epithelial marker E-cadherin (CDH1) and mesenchymal markers N-cadherin (CDH2), Vimentin (VIM), and TWIST was compared. The breast cancer cell lines showed different invasive behavior from hardly any invasion to a stronger cell movement. Coculture with osteoblast-like MG63 cells led to significantly increased cell invasion rates. The highest increase was shown using MCF-7 WT cells. Generated MCF-7-EMT cells showed significantly increased invasion as compared to MCF-7 WT cells. In 8 of 10 mice bearing orthotopically growing MCF-7-EMT tumors, we could detect metastases in liver and lung. In mice bearing MCF-7 WT tumors (n = 10), no metastases were found. MCF-7 WT cells and MCF-7-EMT cells were different in expression of 325 genes. Forty-four of the most regulated 50 invasion and/or EMT-related genes were upregulated and 6 genes were downregulated in MCF-7-EMT cells. Protein expression of mesenchymal markers CDH2, VIM, and TWIST was clearly increased in MCF-7-EMT cells. Protein expression of epithelial marker CDH1 was clearly decreased. With the breast cancer cell lines, MCF-7-EMT and MCF-7 WT cells, we have an excellent model of cells for further studies of EMT and invasion in vitro and in vivo.

Inhibition of GPR30 by estriol prevents growth stimulation of triple-negative breast cancer cells by 17ß-estradiol.

BACKGROUND: Due to the lack of ERα, triple negative breast cancers (TNBCs) are not susceptible to endocrine therapy using antiestrogens. However, the majority of TNBCs express the membrane bound estrogen receptor GPR30. We have recently shown that knock-down of GPR30 expression prevented growth stimulation of TNBC cell lines by 17ß-estradiol. Now we analyzed whether specific inhibition of GPR30 represents a new option for therapy of TNBC. METHODS: Growth of TNBC cells was assessed using Alamar-blue colorimetric assay. Activation of c-Src and EGF-receptor was assessed using Western blots. Expression of c-fos, cyclin D1 and aromatase was quantified by RT-PCR. Gα-specific signaling of GPR30 was analyzed by electrophoretic mobility shift assay. RESULTS: HCC1806 cells showed the highest GPR30 expression, in HCC70 cells it was clearly lower, in MDA-MB-231 cells it was lowest. 10-8 M 17ß-estradiol significantly increased proliferation of HCC1806 cells to 134 ± 12% of control (p < 0.01). Proliferation of HCC70 cells was slightly increased to 116 ± 8% of control. Estriol significantly reduced cell number of HCC1806 cells to 16 ± 12% (p < 0.01). Cell number of HCC70 cells and of MDA-MB-231 cells was reduced to 68 ± 25% and to 61 ± 10%, respectively.Activity of Src kinase increased to 150 ± 10% (p < 0.05) by 10-8 M 17ß-estradiol treatment in HCC1806 and to 220 ± 20% in HCC70 cells (p < 0.01). Estriol treatment completely inhibited 17ß-estradiol-induced p-src activation. Transactivation of EGF-receptor increased by estradiol treatment to 350% in HCC1806 and to 280% in HCC70 cells. Estriol completely suppressed EGF-receptor transactivation. c-fos expression increased to 260% and to 190%, respectively. Estriol reduced this induction to 160% (HCC1806) and below control in HCC70 cells. Cyclin D1 was induced to 290% (HCC1806) and 170% (HCC70) and completely inhibited by estriol. 17ß-estradiol increased CREB-phosphorylation to 400%. Binding of phospho-CREB to a CRE of cyclin D1 was enhanced to 320%. CONCLUSION: Specific pharmacological inhibition of GPR30 might become a promising targeted therapy for TNBC in future.

Efficacy and safety of AEZS-108 (INN: zoptarelin doxorubicin acetate) an LHRH agonist linked to doxorubicin in women with platinum refractory or resistant ovarian cancer expressing LHRH receptors: a multicenter phase II trial of the ago-study group (AGO GYN 5).

OBJECTIVES: To evaluate the activity and toxicity of AEZS-108 (Zoptarelin Doxorubicin Acetate) an LHRH agonist linked to doxorubicin in women with platinum refractory or resistant ovarian cancer expressing LHRH receptors. METHODS: Women with epithelial ovarian, fallopian tube or primary peritoneal cancer, expressing LHRH receptors were eligible for this trial, when they had progression during treatment with a platinum based regimen or within 6months after receiving a platinum based regimen and a previous taxane treatment. At least one measurable target lesion (RECIST) or CA-125 levels higher than twice the upper limit of normal range (GCIG-criteria) were required. Patients received AEZS-108 (267mg/m(2) equimolar to 76.8mg/m(2) of free doxorubicin) every 3weeks as a two hour i.v. infusion. RESULTS: Fifty-five of 59 (93%) of ovarian cancer samples screened expressed LHRH receptors. 42 patients were enrolled in this study and received at least 1 infusion of AEZS-108 (ITT population). Of these 42 patients 6 (14.3%) had a partial response, 16 (38%) stable disease, 16 (38%) progressive disease and 4 patients were not evaluable. Median time to progression was 12weeks (95% CI: 8-20weeks), and median overall survival was 53weeks (95% CI: 39-73weeks). Toxicity profile was favorable. CONCLUSION: AEZS-108 has a clinical activity in platinum refractory/resistant ovarian cancer which seems to be comparable to that of pegylated liposomal doxorubicin or to topotecan. Toxicity was comparably low. These data support the concept of a targeted chemotherapy for tumors expressing LHRH receptors.

Kisspeptin-10 inhibits stromal-derived factor 1-induced invasion of human endometrial cancer cells.

OBJECTIVES: The cross talk between metastatic cancer cells and target sites is critical for the development and progression of metastases. Disruption of this interaction will allow to design mechanism-based effective and specific therapeutic interventions for metastases. We have established a coculture system of cells derived from different tumor entities and MG63 human osteoblastlike cells to analyze tumor cell invasion. Recently, we have shown that breast cancer cell invasion was dramatically increased when cocultured with MG63 cells.Using this model, we have now analyzed whether stromal-derived factor 1 (SDF-1) is responsible for human endometrial cancer cell invasion and whether kisspeptin-10 (KP-10) treatment affects SDF-1-induced invasion of endometrial cancer cells in vitro. METHODS: Invasion was quantified by assessment of endometrial cancer cell migration rate through an artificial basement membrane in a modified Boyden chamber during coculture with MG63 cells or after treatment with SDF-1α, SDF-1ß, or the combination of both SDF-1 isoforms. In addition, the role of SDF-1 in invasion of endometrial cancer cells was analyzed by blocking SDF-1 secretion during coculture with MG64 cells. Furthermore, the effects of KP-10 treatment on MG63 coculture-driven and SDF-1-induced invasion were analyzed. RESULTS: Endometrial cancer cell invasion was significantly increased when cocultured with MG63 cells. Treatment with KP-10 reduced the ability to invade a reconstituted basement membrane and to migrate in response to the cellular stimulus. This effect was significant in a dose window of 10(-13) to 10(-11) mol/L. During coculture, SDF-1 protein expression of MG63 cells was significantly increased. The MG63 coculture-induced increase of endometrial cancer cell invasion could be blocked by anti-SDF-1 antibodies. Treatment of endometrial cancer cells in monoculture (without MG63) with SDF-1α, SDF-1ß, or the combination of both isoforms resulted in a significant increase of endometrial cancer cell invasion. The SDF-1-induced increase of endometrial cancer cell invasion was significantly reduced after treatment with KP-10. CONCLUSIONS: Our findings suggest that SDF-1 plays a major role in endometrial cancer invasion. Stromal-derived factor 1-induced invasion can be inhibited by KP-10 treatment.

Efficacy and safety of AEZS-108 (LHRH agonist linked to doxorubicin) in women with advanced or recurrent endometrial cancer expressing LHRH receptors: a multicenter phase 2 trial (AGO-GYN5).

OBJECTIVE: Advanced or recurrent endometrial cancer (EC) no longer amenable to surgery or radiotherapy is a life-threatening disease with limited therapeutic options left. Eighty percent of ECs express receptors for luteinizing hormone-releasing hormone (LHRH), which can be targeted by AEZS-108 (zoptarelin doxorubicin acetate). This phase 2 trial was performed to assess the efficacy and safety of AEZS-108 in this group of patients. METHODS: Patients had FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) III or IV or recurrent EC, LHRH receptor-positive tumor status, and at least had 1 measurable lesion (Response Evaluation Criteria in Solid Tumors). Prior anthracycline therapy was not allowed. Patients received AEZS-108 as a 2-hour infusion on day 1 of a 21-day cycle. The treatment was continued for a maximum of 6 to 8 cycles. The primary end point was the response rate determined by the Response Evaluation Criteria in Solid Tumors. RESULTS: From April 2008 to November 2009, 44 patients were included in the study at 8 centers in Germany (AGO) and 3 centers in Bulgaria. Forty-three of these patients were eligible. Two (5%) patients had a complete remission, and 8 (18%) achieved a partial remission. Stable disease for at least 6 weeks was observed in 44%. The median time to progression was 7 months, and the median overall survival was 15 months. The most frequently reported grade 3 or 4 adverse effects were neutropenia (12%) and leucopenia (9%). CONCLUSIONS: AEZS-108, an LHRH-agonist coupled to doxorubicin, has significant activity and low toxicity in women with advanced or recurrent LHRH receptor-positive EC, supporting the principle of receptor-mediated targeted chemotherapy.

ARHGAP29 Is Involved in Increased Invasiveness of Tamoxifen-resistant Breast Cancer Cells and its Expression Levels Correlate With Clinical Tumor Parameters of Breast Cancer Patients.

BACKGROUND/AIM: Aggressive breast cancer (BC) cells show high expression of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. In breast cancer cells in which mesenchymal transformation was induced, ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression increased significantly. Therefore, we investigated whether there is a correlation between expression of ARHGAP29 and tumor progression in BC. Since tamoxifen-resistant BC cells exhibit increased mesenchymal properties and invasiveness, we additionally investigated the relationship between ARHGAP29 and increased invasion rate in tamoxifen resistance. The question arises as to whether ARHGAP29 is a suitable prognostic marker for the progression of BC. MATERIALS AND METHODS: Tissue microarrays were used to investigate expression of ARHGAP29 in BC and adjacent normal breast tissues. Knockdown experiments using siRNA were performed to investigate the influence of ARHGAP29 and the possible downstream actors RhoC and pAKT1 on invasive growth of tamoxifen-resistant BC spheroids in vitro. RESULTS: Expression of ARHGAP29 was frequently increased in BC tissues compared to adjacent normal breast tissues. In addition, there was evidence of a correlation between high ARHGAP29 expression and advanced clinical tumor stage. Tamoxifen-resistant BC cells show a significantly higher expression of ARHGAP29 compared to their parental wild-type cells. After knockdown of ARHGAP29 in tamoxifen-resistant BC cells, expression of RhoC was significantly reduced. Further, expression of pAKT1 decreased significantly. Invasive growth of three-dimensional tamoxifen-resistant BC spheroids was reduced after knockdown of ARHGAP29. This could be partially reversed by AKT1 activator SC79. CONCLUSION: Expression of ARHGAP29 correlates with the clinical tumor parameters of BC patients. In addition, ARHGAP29 is involved in increased invasiveness of tamoxifen-resistant BC cells. ARHGAP29 alone or in combination with its downstream partners RhoC and pAKT1 could be suitable prognostic markers for BC progression.

Starving tumors: inhibition of glycolysis reduces viability of human endometrial and ovarian cancer cells and enhances antitumor efficacy of GnRH receptor-targeted therapies.

OBJECTIVE: Increased glycolysis for energy production is necessary for survival of tumor cells and thus represents a selective therapeutic target. We have analyzed in vitro whether inhibition of glycolysis can reduce the viability of human endometrial and ovarian cancer cells and whether it can enhance the antitumor efficacy of GnRH receptor-targeted therapies. MATERIALS AND METHODS: Cell viability of ovarian and endometrial cancer cells treated without or with glycolysis inhibitor 2-Deoxy-D-Glucose (2DG) alone or in combination with GnRH-II antagonist [Ac-D2Nal(1), D-4Cpa(2), D-3Pal(3,6)(8),Leu, D-Ala(10)]GnRH-II or with cytotoxic GnRH-I agonist AEZS-108 (AN-152) was measured using alamar blue assay. Induction of apoptosis was analyzed using TUNEL assay and quantified by measurement of loss of mitochondrial membrane potential. Apoptotic signaling was measured by quantification of activated caspase-3 by using the Western blot technique. RESULTS: Treatment of endometrial and ovarian cancer cells with glycolysis inhibitor 2DG resulted in a significant decrease of cell viability and a significant increase of apoptosis. Treatment with 2DG in combination with the GnRH-II antagonist or with AEZS-108 resulted in a significant reduced viability compared with single-agent treatments. The observed reduction in viability was due to induction of apoptosis. Also for apoptosis induction, a significant stronger effect in the case of cotreatments compared with single-agent treatments could be observed. These additive effects could be correlated to increased activation of caspase-3. CONCLUSIONS: The glycolytic phenotype of human endometrial and ovarian cancer cells can be targeted for therapeutic intervention. In addition, cotreatment of a glycolysis inhibitor with GnRH receptor-targeted therapies might be a suitable therapy for GnRH receptor-positive human endometrial and ovarian cancers.

Inhibition of Increased Invasiveness of Breast Cancer Cells With Acquired Tamoxifen Resistance by Suppression of CYR61.

BACKGROUND/AIM: Hormone sensitivity-targeted therapy with selective estrogen receptor modulators (SERMs), such as 4-hydroxytamoxifen (4-OHT), is the mainstay of treatment for breast cancers (BCs) that express estrogen receptor α (ERα). However, development of resistance limits this therapy approach. The question arises whether changes associated with 4-OHT resistance could be exploited therapeutically. MATERIALS AND METHODS: First, 4-OHT-resistant sublines of ERα-positive breast carcinoma cell lines MCF-7 and T47D were generated. Viability was assessed by the Alamar Blue assay. Cell invasion was quantified in modified Boyden chambers with Matrigel. Changes in expression of CYR61, S100A4, and ERα were examined by RT-qPCR. Expression of CYR61 was suppressed by transient gene silencing using siRNA. Successful suppression was verified by western blot. Efficacy of 4-OHT treatment was analyzed by quantification of viability using Alamar Blue assay. Correlation of CYR61 levels in patients with luminal A BC to distant metastases-free survival was determined by Kaplan-Meier analysis. RESULTS: ERα-positive MCF-7 and T47D BC cells exhibit an extremely weak invasion rate. Acquired tamoxifen resistance significantly increased the invasive behavior of both tamoxifen-resistant MCF-7-TR and T47D-TR sublines. In addition, expression of CYR61 and S100A4 showed significantly increased levels, whereas expression of ERα was decreased. Suppression of CYR61 expression resulted in a significant decreased invasion rate. In addition, expression of S100A4 was reduced, whereas expression of ERα was increased. Furthermore, suppression of CYR61 resulted in re-sensitization to 4-OHT. High CYR61 levels in patients with luminal A BC resulted in reduced distant metastases-free survival. CONCLUSION: The prometastatic factor CYR61 appears to play an important role in the increased invasiveness of tamoxifen-resistant ERα-positive BC cells. Its suppression leads to a lower invasion rate. Given the few therapeutic options available for tamoxifen-resistant BC, therapy that reduces CYR61 may improve its treatability in future.

Role of Estrogen Receptor ß, G-Protein Coupled Estrogen Receptor and Estrogen-Related Receptors in Endometrial and Ovarian Cancer.

Ovarian and endometrial cancers are affected by estrogens and their receptors. It has been long known that in different types of cancers, estrogens activate tumor cell proliferation via estrogen receptor α (ERα). In contrast, the role of ERs discovered later, including ERß and G-protein-coupled ER (GPER1), in cancer is less well understood, but the current state of knowledge indicates them to have a considerable impact on both cancer development and progression. Moreover, estrogen related receptors (ERRs) have been reported to affect pathobiology of many tumor types. This article provides a summary and update of the current findings on the role of ERß, GPER1, and ERRs in ovarian and endometrial cancer. For this purpose, original research articles on the role of ERß, GPER1, and ERRs in ovarian and endometrial cancers listed in the PubMed database have been reviewed.

Knockdown of G Protein-coupled Estrogen Receptor 1 (GPER1) Enhances Tumor-supportive Properties in Cervical Carcinoma Cells.

BACKGROUND/AIM: A wide variety of answers can be found regarding the question of whether G-protein-coupled estrogen receptor 1 (GPER1) is tumor supportive or tumor suppressive. In cervical carcinoma (CC), the function of GPER1 is poorly understood. In this work, we aimed to clarify what role GPER1 plays in CC, tumor promoting of tumor suppressive. MATERIALS AND METHODS: Transient GPER1 silencing was conducted using RNAi and approved by RT-qPCR. Clonogenic potential was tested by colony and sphere formation. Expression of SERPINE1/PAI-1 was quantified by RT-qPCR and Western blot. Morphological changes were analyzed using Phalloidin staining. Localization of GPER1 in tumor spheres was examined by immunofluorescence. RESULTS: After GPER1 knockdown, more colonies formed in HeLa and SiHa, and larger colonies formed in C33-A and SiHa CC cells. Size of HeLa and SiHa tumor spheres was also increased. In addition, number of HeLa tumor spheres was elevated, and larger secondary colonies were present. C33-A only formed tumor sphere-like clusters showing no differences in number and size. Phalloidin staining revealed greater cellular length-to-width ratio and increased average filopodia length. Expression of SERPINE1/PAI-1 was increased in HeLa and decreased in C33-A. In SiHa cells, SERPINE1 was slightly decreased, whereas the protein PAI-1 was increased. Strong expression of GPER1 was detectable in peripheral areas and in sprouts of tumor spheres. CONCLUSION: GPER1 appears to be tumor suppressive in CC, as GPER1 knockdown provoked increased stem cell properties and increased migration/invasion. EMT also appears to be enhanced. Of interest is the increase in SERPINE1/PAI-1 expression after GPER1 knockdown.

Inactivation of GPR30 reduces growth of triple-negative breast cancer cells: possible application in targeted therapy.

Triple-negative breast cancers lack estrogen receptor α (ERα), progesterone receptor, and do not overexpress human epidermal growth factor receptor 2 (Her-2). They are neither susceptible to endocrine therapy nor to a therapy using the anti-Her-2 antibody, trastuzumab. Therefore, an efficient targeted therapy is warranted. Triple-negative breast tumors frequently express membrane bound estrogen receptor G-protein coupled receptor (GPR30). As proof of principle, we analyzed the consequences of a knock-down of GPR30 expression on the growth regulation of triple-negative breast cancer cell lines. Cells of triple-negative breast cancer cell lines were transfected with siRNA against GPR30 or control siRNA, and cell growth was stimulated either with 10(⁻9) M 17ß-estradiol or 10(⁻6) M 4-hydroxytamoxifen. Cell proliferation was measured using Alamar blue staining. Activation of c-Src and epidermal growth factor (EGF)-receptor was assessed using western blot. Expression of c-fos was quantified by reverse transcription polymerase chain reaction. Seven days after transfection with siRNA, GPR30 mRNA in triple-negative breast cancer cell lines MDA-MB-435 and HCC1806 was reduced by 74 and 90%, respectively. 10(⁻8) M 17ß-estradiol enhanced proliferation of MDA-MB-435 to 129.6±5.4% of control (p<0.05) and HCC1806 to 156.9±15.4% of control (p<0.05), respectively. 10(⁻6) M 4-hydroxytamoxifen increased cell number of MDA-MB-435 to 121.0±6.9% of control (p<0.05) and HCC1806 to 124.5±12.1% of control (n.s.), respectively. This increased proliferation by the two estrogenic compounds was completely prevented by knock-down of GPR30 expression in both cell lines. In control cells, activity of Src kinase was increased 3-fold by estradiol and 3.8-fold using 4-hydroxytamoxifen. Transactivation of the EGF-receptor was similarly increased in both cell lines by 17ß-estradiol and 4-hydroxytamoxifen. Both compounds increased c-fos expression 1.5- and 3.1-fold, respectively. Knock-down of GPR30 expression completely abolished activation of all these signaling pathways responsible for enhanced proliferation. A pharmacological inhibition of GPR30 by specific small molecular inhibitors might prove to be an appropriate targeted therapy of triple-negative breast cancer in the future.

Suppression of G Protein-coupled Estrogen Receptor 1 (GPER1) Enhances the Anti-invasive Efficacy of Selective ERß Agonists.

BACKGROUND/AIM: G protein-coupled estrogen receptor 1 (GPER1) is often over-expressed in triple negative breast cancer (TNBC). GPER1 is responsible for many of the non-genomic, membrane-initiated effects of estrogens. Therefore, we have analyzed the effects of GPER1 knockdown using specific siRNA. MATERIALS AND METHODS: Transient GPER1 silencing was conducted using RNA interference and confirmed by RT-PCR and western blot. Viability of human breast cancer cell lines MDA-MB 231 and HCC 1806 was tested using AlamarBlue assay. Cell invasion was analyzed by assessment of cell migration rate through an artificial basement membrane in a modified Boyden chamber. RESULTS: Viability of both cell lines was slightly decreased after suppression of GPER1 expression. Knockdown of GPER1 resulted in a significantly reduced invasion of the TNBC cells. The anti-invasive effect of selective ERß agonists was significantly stronger after knockdown of GPER1 expression. In addition, the efficacy of tamoxifen treatment was significantly increased after suppression of GPER1 expression. CONCLUSION: Suppression of GPER1 reduced the metastatic behavior of TNBC cells, improved the anti-invasive efficacy of selective ERß agonists and sensitized cells to 4OH-tamoxifen.

Agonists and antagonists of GnRH-I and -II reduce metastasis formation by triple-negative human breast cancer cells in vivo.

Metastasis to bone is a frequent problem of advanced breast cancer. Particularly breast cancers, which do not express estrogen and progesterone receptors and which have no overexpression/amplification of the HER2-neu gene, so called triple-negative breast cancers, are considered as very aggressive and possess a bad prognosis. About 60% of all human breast cancers and about 74% of triple-negative breast cancers express receptors for gonadotropin-releasing hormone (GnRH), which might be used as a therapeutic target. Recently, we could show that bone-directed invasion of human breast cancer cells in vitro is time- and dose-dependently reduced by GnRH analogs. In the present study, we have analyzed whether GnRH analogs are able to reduce metastases of triple-negative breast cancers in vivo. In addition, we have evaluated the effects of GnRH analogs on tumor growth. To quantify formation of metastasis by triple-negative MDA-MB-435 and MDA-MB-231 human breast cancers, we used a real-time PCR method based on detection of human-specific alu sequences measuring accurately the amount of human tumor DNA in athymic mouse organs. To analyze tumor growth, the volumes of breast cancer xenotransplants into nude mice were measured. We could demonstrate that GnRH analogs significantly reduced metastasis formation by triple-negative breast cancer in vivo. In addition, we could show that GnRH analogs significantly inhibited the growth of breast cancer into nude mice. Side effects were not detectable. In conclusion, GnRH analogs seem to be suitable drugs for an efficacious therapy for triple-negative, GnRH receptor-positive human breast cancers to prevent metastasis formation.

CD36-mediated activation of endothelial cell apoptosis by an N-terminal recombinant fragment of thrombospondin-2 inhibits breast cancer growth and metastasis in vivo.

Thus far the clinical benefits seen in breast cancer patients treated with drugs targeting the vascular endothelial growth factor (VEGF) pathway are only modest. Consequently, additional antiangiogenic approaches for treatment of breast cancer need to be investigated. Thrombospondin-2 (TSP-2) has been shown to inhibit tumor growth and angiogenesis with a greater potency than the related molecule TSP-1. The systemic effects of TSP-2 on tumor metastasis and the underlying molecular mechanisms of the antiangiogenic activity of TSP-2 have remained poorly understood. We generated a recombinant fusion protein consisting of the N-terminal region of TSP-2 and the IgG-Fc1 fragment (N-TSP2-Fc) and could demonstrate that the antiangiogenic activity of N-TSP2-Fc is dependent on the CD36 receptor. We found that N-TSP2-Fc inhibited VEGF-induced tube formation of human dermal microvascular endothelial cells (HDMEC) on matrigel in vitro and that concurrent incubation of anti-CD36 antibody with N-TSP2-Fc resulted in tube formation that was comparable to untreated control. N-TSP2-Fc potently induced apoptosis of HDMEC in vitro in a CD36-dependent manner. Moreover, we could demonstrate a CD36 receptor-mediated loss of mitochondrial membrane potential and activation of caspase-3 in HDMEC in vitro. Daily intraperitoneal injections of N-TSP2-Fc resulted in a significant inhibition of the growth of human MDA-MB-435 and MDA-MB-231 tumor cells grown in the mammary gland of immunodeficient nude mice and in reduced tumor vascularization. Finally, increased serum concentrations of N-TSP2-Fc significantly inhibited regional metastasis to lymph nodes and distant metastasis to lung as shown by quantitative real-time alu PCR. These results identify N-TSP2-Fc as a potent systemic inhibitor of tumor metastasis and provide strong evidence for an important role of the CD36 receptor in mediating the antiangiogenic activity of TSP-2.

The Role of Gonadotropin-Releasing Hormone (GnRH) in Endometrial Cancer.

Endometrial cancer (EC) is one of the most common gynecological malignancies. Gonadotropin releasing hormone (GnRH) is a decapeptide first described to be secreted by the hypothalamus to regulate pituitary gonadotropin secretion. In this systematic review, we analyze and summarize the data indicating that most EC express GnRH and its receptor (GnRH-R) as part of an autocrine system regulating proliferation, the cell cycle, and apoptosis. We analyze the available data on the expression and function of GnRH-II, its putative receptor, and its signal transduction. GnRH-I and GnRH-II agonists, and antagonists as well as cytotoxic GnRH-I analogs, have been shown to inhibit proliferation and to induce apoptosis in human EC cell lines in pre-clinical models. Treatment with conventional doses of GnRH-agonists that suppress pituitary gonadotropin secretion and ovarian estrogen production has become part of fertility preserving therapy of early EC or its pre-cancer (atypical endometrial hyperplasia). Conventional doses of GnRH-agonists had marginal activity in advanced or recurrent EC. Higher doses or more potent analogs including GnRH-II antagonists have not yet been used clinically. The cytotoxic GnRH-analog Zoptarelin Doxorubicin has shown encouraging activity in a phase II trial in patients with advanced or recurrent EC, which expressed GnRH-R. In a phase III trial in patients with EC of unknown GnRH-R expression, the cytotoxic GnRH doxorubicin conjugate was not superior to free doxorubicin. Further well-designed clinical trials exploiting the GnRH-system in EC might be useful.