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.

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.

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.

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.

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.

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 Metabolism as a Therapeutic Option for Tamoxifen-Resistant Breast Cancer Cells.

Cancer cells have an increased need for glucose and, despite aerobic conditions, obtain their energy through aerobic oxidation and lactate fermentation, instead of aerobic oxidation alone. Glutamine is an essential amino acid in the human body. Glutaminolysis and glycolysis are crucial for cancer cell survival. In the therapy of estrogen receptor α (ERα)-positive breast cancer (BC), the focus lies on hormone sensitivity targeting therapy with selective estrogen receptor modulators (SERMs) such as 4-hydroxytamoxifen (4-OHT), although this therapy is partially limited by the development of resistance. Therefore, further targets for therapy improvement of ERα-positive BC with secondary 4-OHT resistance are needed. Hence, increased glucose requirement and upregulated glutaminolysis in BC cells could be used. We have established sublines of ERα-positive MCF7 and T47D BC cells, which were developed to be resistant to 4-OHT. Further, glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) and glutaminase inhibitor CB-839 were analyzed. Co-treatments using 4-OHT and CB-839, 2-DG and CB-839, or 4-OHT, 2-DG and CB-839, respectively, showed significantly stronger inhibitory effects on viability compared to single treatments. It could be shown that tamoxifen-resistant BC cell lines, compared to the non-resistant cell lines, exhibited a stronger reducing effect on cell viability under co-treatments. In addition, the tamoxifen-resistant BC cell lines showed increased expression of proto-oncogene c-Myc compared to the parental cell lines. This could be reduced depending on the treatment. Suppression of c-Myc expression using specific siRNA completely abolished resistance to 4OH-tamoxifen. In summary, our data suggest that combined treatments affecting the metabolism of BC are suitable depending on the cellularity and resistance status. In addition, the anti-metabolic treatments affected the expression of the proto-oncogene c-Myc, a key player in the regulation of cancer cell metabolism.

HPV and Other Microbiota; Who's Good and Who's Bad: Effects of the Microbial Environment on the Development of Cervical Cancer-A Non-Systematic Review.

Cervical cancer is responsible for around 5% of all human cancers worldwide. It develops almost exclusively from an unsolved, persistent infection of the squamocolumnar transformation zone between the endo- and ecto-cervix with various high-risk (HR) human papillomaviruses (HPVs). The decisive turning point on the way to persistent HPV infection and malignant transformation is an immune system weakened by pathobionts and oxidative stress and an injury to the cervical mucosa, often caused by sexual activities. Through these injury and healing processes, HPV viruses, hijacking activated keratinocytes, move into the basal layers of the cervical epithelium and then continue their development towards the distal prickle cell layer (Stratum spinosum). The microbial microenvironment of the cervical tissue determines the tissue homeostasis and the integrity of the protective mucous layer through the maintenance of a healthy immune and metabolic signalling. Pathological microorganisms and the resulting dysbiosis disturb this signalling. Thus, pathological inflammatory reactions occur, which manifest the HPV infection. About 90% of all women contract an HPV infection in the course of their lives. In about 10% of cases, the virus persists and cervical intra-epithelial neoplasia (CIN) develops. Approximately 1% of women with a high-risk HPV infection incur a cervical carcinoma after 10 to 20 years. In this non-systematic review article, we summarise how the sexually and microbial mediated pathogenesis of the cervix proceeds through aberrant immune and metabolism signalling via CIN to cervical carcinoma. We show how both the virus and the cancer benefit from the same changes in the immune and metabolic environment.

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

The hypothalamus-pituitary-gonadal (HPG) axis is the endocrine regulation system that controls the woman's cycle. The gonadotropin-releasing hormone (GnRH) plays the central role. In addition to the gonadotrophic cells of the pituitary, GnRH receptors are expressed in other reproductive organs, such as the ovary and in tumors originating from the ovary. In ovarian cancer, GnRH is involved in the regulation of proliferation and metastasis. The effects on ovarian tumors can be indirect or direct. GnRH acts indirectly via the HPG axis and directly via GnRH receptors on the surface of ovarian cancer cells. In this systematic review, we will give an overview of the role of GnRH in ovarian cancer development, progression and therapy.

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.

Influence of ARHGAP29 on the Invasion of Mesenchymal-Transformed Breast Cancer Cells.

Aggressive and mesenchymal-transformed breast cancer cells show high expression levels of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression significantly increased after the induction of mesenchymal transformation in breast cancer cells. Therefore, we investigated the influence of ARHGAP29 on the invasiveness of aggressive and mesenchymal-transformed breast cancer cells. After knock-down of ARHGAP29 using siRNA, invasion of HCC1806, MCF-7-EMT, and T-47D-EMT breast cancer cells was significantly reduced. This could be explained by reduced inhibition of RhoA and a consequent increase in stress fiber formation. Proliferation of the breast cancer cell line T-47D-EMT was slightly increased by reduced expression of ARHGAP29, whereas that of HCC1806 and MCF-7-EMT significantly increased. Using interaction analyses we found that AKT1 is a possible interaction partner of ARHGAP29. Therefore, the expression of AKT1 after siRNA knock-down of ARHGAP29 was tested. Reduced ARHGAP29 expression was accompanied by significantly reduced AKT1 expression. However, the ratio of active pAKT1 to total AKT1 remained unchanged or was significantly increased after ARHGAP29 knock-down. Our results show that ARHGAP29 could be an important factor in the invasion of aggressive and mesenchymal-transformed breast cancer cells. Further research is required to fully understand the underlying mechanisms.

Identification of drivers of breast cancer invasion by secretome analysis: insight into CTGF signaling.

An altered consistency of tumor microenvironment facilitates the progression of the tumor towards metastasis. Here we combine data from secretome and proteome analysis using mass spectrometry with microarray data from mesenchymal transformed breast cancer cells (MCF-7-EMT) to elucidate the drivers of epithelial-mesenchymal transition (EMT) and cell invasion. Suppression of connective tissue growth factor (CTGF) reduced invasion in 2D and 3D invasion assays and expression of transforming growth factor-beta-induced protein ig-h3 (TGFBI), Zinc finger E-box-binding homeobox 1 (ZEB1) and lysyl oxidase (LOX), while the adhesion of cell-extracellular matrix (ECM) in mesenchymal transformed breast cancer cells is increased. In contrast, an enhanced expression of CTGF leads to an increased 3D invasion, expression of fibronectin 1 (FN1), secreted protein acidic and cysteine rich (SPARC) and CD44 and a reduced cell ECM adhesion. Gonadotropin-releasing hormone (GnRH) agonist Triptorelin reduces CTGF expression in a Ras homolog family member A (RhoA)-dependent manner. Our results suggest that CTGF drives breast cancer cell invasion in vitro and therefore could be an attractive therapeutic target for drug development to prevent the spread of breast cancer.

Shedding New Light on Cancer Metabolism: A Metabolic Tightrope Between Life and Death.

Since the earliest findings of Otto Warburg, who discovered the first metabolic differences between lactate production of cancer cells and non-malignant tissues in the 1920s, much time has passed. He explained the increased lactate levels with dysfunctional mitochondria and aerobic glycolysis despite adequate oxygenation. Meanwhile, we came to know that mitochondria remain instead functional in cancer cells; hence, metabolic drift, rather than being linked to dysfunctional mitochondria, was found to be an active act of direct response of cancer cells to cell proliferation and survival signals. This metabolic drift begins with the use of sugars and the full oxidative phosphorylation via the mitochondrial respiratory chain to form CO2, and it then leads to the formation of lactic acid via partial oxidation. In addition to oncogene-driven metabolic reprogramming, the oncometabolites themselves alter cell signaling and are responsible for differentiation and metastasis of cancer cells. The aberrant metabolism is now considered a major characteristic of cancer within the past 15 years. However, the proliferating anabolic growth of a tumor and its spread to distal sites of the body is not explainable by altered glucose metabolism alone. Since a tumor consists of malignant cells and its tumor microenvironment, it was important for us to understand the bilateral interactions between the primary tumor and its microenvironment and the processes underlying its successful metastasis. We here describe the main metabolic pathways and their implications in tumor progression and metastasis. We also portray that metabolic flexibility determines the fate of the cancer cell and ultimately the patient. This flexibility must be taken into account when deciding on a therapy, since singular cancer therapies only shift the metabolism to a different alternative path and create resistance to the medication used. As with Otto Warburg in his days, we primarily focused on the metabolism of mitochondria when dealing with this scientific question.

Inhibition of CYR61-S100A4 Axis Limits Breast Cancer Invasion.

Background and Objective: Matricellular proteins modulate the micro environment of tumors and are recognized to contribute to tumor cell invasion and dissemination. The cysteine-rich angiogenic inducer 61 (CYR61) is upregulated in mesenchymal transformed and invasive breast cancer cells. CYR61 correlates with poor prognosis of breast cancer patients. The signaling mechanism that causes invasive properties of cancer cells regarding to epithelial-mesenchymal transition (EMT) needs further research. In this study, we investigated the signaling mechanism, which is responsible for reduced cell invasion after suppression of CYR61 in mesenchymal transformed breast cancer cells and in triple negative breast cancer cells. Methods: We addressed this issue by generating a mesenchymal transformed breast cancer cell line using prolonged mammosphere cultivation. Western blotting and quantitative PCR were used to analyze gene expression alterations. Transient gene silencing was conducted using RNA interference. Proliferation was assessed using AlamarBlue assay. Invasiveness was analyzed using 2D and 3D invasion assays. Immune-histochemical analysis of patient tissue samples was performed to examine the prognostic value of CYR61 expression. Results: In this study, we investigated whether CYR61 could be used as therapeutic target and prognostic marker for invasive breast cancer. We discovered an interaction of CYR61 with metastasis-associated protein S100A4. Suppression of CYR61 by RNA interference reduced the expression of S100A4 dependent on ERK1/2 activity regulation. Non-invasive breast cancer cells became invasive due to extracellular CYR61 supplement. Immune-histochemical analysis of 239 patient tissue samples revealed a correlation of higher CYR61 and S100A4 expression with invasive breast cancer and metastasis. Conclusion: Our data suggest that suppression of CYR61 impedes the formation of an invasive cancer cell phenotype by reducing ERK1/2 phosphorylation thereby suppressing S100A4. These findings identify mechanisms by which CYR61 suppresses cell invasion and suggest it to be a potential therapeutic target and prognostic marker for invasive breast cancer and metastasis.

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.

Mechanisms of Metastasis and Cell Mobility - The Role of Metabolism.

Tumour metastasis is responsible for more than 90% of tumour-associated mortality. About one third of breast cancer patients in the early stage develop metastases. The transformation in tumour development referred to as the "metastatic cascade" or "metastatic cycle" is a complex and multi-stage event. While it is generally recognised that epithelial-mesenchymal transformation (EMT) plays a crucial role in cancer progression and metastasis, the metabolic events in this process have received little attention to date. We would therefore like to provide a brief overview here of the influence of the metabolism on the progression and metastasis of tumours.

Inhibition of growth hormone receptor by Somavert reduces expression of GPER and prevents growth stimulation of triple-negative breast cancer by 17ß-estradiol.

Currently, conventional chemotherapy is the only treatment option for triple-negative breast cancers (TNBC) due to a lack of a unique target. In TNBC, a high expression of the membrane bound G protein-coupled estrogen receptor (GPER), correlates with a worse outcome. There is a potential for an association between growth hormone receptor (GHR) and GPER expression. To confirm this hypothesis, GHR was inhibited in TNBC cells with Somavert, and GPER expression levels, and the effect on signal transduction and proliferation induction in TNBC cells were analyzed. Proliferation of TNBC cells was measured using an Alamar-blue assay. Expression of GPER and activation of c-src and epidermal growth factor receptor (EGFR) by 17ß-estradiol was analyzed by western blotting. Induction of c-fos, cyclin D1 and aromatase expression was determined by reverse transcription-semi-quantitative polymerase chain reaction. The expression of GPER was concentration- and time-dependently reduced by Somavert down to 46±7% (P<0.01) of the control. Furthermore, 17ß-estradiol significantly increased the cell number of HCC1806 cells to 128±14% (P<0.05), and that of MDA-MB-453 cells to 115±3%. This increase in cell number was reduced to 103±11% in HCC1806 cells in which GPER expression was downregulated by Somavert, and to 102±3% in MDA-MB-453 cells. In addition, 17ß-estradiol increased the activation of c-src in HCC1806 cells by 1.8-fold, and Somavert reduced p-src to 63% of control. In MDA-MB-453 cells src phosphorylation increased by 7-fold upon stimulation with estradiol, but after treatment with Somavert only a 4-fold increase was observed. Phosphorylation of EGFR was increased by 2.2-fold of control in HCC1806 cells by 17ß-estradiol, and by 1.4-fold in MDA-MD-453 cells. Somavert completely prevented this activation. Induction of cyclin D1 and aromatase expression by 17ß-estradiol was also prevented by Somavert. Somavert reduces GPER expression in triple negative breast cancer cells. Treatment with Somavert prevents induction of genes regulating proliferation by 17ß-estradiol. Inhibition of GPER expression is a promising therapeutic intervention for TNBC.

Estrogen Signaling in ERα-Negative Breast Cancer: ERß and GPER.

Estrogen receptors are important regulators of the growth of breast tumors. Three different receptors for estrogens have been identified in breast tumors, two nuclear receptors, ERα and ERß, and a G-protein coupled estrogen receptor 1 (GPER) that initiates non-genomic effects of estrogens in the cytosol. Recent findings show that the stimulation of cytoplasmic ERα and ERß also triggers non-genomic signaling pathways. The treatment of breast cancer with anti-estrogens depends on the presence of ERα. About 40% of all breast cancers, however, do not express ERα. One subgroup of these tumors overexpress Her-2, another important group is designated as triple-negative breast cancer, as they neither express ERα, nor progesterone receptors, nor do they overexpress Her-2. This review addresses the signaling of ERß and GPER in ERα-negative breast tumors. In addition to the well-established EGF-receptor transactivation pathways of GPER, more recent findings of GPER-dependent activation of FOXO3a, the Hippo-pathway, and HOTAIR-activation are summarized.

The Role of Gonadotropin-Releasing Hormone in Cancer Cell Proliferation and Metastasis.

In several human malignant tumors of the urogenital tract, including cancers of the endometrium, ovary, urinary bladder, and prostate, it has been possible to identify expression of gonadotropin-releasing hormone (GnRH) and its receptor as part of an autocrine system, which regulates cell proliferation. The expression of GnRH receptor has also been identified in breast cancers and non-reproductive cancers such as pancreatic cancers and glioblastoma. Various investigators have observed dose- and time-dependent growth inhibitory effects of GnRH agonists in cell lines derived from these cancers. GnRH antagonists have also shown marked growth inhibitory effects on most cancer cell lines. This indicates that in the GnRH system in cancer cells, there may not be a dichotomy between GnRH agonists and antagonists. The well-known signaling mechanisms of the GnRH receptor, which are present in pituitary gonadotrophs, are not involved in forwarding the antiproliferative effects of GnRH analogs in cancer cells. Instead, the GnRH receptor activates a phosphotyrosine phosphatase (PTP) and counteracts with the mitogenic signal transduction of growth factor receptors, which results in a reduction of cancer cell proliferation. The PTP activation, which is induced by GnRH, also inhibits G-protein-coupled estrogen receptor 1 (GPER), which is a membrane-bound receptor for estrogens. GPER plays an important role in breast cancers, which do not express the estrogen receptor α (ERα). In metastatic breast, ovarian, and endometrial cancer cells, GnRH reduces cell invasion in vitro, metastasis in vivo, and the increased expression of S100A4 and CYR61. All of these factors play important roles in epithelial-mesenchymal transition. This review will summarize the present state of knowledge about the GnRH receptor and its signaling in human cancers.