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 17β-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 17β-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 17β-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

Effekte des LHRH-Agonisten Triptorelin auf die in vitro Proliferation verschiedener Ovarial- und Endometriumkarzinomzellinien

5. Zusammenfassung Ovarial- und Endometriumkarzinome sind in den weiter fortgeschrittenen, rezidivierten oder gar metastasierten Stadien schwierig zu behandeln. Seit Ende der achtziger Jahre konnten antiproliferative Effekte von LHRH und seinen Analoga auf Ovarial- und Endometriumkarzinomzellen gezeigt werden. Daneben wurden sowohl in verschiedenen Karzinomzellinien als auch in Tumorgewebeproben die Expression von LHRH und seinem Rezeptor nachgewiesen. Diese Ergebnisse sprechen für ein lokal autokrines System, welches auf LHRH basierend direkten Einfluß auf Proliferation und Differenzierung der Tumorzellen nimmt. Die Experimente und die davon abgeleiteten Überlegungen der vorliegenden Arbeit lassen den antiproliferativen Effekt des LHRH-Analogons Triptorelin in direktem Zusammenhang mit der Expression des LHRH-Rezeptors erscheinen. An einer Reihe von neun bislang nicht untersuchten, etablierten Tumorzellinien konnte hier ein eindeutiger antiproliferativer Effekt des LHRH-Agonisten Triptorelin in Zellinien gezeigt werden, die den LHRH-Rezeptor exprimierten. In Zellinien ohne Expression des LHRH-Rezeptors war Triptorelin wirkungslos. Bei den Ovarialkarzinomzellinien NIH:OVCAR-3 und BG-1, sowie den Endometriumkarzinomzellinien HEC-1B, KLE und AN-3-CA konnten deutliche Zellzahlreduktionen durch das Einwirken von Triptorelin erreicht werden. Im zeitabhängigen Versuch ließen sich die Zahlen der Ovarialkarzinomzellen mit einer Dosis von 10-5 mol/L Triptorelin über 5 Tage um bis zu 30,3% reduzieren (p<0,01), die dosisabhängige Reduktion belief sich sogar auf 41,6% (p<0,001). Die Zellzahl der Endometriumkarzinomzellinie HEC-1B konnte am vierten Tag um 47,5% (p<0,001) reduziert werden, dosisabhängig gelang dies um 43,3% (p<0,001) mit der höchsten Konzentration Triptorelin von 10-5 mol/L. Auch konnte bereits in niedrigen Dosierungen von 1 nmol/L Triptorelin bei vier dieser fünf Zellinien eine signifikannte Wachstumshemmung nachvollzogen werden. Bei drei der fünf Ovarialkarzinomzellinien (SK-OV-3, CA-OV-3 und SW 626) sowie bei einer der vier Endometriumkarzinomzellinien (MFE-296) konnte keine signifikante antiproliferative Wirkung durch Triptorelin gefunden werden. Werden diese Ergebnisse mit denen anderer Mitarbeiter korreliert, so kann festgestellt werden, daß die Zellinien, die LHRH-Bindungsstellen und mRNA für LHRH und seinen Rezeptor exprimieren, auch durch den LHRH-Agonisten gehemmt wurden. Diese Ergebnisse erklären möglicherweise die unterschiedlichen Auffassungen der verschiedenen Arbeitsgruppen bezüglich der antiproliferativen Wirkung von LHRH-Analoga auf Ovarial- und Endometriumkarzinomzellinien. Zusammenfassend lassen diese Erkenntnisse annehmen, dass Tumoren, die LHRH-Rezeptoren exprimieren können, möglicherweise in naher Zukunft mit LHRH-Analoga zu therapieren sind

Prevalence of pathogenic BRCA1/2 germline mutations among 802 women with unilateral triple-negative breast cancer without family cancer history

Background: There is no international consensus up to which age women with a diagnosis of triple-negative breast cancer (TNBC) and no family history of breast or ovarian cancer should be offered genetic testing for germline BRCA1 and BRCA2 (gBRCA) mutations. Here, we explored the association of age at TNBC diagnosis with the prevalence of pathogenic gBRCA mutations in this patient group. Methods: The study comprised 802 women (median age 40 years, range 19–76) with oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor type 2 negative breast cancers, who had no relatives with breast or ovarian cancer. All women were tested for pathogenic gBRCA mutations. Logistic regression analysis was used to explore the association between age at TNBC diagnosis and the presence of a pathogenic gBRCA mutation. Results: A total of 127 women with TNBC (15.8%) were gBRCA mutation carriers (BRCA1: n = 118, 14.7%; BRCA2: n = 9, 1.1%). The mutation prevalence was 32.9% in the age group 20–29 years compared to 6.9% in the age group 60–69 years. Logistic regression analysis revealed a significant increase of mutation frequency with decreasing age at diagnosis (odds ratio 1.87 per 10 year decrease, 95%CI 1.50–2.32, p &lt; 0.001). gBRCA mutation risk was predicted to be &gt; 10% for women diagnosed below approximately 50 years. Conclusions: Based on the general understanding that a heterozygous mutation probability of 10% or greater justifies gBRCA mutation screening, women with TNBC diagnosed before the age of 50 years and no familial history of breast and ovarian cancer should be tested for gBRCA mutations. In Germany, this would concern approximately 880 women with newly diagnosed TNBC per year, of whom approximately 150 are expected to be identified as carriers of a pathogenic gBRCA mutation

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

Hormone-Dependent Cancers: Molecular Mechanisms and Therapeutical Implications

Hormone-dependent cancers of the breast and prostate are the most common cancers in women and men, respectively [...