Damiano Cosimo Rigiracciolo

// Damiano Cosimo Rigiracciolo 1 , Andrea Scarpelli 1 , Rosamaria Lappano 1 , Assunta Pisano 1 , Maria Francesca Santolla 1 , Paola De Marco 1 , Francesca Cirillo 1 , Anna Rita Cappello 1 , Vincenza Dolce 1 , Antonino Belfiore 2 , Marcello Maggiolini 1 and Ernestina Marianna De Francesco 1 1 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy 2 Endocrinology, Department of Health, University Magna Graecia of Catanzaro, Catanzaro, Italy Correspondence to: Marcello Maggiolini, email: // Keywords : copper, cancer, angiogenesis, GPER, HIF-1α, VEGF, Pathology Section Received : June 15, 2015 Accepted : August 31, 2015 Published : September 22, 2015 Abstract Copper promotes tumor angiogenesis, nevertheless the mechanisms involved remain to be fully understood. We have recently demonstrated that the G-protein estrogen receptor (GPER) cooperates with hypoxia inducible factor-1α (HIF-1α) toward the regulation of the pro-angiogenic factor VEGF. Here, we show that copper sulfate (CuSO 4 ) induces the expression of HIF-1α as well as GPER and VEGF in breast and hepatic cancer cells through the activation of the EGFR/ERK/c-fos transduction pathway. Worthy, the copper chelating agent TEPA and the ROS scavenger NAC prevented the aforementioned stimulatory effects. We also ascertained that HIF-1α and GPER are required for the transcriptional activation of VEGF induced by CuSO 4 . In addition, in human endothelial cells, the conditioned medium from breast cancer cells treated with CuSO 4 promoted cell migration and tube formation through HIF-1α and GPER. The present results provide novel insights into the molecular mechanisms involved by copper in triggering angiogenesis and tumor progression. Our data broaden the therapeutic potential of copper chelating agents against tumor angiogenesis and progression.

Abstract The saturated medium-chain fatty-acid lauric acid (LA) has been associated to certain health-promoting benefits of coconut oil intake, including the improvement of the quality of life in breast cancer patients during chemotherapy. As it concerns the potential to hamper tumor growth, LA was shown to elicit inhibitory effects only in colon cancer cells. Here, we provide novel insights regarding the molecular mechanisms through which LA triggers antiproliferative and pro-apoptotic effects in both breast and endometrial cancer cells. In particular, our results demonstrate that LA increases reactive oxygen species levels, stimulates the phosphorylation of EGFR, ERK and c-Jun and induces the expression of c-fos. In addition, our data evidence that LA via the Rho-associated kinase-mediated pathway promotes stress fiber formation, which exerts a main role in the morphological changes associated with apoptotic cell death. Next, we found that the increase of p21 Cip1/WAF1 expression, which occurs upon LA exposure in a p53-independent manner, is involved in the apoptotic effects prompted by LA in both breast and endometrial cancer cells. Collectively, our findings may pave the way to better understand the anticancer action of LA, although additional studies are warranted to further corroborate its usefulness in more comprehensive therapeutic approaches.

BACKGROUND: Focal adhesion kinase (FAK) is a cytoplasmatic protein tyrosine kinase that associates with both integrins and growth factor receptors toward the adhesion, migration and invasion of cancer cells. The G-protein coupled estrogen receptor (GPER) has been involved in the stimulatory action of estrogens in breast tumor. In this study, we have investigated the engagement of FAK by GPER signaling in triple negative breast cancer (TNBC) cells. METHODS: Publicly available large-scale database and patient data sets derived from "The Cancer Genome Atlas" (TCGA; www.cbioportal.org ) were used to assess FAK expression in TNBC, non-TNBC tumors and normal breast tissues. MDA-MB 231 and SUM159 TNBC cells were used as model system. The levels of phosphorylated FAK, other transduction mediators and target genes were detected by western blotting analysis. Focal adhesion assay was carried out in order to determine the focal adhesion points and the formation of focal adhesions (FAs). Luciferase assays were performed to evaluate the promoters activity of c-FOS, EGR1 and CTGF upon GPER activation. The mRNA expression of the aforementioned genes was measured by real time-PCR. Boyden chamber and wound healing assays were used in order to evaluate cell migration. The statistical analysis was performed by ANOVA. RESULTS: We first determined by bioinformatic analysis that the mRNA expression levels of the gene encoding FAK, namely PTK2, is higher in TNBC respect to non-TNBC and normal breast tissues. Next, we found that estrogenic GPER signaling triggers Y397 FAK phosphorylation as well as the increase of focal adhesion points (FAs) in TNBC cells. Besides, we ascertained that GPER and FAK activation are involved in the STAT3 nuclear accumulation and gene expression changes. As biological counterpart, we show that FAK inhibition prevents the migration of TNBC cells upon GPER activation. CONCLUSIONS: The present data provide novel insights regarding the action of FAK in TNBC. Moreover, on the basis of our findings estrogenic GPER signaling may be considered among the transduction mechanisms engaging FAK toward breast cancer progression.

Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype that currently lacks targeted treatment options. The role played by the insulin-like growth factor-1 (IGF-1) and its cognate receptor IGF-1R in TNBC has been reported. Nevertheless, the molecular mechanisms by which the IGF-1/IGF-1R system may contribute to TNBC progression still remains to be fully understood. By computational analysis of the vast cancer genomics information in public databases (TCGA and METABRIC), we obtained evidence that high IGF-1 or IGF-1R levels correlate with a worse clinical outcome in TNBC patients. Further bioinformatics analysis revealed that both the focal adhesion and the Hippo pathways are enriched in TNBC harboring an elevated expression of IGF-1 or IGF-1R. Mechanistically, we found that in TNBC cells, the IGF-1/IGF-1R system promotes the activation of the FAK signal transduction pathway, which in turn regulates the nuclear accumulation of YAP (yes-associated protein/yes-related protein) and the expression of its target genes. At the biological level, we found that the IGF-1/IGF-1R-FAK-YAP network cascade triggers the growth potential of TNBC cells, as evaluated in different experimental systems. Overall, our results suggest that the IGF-1/IGF-1R/FAK/YAP axis may contribute to the progression of the aggressive TNBC subtype.