Tesi di Dottorato
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Item Activated FXR inhibits leptin signaling and counteracts tumor-promoting activities of cancer-associated fibroblasts in breast malignancy(2017-06-12) Vircillo, Valentina; Andò, Sebastiano; Catalano, StefaniaCancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. In this study, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. We further extend our data investigating whether FXR agonist may directly influence CAF phenotype. We demonstrated that FXR is expressed in different CAFs and treatment with GW 4064 is able to induce the transcription of key FXR target genes, including SHP (Small Heterodimer Partner) and BSEP (Bile Salt Export Pump). Interestingly, FXR activation is able to significantly reduce CAF motility, without influencing their proliferation capabilities. Accordingly, IPA (Ingenuity Pathway Analysis) on FXR-modulated genes highlighted cellular movement as the most significantly represented biologic process and evidenced a marked reduction in the activity of Rho signaling and Integrin proteins, with activation z-score of -1, -0,5 respectively. Moreover, our data showed a reduction in stress fibers formation in GW 4064 -treated CAFs. Activated FXR is able to reduce tumor promoting effects of CAFs on breast cancer cells, due to the ability of GW 4064 to reduce CAF secreted soluble factors, including IGF-1 (Insulin Growth Factor-1), FGF-9 (Fibroblast Growth Factor 9), TGF-3 (Transforming Growth Factor Beta 3) and others key mediators involved in the crosstalk tumor-stroma. Indeed, our data demonstrate how ER-breast cancer cell lines, MCF-7 and T47D, cocoltured with conditioned media derived from GW4064-treated CAFs, exhibit a significantly reduced anchorage-independent growth and migration. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironmentItem Role of ERalpha/NOTCH4 axis in sustaining stemness in breast cancer cells(2018-02-27) Elena Spina, Elena Spina; Andò, SebastianoEarly detection and new therapeutic strategies have improved breast cancer patient outcome and survival rates in the last years. However, breast cancer still remains the second leading cause of cancer-related deaths among women worldwide, and approximately 30% of patients eventually experience a tumor relapse. Treatment failure is mainly due to metastatic process and resistance to conventional therapy. Over the past decade it has been established the existence of a subpopulation of cancer stem cell (CSC) within breast cancers that is responsible for tumor initiation, progression and resistance to endocrine therapies. It is well known the “driving role” of oestrogens and its receptor alpha (ERα), in development and progression of breast cancer disease, but still unknown their role in regulating breast CSCs (BCSCs). In the past few years, several studies revealed the presence of gain-of-function mutation in ESR1, gene encoding for ERα, in metastatic breast cancer patients after long-term endocrine therapies treatment. Particularly, Y537N, Y537S and D538G are the most frequent “hot spot” mutations within ERα hormone-binding domain (HBD) that lead to ligand-independent ERα activity and consequently, resistance to endocrine therapy. Here, we studied how HBD-ESR1 mutations might account for a mechanism of metastatic process and endocrine resistance, sustaining stem cell-like phenotype. As experimental model, we used breast cancer cell lines expressing wild-type and HBDESR1 mutations. Our results, using in vivo and in vitro experiment (mammosphereforming assay and CD44+/CD24- phenotype analysis) have suggested an enrichment of BCSCs activity by HBD-ESR1 mutations, that seems to be sustained by Notch4 signaling through constitutive hyper phosphorylation of Serine 118 residue of ERα that has been demonstrated related to stem cell phenotype and tumor initiation, in mutant-expressing cells. Experiments conducted using CRISPR-Cas9 knock-in of Y537S-ERα mutation confirmed the role of this mutation in tumor initiation and progression as obtained using HBD-ESR1 stable clones. We propose a potential novel role of HBD-ESR1 mutations in sustaining BCSCs activity, that could have clinical relevance, suggesting new molecular biomarker and target to aim better therapeutic strategies for ERα-positive breast cancer metastatic patients.Item Estrogen receptor alpha interferes with LKBl/AMPK/mTOR signaling activation in adiponectin-treated breast cancer cells(2018-02-27) Naimo, Giuseppina Daniela; Andò, Sebastiano; Panno, Maria Luisa; Mauro, LoredanaBreast cancer is the most common type of tumor and the leading cause of cancer-related deaths in women, worldwide. The cause of breast cancer is multifactorial and includes hormonal, genetic and environmental cues. Obesity is now an accepted risk factor for breast cancer in postmenopausal women, particularly for the hormone-dependent subtype of mammary tumor. Obesity has regarded as a multifactorial disorder characterized by an increased number and size of adipocytes. Adipose tissue is an active metabolic and endocrine organ that secretes many adipocytokines, which act as key mediators in several obesity-associated diseases. Among these, adiponectin represents the most abundant adipose tissue-excreted protein, which exhibits insulin sensitizing, antiinflammatory, and antiatherogenic properties Adiponectin has been proposed as having a key role in the pathogenesis of cardiovascular disease and type 2 diabetes along with obesity-associated malignancies, such as breast cancer. An inverse correlation is reported between obesity and adiponectin, for which low levels of adiponectin represent a risk factor for breast cancer. The role of adiponectin on breast tumorigenesis seems to be dependent on cell phenotypes. Indeed, several in vitro and in vivo studies demonstrated that low adiponectin levels repressed growth in ER-negative breast cancer cells whereas increased proliferation in ER- positive cells. Adiponectin interacts with specific receptors and exerts its effects, including regulation of cell survival, apoptosis and metastasis, via a plethora of signaling pathways. The key molecule of adiponectin action is AMP-activated protein kinase (AMPK), which is mainly activated by liver kinase B1 (LKB1). On the basis of this observations, the aim of the present study was to investigate the effect of adiponectin on LKB1/AMPK signaling in ER-negative (MDA-MB-231) and positive (MCF-7) breast cancer cells. In MCF-7 cells, upon low adiponectin levels, ER impaired LKB1/AMPK interaction by recruiting LKB1 as coactivator at nuclear level, sustaining breast tumor growth. In this condition, AMPK signaling was not working, letting fatty acid synthesis still active. In contrast, in MDA-MB-231 cells the phosphorylated status of AMPK and ACC appeared enhanced, with consequent inhibition of both lipogenesis and cell growth. Thus, in the presence of adiponectin, ERα signaling switched energy balance of breast cancer cells towards a lipogenic phenotype. The same results on tumor growth were reproduced in a xenograft model. These results emphasize how adiponectin action in obese patients is tightly dependent on ERα, addressing that adiponectin may work as growth factor in ERα- positive breast cancer cells.