Dipartimento di Farmacia e Scienze della Salute e della Nutrizione

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Novel insight into the protective role of androgen receptor in ER-positive breast cancer
(Università della Calabria, 2020-04-17) Chiodo, Chiara; Andò, Sebastiano; Lanzino, Marilena
Introduction: Androgen receptor (AR) role in breast cancer appears to be clinically relevant and disease context specific. In estrogen receptor (ER) α-positive primary breast cancers, AR positivity correlates with low tumor grade and a better clinical outcome. These clinical-pathological findings mirror androgen capability to counteract ERα-dependent proliferation in both normal and tumor mammary epithelium. Tumor microenvironment is a key factor in cancer development and progression since the physical and hormonal paracrine exchanges with the epithelial compartment promote tumor cell proliferation and metastasis. This research project reports additional molecular mechanisms in the dynamic interplay between AR and ER-α signaling pathway in breast cancer cells and showed AR expression and role in the stroma of ER-positive breast cancers. Material and methods: MCF-7, T47D, SKBR3 breast cancer cells and cancer-associated fibroblats (CAFs) from biopsies of primary breast tumors (n=3); cell proliferation assay, transient transfection, quantitative Real Time PCR (qRT-PCR), western blotting (WB), immunoprecipitation assay (IP), chromatin immunoprecipitation assay (ChIP), RNA silencing, tunel assay, DNA affinity precipitation assay (DAPA), immunofluorescence analysis (IF), immunocytochemical staining (IHC), wound-healing scratch assay, F-actin staining assay, Boyden-chamber transmigration assay, matrigel-based invasion assay, cytokine array, zymography assay, mammosphere forming efficiency assay. Data were analyzed by ANOVA. Results: This study demonstrated an androgen-dependent mechanism through which ligand-activated AR decreased estradiol-induced cyclin D1 protein, mRNA and gene promoter activity in MCF-7 cells. This mechanism involved the competition of AR and ERα for the steroid receptor coactivator AIB1, a limiting factor in the functional coupling of ERα with the cyclin D1 promoter. Indeed, AIB1 overexpression was able to reverse the down-regulatory effects exerted by AR. Co-immunoprecipitation studies showed that AIB1 preferentially interacted with ERα or AR in relation to their intracellular levels. In addition, ChIP analysis evidenced that androgen administration decreased E2-induced recruitment of AIB1 at the AP-1 site on the cyclin D1 promoter gene. Moreover, this research project showed an increased expression of the pro-apoptotic protein BAD following androgen treatment while the levels of anti-apoptotic protein Bcl-2 as well as of the pro-apoptotic proteins BID and BAX remained unchanged. As consequence, the Bcl-2/BAD ratio was reduced, shifting the delicate balance between inhibitors and inducers of cell death. Androgen stimulation increased also BAD levels into the nuclear compartment in ERα/AR-positive MCF-7 as well as in ERα negative/AR-positive SKBR3 cells. The androgen-regulated intracellular localization of BAD involved an AR/BAD physical interaction, suggesting a nuclear role for BAD upon androgen stimulation. Indeed, androgens induced both AR and BAD recruitment at the AP-1 and the ARE sites within the cyclin D1 promoter region, contributing to explain the anti-proliferative effect of androgens in breast cancer cells. Finally, the study demonstrated AR expression and functionally activation upon androgen treatment in primary human breast CAFs. Androgen-activated AR affected CAFs secretory phenotype as evidenced by cytokine array performed on CAFs conditioned medium (CM). Co-culture experiments showed that CM from androgen-treated CAFs was less effective in stimulating MCF-7 and T47D cells motility and invasion compared to CM from untreated ones, indicating that androgens, via AR, may influence CAFs secretion of paracrine soluble factors involved in tumor cell motility and invasiveness sustainment. Conclusions: Taken together all these data showed that ligand activated AR exerts a protective role in E2-dependent breast cancer development and progression by inhibiting CD1 expression through the squelching of the steroid receptor co-activator AIB1 and the overexpression of the pro-apoptotic protein BAD. Furthermore, the study highlighted the protective role of AR in the tumor microenvironment, since activated-stromal AR affects the paracrine factors secreted by CAFs reducing ER-positive breast cancer cell migration and invasiveness. Thus, these findings reinforce the possibility to couple the androgen-based therapy with therapies targeting other important pathways in ERα-positive breast cancer patient treatment.
Stromal cells in breast cancer microenvironment: molecular mechanisms involved in tumor progression and potential therapeutic targets
(Università della Calabria, 2021-03-26) Augimeri, Giuseppina; Andò, Sebastiano; Bonofiglio, Daniela; Kleer, Celina
Stromal cells in the tumor microenvironment (TME) play an important role in breast cancer progression, metastasis and therapeutic outcome. Among stromal cells, Tumor-Associated Macrophages (TAMs) and Mesenchymal Stem Cells (MSCs) have been shown to sustain breast tumor progression and worsen breast cancer prognosis. Elucidating the molecular mechanisms of epithelial/stromal cell interactions and discovering new therapeutic targets within the breast TME represent the main challenge of current research to increase the chances of successful treatment of breast cancer patients. Here, we firstly investigated the role of ligand-activated Peroxisome Proliferator Activator Receptor γ (PPARγ), a well-known tumor suppressor gene, to modulate breast TAM functional phenotype. We found that the treatment with natural and synthetic PPARγ ligands reduced the cytokine secretion by TAMs generated by exposure of conditioned media (CM) from breast cancer cells (BCCs). Interestingly, this effect was reversed by the PPARγ antagonist GW9662, suggesting the potential involvement of PPARγ in the attenuation of TAM polarization. Next, since it has been reported that soluble factors released in the TME mediate the tumor/stroma interactions, we mainly focused on the role of leptin which has been reported to sustain macrophage recruitment. Thus, we explored the impact of the leptin receptor knockdown (ObR sh) on BCCs in mediating the interaction between tumor cells and macrophages. In co-culture experiments between monocytes and BCCs, the absence of ObR reduced the recruitment of macrophages and affected their cytokine mRNA expression profile toward a less aggressive phenotype. We confirmed a decreased macrophage infiltration and reduced breast cancer growth in xenograft tumors of mice injected with ObR sh BCC. Furthermore, we explored the interaction between BCCs and MSCs within the breast TME. To this aim, we generated BCCs engulfing MSCs which result in hybrid cancer cells characterized by a multinucleated phenotype with increased dormancy and chemoresistance. In mouse models of breast cancer metastasis, hybrid cells had a reduced ability to form metastasis, but upon doxorubicin treatment they acquired resistance, inducing the metastatic spread of breast cancer. Collectively, our findings provide novel insights into the role of PPARγ and leptin signaling in modulating TAM polarization, opening new avenues for therapeutic intervention in breast cancer. Moreover, we identified and characterized a hybrid cell population, generated through. MSC engulfment by BBCs, with phenotypic features of malignancy, highlighting the potential of targeting stromal cells, to overcome drug resistance and metastasis in breast cancer.

Dipartimento di Farmacia e Scienze della Salute e della Nutrizione - Tesi di Dottorato

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