FLNA is implicated in pulmonary neuroendocrine tumors aggressiveness and progression FLNA is implicated in pulmonary neuroendocrine tumors aggressiveness and progression

Introduction: Treatment options for neuroendocrine tumors (NETs) are rarely curative, as NETs frequently show resistance to medical therapy. The use of everolimus, an mTOR inhibitor, is limited by the development of resistance, probably due to the activation of Akt signaling. In this context, the antidiabetic drug metformin is able to inhibit mTOR, providing a rationale for the use of metformin and everolimus in combination. Methods: We investigated the effects of the metformin and everolimus combination on NET cell proliferation, apoptosis, colony formation, cell viability, NET spheroids growth and the involvement of the Akt and mTOR pathways, and also developed everolimus-resistant NET cells to further study this combination. Results: Metformin and everolimus in combination are more effective than monotherapy in inhibiting pancreatic NET (PAN-NET) cell proliferation (−71% ± 13%, p < 0.0001 vs. basal), whereas no additive effects were observed on pulmonary neuroendocrine tumor (PNT) cell proliferation. The combinatorial treatment is more effective than monotherapy in inhibiting colony formation, cell viability, NET spheroids growth rate and mTOR phosphorylation in both NET cell lines. In a PAN-NET cell line, metformin did not affect Akt phosphorylation; conversely, it significantly decreased Akt phosphorylation in a PNT cell line. Using everolimus-resistant NET cells, we confirmed that metformin maintained its effects, acting by two different pathways: Akt-dependent or independent, depending on the cell type, with both leading to mTOR suppression. Conclusions: Considering the promising effects of the everolimus and metformin combination in NET cells, our results provide a rationale for its use in NET patients.

Autologous fat grafting (AFG) is a safe and minimally invasive procedure to correct soft tissue defects. The benefit of AFG is attributed to adipose-derived stem cells (ASCs) in fat tissue graft. This technique is useful also in patients undergoing reconstructive surgery following quadrantectomy for breast cancer. However, these patients are frequently treated with tamoxifen. We evaluated the ex vivo effects of tamoxifen on ASCs to understand if cellular functions of ASCs are affected. We selected 24 female patients; 10 of which were breast cancer patients treated with quadrantectomy and tamoxifen. As control group, we selected 14 healthy female subjects (9 premenopausal and 5 menopausal). We found that tamoxifen has no effect on cellular proliferation, VEGF secretion or apoptosis of ASCs. The gene expression assessment demonstrated no impairment in differentiation capacity of ASCs. Our results showed that tamoxifen has no effect on cellular functions of ASCs for the first time in an ex vivo single-center study.

Neuroendocrine tumors (NETs) are rare neoplasms showing a wide spectrum of clinical behaviors. The therapeutic options available for NET treatment are rarely curative and most are palliative, as NETs frequently show resistance to pharmacological therapy. Cancers develop in complex tissue environments, which they depend upon for sustained growth, invasion and metastasis. A major characteristic of the tumor microenvironment is inflammatory cell infiltration, where tumor-associated macrophages (TAMs) are a major cellular component. The polarized state of macrophages is classified into two subsets: classically activated M1 (anti-tumor promoting effects) and alternatively activated M2 (pro-tumor promoting effects). Thus the restoration of an M1 phenotype may provide therapeutic value by promoting anti-tumor behavior. The importance of immune microenvironment and the role of macrophages in NET are poorly understood. Aims of the study are: to investigate the effects of the different macrophages subtypes (M1, M2) on biological behavior of primary NET cells and QGP1 (pancreatic-NET) and H727 (pulmonary-NET) cell lines. We found that M1 macrophages significantly decreased the viability of QGP1 cell line (−40±14% P<0.01 vs basal) and H727 cell line (−43±8 P<0.01 vs basal). Moreover, CM (conditioned medium) of M1 macrophages strongly decreased colony formation, colony average area and cell number (QGP1: −42±17% P<0.05 vs basal; H727: −60±15% P<0,001 vs basal) after 7 days of incubation. As shown by flow cytometry analysis, after CM of M1 macrophages treatment, G0/G1 phase was increased in H727 cells (+18±7% P<0.05), whereas G2/M phase was decreased in QGP1 cell (−44±4% P<0.05). Interestingly, as shown by real time analysis, the CM of NET cell lines promoted the differentiation of macrophages into an M2 phenotype, after 24 h of culture. Moreover, as shown in preliminary data of primary pancreatic NET cells, CM of M1 macrophages strongly decreased cell proliferation, whereas CM of M2 macrophages promoted it. In conclusion, M1 macrophages have a potent anti-tumor effect, able to affect the proliferation and the tumorigenicity of NET cell lines. Interestingly, NET cell lines contribute to promote a M2 phenotype of macrophages, suggesting a potential involvement of tumor microenvironment in the behaviour of pulmonary and pancreatic NETs.

Metformin (1.1-dimethylbiguanide hydrochloride), a widely used antidiabetic drug, has been reported to display potent anticancer properties in various types of cancers, including neuroendocrine tumors. Recently, a potential synergistic activity between metformin and octreotide (SSA) in Pancreatic Neuroendocrine Tumors (P-NETs) has been proposed. AIP (aryl hydrocarbon receptor-interacting protein) acts as tumor suppressor gene in neuroendocrine tumors at pituitary level and it is up-regulated by SSA. We investigated the effects of metformin on P-NETs cell proliferation, apoptosis and colony formation, the potential synergistic effect between metformin and octreotide and the possible role of AIP pathway in mediating these effects. We found that in QGP-1 cells metformin significantly inhibited cell proliferation (−37±2% P<0.001 vs basal at 10 mM). We showed no additive effect between Metformin and Octreotide on P-NETs cell proliferation. Moreover, metformin incubation reduced colony formation, cells amount after 7 days (−66±6% P<0.001 vs basal) and it promoted apoptosis (+150±8% P<0.05 vs basal). As expected, octreotide induced AIP up-regulation (+60±11% P<0.05 vs basal), and surprisingly, AIP expression levels were also increased by metformin (+90±13% P<0.05 vs basal). In addition, AIP silencing abolished the antiproliferative and proapoptotic effects of metformin, confirming its involvement in mediating metformin effects. Interestingly, metformin decreased HSP70 expression, increased Zac1 and AhR expression levels. These effects were totally abolished in QGP-1 cells lacking AIP. In conclusion, metformin acts as anticancer agent in P-NET cells, its activity is mediated by AIP and its interacting proteins, HSP70, AHR and Zac1. These findings provide a novel insight into the antitumorigenic mechanism of metformin.