Pulmonary neuroendocrine tumors (PNTs) comprise different neoplasms, ranging from low grade carcinoids to the highly malignant small cell lung cancers. Several studies identified the cytoskeleton protein Filamin A (FLNA) as determinant in cancer progression and metastasis, but the role of FLNA in PNT aggressiveness and progression is still unknown.We evaluated FLNA expression in PNTs with different grade of differentiation, the role of FLNA in cell proliferation, colony formation, angiogenesis, cell adhesion and migration in PNT cell line (H727 cells) and primary cultures and the possible interaction between FLNA and Rap1-GTPase. FLNA is highly expressed in PNTs with high malignant grade. FLNA silencing reduces cyclin D1 levels (-51±5, p<0.001) and cell proliferation in PNT cells (-37±4, p<0.05), colony formation and VEGF expression (-39±9%, p<0.01) in H727 cells.FLNA and Rap1 co-localize in cellular protrusions and FLNA silencing up-regulates Rap1 expression (+73±18%, p<0.01).Rap1 silencing prevents cell adhesion increase (+43%±18%, p<0.01) and cell migration decrease (-56±7%, p<0.01) induced by FLNA silencing, without affecting cell proliferation reduction. In conclusion, FLNA is implicated in PNT progression, in part through Rap1, thus providing a potential diagnostic and therapeutic target.

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.

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