Metformin and everolimus: a promising combination for neuroendocrine tumors treatment Giulia Tarantola, Humanitas University Nurcin Liman, Università degli Studi di Milano

Neuroendocrine tumors (NETs) Heterogeneous group of tumors Arise from cells of the neuroendocrine system Sites of primary tumors LUNG (1,35) THYMUS (0,02) Other/Unknown (0.74) GI SYSTEM (2.89) Pancreas (0.32) Liver (0.04) Stomach (0.30) Duodenum (0.19) Jejunum/ileum (0.67) Cecum (0.16) Appendix (0.15) Colon (0.20) Rectum (0.86) A recent reclassification of pituitary adenoma as pituitary NET (Pi-NET) for their analogy with other NETs has been approved

Neuroendocrine tumors (NETs) Wide variety of clinical presentations Late presentation: - Over 60% of NETs are advanced at the time of diagnosis - The median survival for patients with advanced NET is 33 months Have metastatic potential Histological diagnosis may be difficult

Incidence 1973-2012: +700% Analysis of 64,971 NET cases in the Surveillance, Epidemiology, and End Results (SEER) database (from Dasari et al, JAMA Oncology, 2017)

Treatment options and their target pathways Surgery Somatostatin analogs (Octreotide) Everolimus  mTOR inhibitor which has been approved for the treatment of advanced P-NETs Sunitinib Peptide receptor radionuclide therapy (PRRT) Chemotherapy

Everolimus resistance Inhibition of mTOR by everolimus may initiate a feedback loop that results in Akt upregulation A vast majority of patients develop resistance Akt/PI3K inhibitors may have a limited clinical role because of unacceptable toxicity in patients

Metformin antihyperglycemic drug commonly used in the treatment of type 2 diabetes associated with a reduced incidence of cancer (2005) possesses anticancer activity in several tumours, in particular we demonstrated that metformin exerts anti-proliferative and pro- apoptotic effect in pancreatic NETs through AIP (Aryl hydrocarbon receptor Interacting Protein) Vitali et al, Oncotarget, 2019 (submitted)

IGF-1 and Insulin Receptors Protein synthesis and cell growth Akt Metformin Insulin, IGF-1 IRS-1 PI3K Raptor mTOR mTORC1 Protein synthesis and cell growth AMPK TSC2 Metformin and everolimus – Mechanism of action Everolimus

Is there a potential role for everolimus and metformin combination in pancreatic and pulmonary NETs treatment?

Aims of the study Observe the effects of metformin and everolimus combination on cell proliferation, colony formation and apoptosis. 2. Investigate the mechanism of action and the molecular pathway of metformin in QGP1 and H727 cell lines and in pancreatic and pulmonary neuroendocrine tumors primary cell lines through western blot analysis. 3. Clarify whether metformin can be effective when NET cells are resistant to everolimus.

The effect of metformin and everolimus on NET cell proliferation P-NETs cell proliferation (% of basal) ** **** ## # PNTs cell proliferation (% of basal) **** ** QGP-1 cell proliferation (% of basal) Metformin (1mM) Metformin (10mM) Everolimus (10 nM) Everolimus (100 nM) - + - - - + + - - - - + - - - - + + - - - + - + - + - - - - - + - + - + ** *** **** ## ° H727 cell proliferation (% of basal) Metformin (1mM) Metformin (10mM) Everolimus (10 nM) Everolimus (100 nM) - + - - - + + - - - - + - - - - + + - - - + - + - + - - - - - + - + - + * **** *** ** The combination is significantly more effective than monotherapy only in P-NETs

Metformin and everolimus in combination are more effective than monotherapy in inhibiting QGP-1 and H727 colony formation *** **** Number of QGP-1 cells (% of basal) quantification after 7 days Bas Met Eve Met + eve # °° Bas Met Eve Met + eve Number of H727 cells (% of basal) quantification after 7 days * **** # ° Basal Metformin 1mM Everolimus 10nM Metformin 1 mM+ everolimus 10nM Basal Metformin 1mM Everolimus 10nM Metformin 1 mM+ everolimus 10nM Metformin 1mM+ Combinatory treatment is significantly more effective in decreasing quantity of colonies, number of cells and colony size

Metformin and everolimus effects on apoptosis in QGP-1 and H727 *** ** Apoptotic QGP-1 cells (% of basal) Basal Metformin Everolimus Metformin + Apoptotic H727 cells (% of basal) ** * Basal Metformin Everolimus Metformin + Only metformin is able to significantly promote apoptosis.

The effect of metformin and everolimus on mTOR and Akt phosphorylation in QGP-1 and H727 P-mTOR Vinculin P-mTOR Vinculin **** ### # pmTOR/vinculin (% of basal) *** **** ### # pmTOR/vinculin (% of basal) pAkt GAPDH pAkt GAPDH pAkt/GAPDH (% of basal) pAkt/GAPDH (% of basal) *

QGP-1 R cell proliferation (% of basal) H727 R cell proliferation *** * **** Metformin (1mM) Metformin (10mM) Everolimus (10 nM) Everolimus (100 nM) - + - - - + + - - - - + - - - - + + - - - + - + - + - - - - - + - + - + Apoptotic QGP-1 R cells ** Met Eve Met + eve Apoptotic H727 R cells Basal Metformin 10 mM Everolimus 10 nM Metformin 10 mM+ Everolimus 10 nM Resistant cells Metformin keeps inhibiting cell proliferation, blocking cell cycle in G0/G1, and inducing apoptosis

mTOR and AKT phosphorylation in everolimus-resistant QGP1-R and H727-R cells after metformin incubation QGP-1 R H727 R P-mTOR P-mTOR Vinculin Vinculin pmTOR/vinculin (% of basa) pmTOR/vinculin (% of basa) * * * * Basal Metformin 10mM Everolimus 10nM Metformin 10mM+ Everolimus 10nM QGP-1 R H727 R pAkt pAkt GAPDH GAPDH ** ** pAkt/GAPDH (% of basa) * pAkt/GAPDH (% of basa) * *

QGP-1 H727

TAKE HOME MESSAGE Everolimus is an effective drug against pancreatic and pulmonary NETs, but patients often develop resistance: combination therapy with metformin helps in both preventing and overcoming the resistance.

Thank you for your attention Thanks to Eleonora Vitali, Ilena Boemi and Andrea Lania