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

2. 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

3. 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

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

5. 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

6. 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

7. 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)

8. 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

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

10. 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.

11. 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

12. 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

13. 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.

14. 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) *

15. 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

16. 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) * *

17. QGP-1
H727

18. 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.

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