Type 2 Diabetes in Neuroendocrine Tumors

Are Biguanides and Statins Part of the Solution?

Aura D. Herrera-Martínez; Sergio Pedraza-Arevalo; Fernando L-López; Manuel D. Gahete; María A. Gálvez-Moreno; Justo P. Castaño; Raúl M. Luque

Disclosures

J Clin Endocrinol Metab. 2019;104(1):57-73. 

In This Article

Discussion

NETs are a widely heterogeneous group of neoplasms that are frequently diagnosed at an advanced stage of disease.[55] Therapeutic options for advanced, metastasized NETs include somatostatin analogs, interferon-α, chemotherapy, and peptide receptor therapy.[55,56] Target-directed therapies have increased the therapeutic spectrum for progressive NETs (e.g., sunitinib as tyrosine kinase inhibitor and everolimus for mTOR pathway inhibition).[57–59] However, despite the improvements in progression-free survival with these therapeutic options, their effect on overall survival is still controversial.[60] Therefore, novel treatments are still required, especially for patients with advanced disease.

The incidence of metabolic syndrome is continuously increasing, reaching almost 35% in some countries, with a consequent increase in the prevalence of some types of cancer.[61] T2DM has been also related to an increased risk of malignancies[62,63] and is frequently developed in patients using everolimus and some somatostatin analogs.[64,65] In this context, metformin is one of the most widely prescribed oral hypoglycemic agents and it has received increased attention because of its potential antitumorigenic effects.[5,66] Likewise, some publications have described an inhibitory effect exerted by statins on tumor-induced angiogenesis and an antitumor effect in cellular and animal models of human cancer.[22,67] However, other studies have also suggested a potential risk of cancer when statins are used.[68] In this sense, the current study, although using a limited number of samples, is, to our knowledge, the first in which (i) the association of T2DM with clinical evolution parameters in patients with different NET types (LCs and GEP-NETs) was assessed; (ii) the expression levels of all the components of two key regulatory systems (SST and ghrelin) in the tumor tissue of patients with these two NET types was evaluated in relation to T2DM and metformin treatment; and (iii) the effects of different biguanides and statins in key functional parameters were analyzed and compared in two representative models of NET cell lines: BON-1 and QGP-1 cells.

T2DM is linked to relevant defects in the INSR signaling pathway, which regulates growth and metabolic responses in insulin-targeted cells and tissues.[69] Some epidemiological studies have described an increased risk for several types of cancer (i.e., breast, colon, rectum, liver, and pancreas) in patients with insulin resistance.[70] Remarkably, in our cohort of patients, a more aggressive pattern in LCs (increased incidence of pleura invasion) and increased tumor size in GEP-NETs were observed in patients with T2DM when compared with patients without diabetes, suggesting a possible association between T2DM and NET pathophysiology.

Because insulin is related to increased risk of cancer, treatment options targeting this pathway could be effective in cancer prevention.[71] Indeed, a meta-analysis showed a 31% reduction in overall cancer incidence and a 34% decline in cancer mortality in patients with diabetes treated with metformin.[72] A retrospective study in patients with T2DM and NETs showed a lower recurrence rate in those treated with metformin compared with patients not treated with metformin or those without diabetes.[72] Moreover, in a cohort of patients with PNETs who were receiving everolimus and octreotide LAR, progression-free survival was longer in patients treated with metformin compared with other drugs.[73] To the best of our knowledge, no other specific reports in NETs have been published yet. In our cohort, metformin appeared to avoid weight loss in patients with LCs and T2DM. Interestingly, the numerical records assessed in patients with LCs receiving metformin suggested that tumors were smaller, incidence of metastasis was lower, and disease-free follow-up was longer than in patients not treated with metformin; however, these results did not reach statistical significance, likely due to the limited size of the groups. In contrast, no association was observed between clinical or histological variables and the use of metformin in the GEP-NETs group. We should underline that the main limitation of this work might be the limited number of patients with T2DM and those treated with metformin included in the analysis, although the size of the total cohort evaluated was large enough for making general comparisons. Therefore, like other studies reporting a limited cohort of samples,[73] the results of this study should be interpreted with caution.

Novel mechanisms of action have been proposed for metformin in recent years. Among them, the induction of the expression of the glucagon-like peptide 1 receptor on pancreas β cells was described.[74] Our report shows that the expression of several SSTR subtypes is reduced in NETs from patients with diabetes (in the LC and GEP-NET groups) compared with those from patients without diabetes, and, most importantly, that the overall expression of SSTR is significantly increased in LCs from patients with diabetes treated with metformin compared with LCs from patients with diabetes without metformin treatment. In fact, these expression levels of SSTR in LCs from patients with diabetes treated with metformin achieved the levels observed in LCs from patients without T2DM. These results provide suggestive evidence that metformin treatment could increase SSTR expression in NETs in patients with diabetes, which might be important from a clinical point of view, in that a previous study has suggested that metformin could have a potential synergistic effect when combined with somatostatin analogs via the inhibition of PI3K/AKT/mTOR axis.[73] Thus, it will be worth elucidating the mechanisms involved in the capacity of metformin to regulate the expression of SSTRs as well as a putative synergistic effect between somatostatin analogs and metformin. In this sense, we analyzed the SSTR expression in BON1 and QGP1 cells after metformin treatment, but we did not observe changes in SSTRs mRNA expression levels, which could be in line with the idea that metformin could reverse the changes previously altered under diabetic conditions and maybe only have reduced potential to modulate basal expression of SSTRs. In addition, it has to be noted that pre- or cotreatment with biguanides and statins was not evaluated in this study, because the antiproliferative in vitro response to somatostatin analogs is limited in these NET cell lines (45, 75, 76).

Biguanides increase insulin sensitivity as well as glucose use by peripheral tissues.[3] Antitumoral effects of metformin and phenformin have been evaluated in in vitroand in vivo studies, and metformin is also being tested as an adjuvant therapy to classic chemotherapeutic regimens.[66,77] Specifically, an earlier study showed that metformin inhibited cell proliferation in pancreatic, bronchopulmonary, and midgut NET cell lines in a dose-dependent manner, wherein these antitumoral effects appeared to be mediated via inhibition of mTORC1 signaling.[12] Metformin also inhibits breast cancer cell growth in vitro in an AMPK-dependent manner in association with a decreased mTOR activation.[78] In our study, we also observed a time-dependent antiproliferative effect of different biguanides in PNET cell lines. Similarly, by measuring other relevant functional end points (i.e., migration capacity and apoptotic rate), our study revealed that biguanides could exert additional, beneficial effects on NET cell function. These results support and extend previous data showing that metformin exerted antitumoral actions in vitro by modulating cell proliferation and apoptosis in breast cancer cells.[79] However, we found that phenformin, but not metformin, increased apoptosis in both NET cell lines, which is partially in agreement with previous data indicating that apoptosis induced by metformin would differ depending on the NET cell type.[12] We also found that metformin and phenformin decreased serotonin secretion in BON-1, but not in QGP-1, cells. Although the exact mechanisms are still to be elucidated, these results could be clinically relevant for patients with carcinoid syndrome, because elevated serotonin levels are directly associated with symptoms in this pathology.[78] In this sense, we should remark that this is not the first time that different results have been observed in the functional response of BON-1 and QGP-1 cells (45, 48, 81, 82), which further emphasizes their potential distinct value to study the intrinsic heterogeneity of NETs. Indeed, the reason for these differences is still unknown but could be related to the distinct expression pattern of key regulatory systems (e.g., SST, ghrelin, IGF-I) (48, 81–83) and/or to the different activation or signaling of these NET cells in response to the same treatment as it has been previously observed, for instance, for SST analogs (i.e., octreotide and pasireotide).[48,81]

Statins can also exert antitumoral actions. Thus, a phase II trial has reported a statin-induced antiproliferative effect in breast cancer.[84] As well, the antiproliferative effect of statins has also been reported in several cancer cell lines, including cervical,[85] leukemic natural killer,[86] cholangiocarcinoma,[87] and prostate.[88] In line with these studies, we observed here that different statins exerted a clear antiproliferative effect in NET cells. In addition, we found that simvastatin significantly increased apoptosis levels in QGP-1 cells, an effect that has been described in cervical cancer, leukemia, natural killer, and cholangiocarcinoma cell lines.[84–86]

It is well known that the PI3K/AKT/mTORC1 pathway exerts important roles in NETs pathogenesis.[89] In LCs, metformin inhibited AKT, ERK, and mTOR pathways, suggesting that its antiproliferative effects can be both AMPK dependent and independent.[90] In fact, Vlotides et al.[12] suggested that the functional effect of metformin is cell-type dependent; they reported that AMPK and AKT phosphorylation was elevated in pancreatic and midgut NET cell lines in response to metformin (after 48 hours of incubation), but this effect was not observed in bronchopulmonary neuroendocrine cells.[12] Interestingly, it was also suggested that the inhibition of the mTOR pathway was associated to the induction of GSK3 phosphorylation following the ERK or AKT pathway. In our study, we observed an inhibition of phosphorylated AKT and ERK pathways after treating cells with biguanides (and also with simvastatin), which also reveals the AMPK-dependent and -independent effects of these drugs in NET cells. It should be mentioned that the differences between our results and those reported by Vlotides et al.[12] may be related to the drug-incubation period (8 minutes vs 48 hours). However, cell inhibition of the ERK pathway has been also reported in non–small lung cancer and cholangiocarcinoma cell lines with concomitant induction of apoptosis.[87,91]

The mechanisms linking T2DM and cancer are a most exciting and interesting research topic. It has been proposed that chronic hyperinsulinemia may promote the development of neoplasms via abnormal stimulation of multiple cellular signaling cascades by insulin, enhancing growth factor–dependent cell proliferation and/or modifying cell metabolism.[66] In our study, we observed changes in the molecular expression of key genes involved in tumor aggressiveness (e.g., INSR, GLUT4) in response to metformin or phenformin, but not simvastatin, suggesting a putative modulatory effect of biguanides in these signaling pathways. In line with this, some studies have suggested that the antiproliferative effect of statins in cancer cell lines might be associated with cell cycle regulatory effects,[84] epigenetic alterations,[92] or with gene expression modifications of cancer signaling.[93] However, the effects of simvastatin treatment on these regulatory end points, as well as whether metformin and simvastatin could have synergistic effects in NETs [which has been demonstrated in different tumor pathologies[94–96]], could not be evaluated in our study, but deserve further attention.

In sum, our study, using a limited cohort of patients, revealed a potential association between key clinical parameters of NET aggressiveness (e.g., incidence of pleura invasion or metastasis, tumor size) and the presence of diabetes and/or treatment with antidiabetic drugs in patients with different NET types (i.e., LCs and GEP-NETs). Moreover, this study provides evidence that the expression of multiple components of two key regulatory systems for the pathophysiology of NETs, the SST and ghrelin systems, are modulated in patients with diabetes with LCs and GEP-NETs compared with patients without diabetes. Finally, our results also showed that different biguanides and statins can directly exert clear antitumoral actions in NET cells, probably due to their effect on cell survival, cell migration, apoptosis, gene expression, and metabolic pathway modifications. Therefore, because metformin and statins are low-cost, commercially available drugs with a safe profile and large experience in their clinical use, our present results invite further exploration of their potential value as adjuvant therapy for the treatment of patients with NETs.

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