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

Results

Clinical Evolution in Patients With LCs and GEP-NETs and Correlations with T2DM

In the LC group, weight loss was more frequently observed in patients with T2DM than in patients without diabetes (36.4% vs 8.6%; P < 0.05; Table 1). Likewise, pleura invasion was also higher in patients with T2DM (37.5% vs 2.2%; P < 0.05; Table 1). Despite the increased incidence of weight loss in patients with diabetes with LC, none of the metformin-treated patients with T2DM exhibited this symptom (P < 0.05; Table 2). In this cohort, the clinical outcome did not differ in those patients receiving metformin or other antidiabetic treatment (Table 2). The death rate tended to be increased in patients with T2DM (P = 0.09; Table 1).

In the GEP-NETs group, an increased incidence of a second neoplasm was observed in the nondiabetic group (25.7% vs 0%; P < 0.05; Table 3). Tumor diameter tended to be greater in patients with T2DM compared with patients without diabetes (3.4 ± 0.5 vs 2.5 ± 0.2 cm; P = 0.06). In addition, the proportion of patients with complete surgical resection was lower in the T2DM group compared with the nondiabetic group (69.2% vs 93.8%; P < 0.05; Table 3). In this cohort, the clinical outcome of patients treated with metformin was also similar to those treated with other antidiabetic drugs or insulin (Table 4).

None of the other clinical parameters evaluated (including functionality or incidental findings), histopathological variables (including necrosis, local invasion, presence of metastasis, and vascular or nerve invasion), tumor grading, or evolution parameters (including relapsed disease, disease-free survival, and mortality) were associated with T2DM or the use of metformin in the cohorts of patients with LCs or GEP-NETs.

No clinical, histological, or molecular variable was associated with the presence of hyperlipidemia in the cohort of patients with LCs (Table 5) or GEP-NETs (Table 6). A higher proportion of patients treated with statins were free of disease during the follow-up (χ 2, 7.07; P < 0.05). None of the other clinical, histological, or evolution parameters were associated with the use of statins in the cohorts of patients with LCs or GEP-NETs.

mRNA Expression of SST and Ghrelin System Components in LCs and GEP-NETs, and Their Correlations With T2DM

The mRNA levels of several genes of interest were measured in the tumor tissue obtained from patients with LCs (Figure 1A) and GEP-NET (Figure 1B). In the LCs group, mRNA levels of SST and several receptor subtypes (i.e., SSTR1, SSTR2, SSTR4, SSTR5, and sst5TMD4), but not of CORT or SSTR3, were numerically, albeit nonsignificantly, decreased in patients with T2DM compared with the patients without diabetes (Figure 1A). A similar pattern of expression was observed in the GEP-NETs group, except for the mRNA levels of SSTR5 (Figure 1B). Interestingly, an overall decrease in the mRNA levels of all SSTRs was found in the LC and GEP-NET groups, but this difference only reached statistical significance in the GEP-NET group (Figure 1C).

Figure 1.

mRNA expression of SST system components in patients with and without T2DM with (A) LCs and (B) GEP-NETs. (C) Total mRNA expression of somatostatin receptors in patients with LCs or GEP-NETs. The absolute mRNA expression of the different components of the SST system was determined by quantitative real-time PCR in tumor samples (values were normalized to BACT in LCs and 18S in GEP-NETs). Data represent the mean ± SEM. **P < 0.01.

Similarly, mRNA levels of all the components of the ghrelin system (i.e., GHRL, In1-ghrelin, GOAT, and the receptors GHSR1a and GHSR1b) displayed nonsignificant lower levels in patients with diabetes in the LC group compared with patients with LCs but not diabetes (Figure 2A). In GEP-NETs, the mRNA levels of In1-ghrelin, GOAT, and GHSR1b, but not GHRL or GHSR1a, also tended to be lower in patients with T2DM (Figure 2B).

Figure 2.

mRNA expression of ghrelin system components in patients with and without T2DM with (A) LCs and (B) GEP-NETs. The absolute mRNA expression of the different components of the ghrelin system was determined by quantitative real-time PCR in tumor samples. Data represent the mean ± SEM.

Interestingly, a subanalysis showed that although the overall expression of SSTRs was significantly lower in the GEP-NETs group with T2DM compared with patients without diabetes, these levels were not decreased in patients with T2DM treated with metformin compared with patients who were not diabetic (Figure 3A). Specifically, a nonsignificant increase in the mRNA levels of SST, CORT, SSTR1, SSTR2, and SSTR3was observed in patients with T2DM treated with metformin compared with patients with T2DM not treated with metformin (Figure 3B), as well as in the mRNA levels of GHRL, In1-ghrelin, and GHSR1a (Figure 3C).

Figure 3.

Effects of metformin in patients with T2DM and GEP-NETs. (A) Total mRNA expression of somatostatin receptors in GEP-NETs. Specific (B) SST and (C) ghrelin system components in GEP-NETs. The absolute mRNA expression of the different components of the SST system was determined by quantitative real-time PCR and normalized to 18S. mRNA expression was assessed in patients with GEP-NET with and without T2DM. Among patients with GEP-NET with and without T2DM, two subgroups were analyzed: those treated with metformin vs those treated with other antidiabetic drugs or insulin. mRNA expression was compared with that in controls. Data represent the mean ± SEM. *P < 0.05.

Cell Survival of PNET Cells After Treatment With Biguanides or Statins

All biguanides tested clearly decreased the survival rate in both BON-1 (Figure 4A) and QGP-1 (Figure 4B) cell lines in a time-dependent manner. The most remarkable effect was observed in BON-1 cells with phenformin (5 × 10−3 M), which decreased the survival rate by 76.6%, 93.1%, and 97.13% after 24, 48, and 72 hours of incubation, respectively. Metformin (10−2 M) decreased the survival rate in these cells by 25.1%, 38.1%, and 49.4%, whereas buformin (5 × 10−3 M) reduced it by 36.9%, 37.1%, and 56.3% after 24, 48, and 72 hours of incubation, respectively (Figure 4A).

Figure 4.

Time-dependent effect on cell viability of biguanides in (A) BON-1 and (B) QGP-1 cell lines; and statins in (C) BON-1 and (D) QGP-1. Cell viability is expressed as cell survival percentage after 24, 48, and 72 hours. Cell proliferation rate compared with that of controls was assessed by multiple comparison tests. *P < 0.05; **P < 0.01.

A similar effect was observed in the QGP-1 cell line. Specifically, phenformin was also the most effective biguanide; survival rates with phenformin decreased by 68.2%, 87.4%, and 96.9% after 24, 48, and 72 hours of incubation, respectively, whereas, metformin decreased the survival rate by 24.9%, 45%, and 60%, respectively, and buformin by 30.7%, 53.0%, and 69.7%, respectively (Figure 4B).

We also analyzed the effect of different statins on cell survival in BON-1 (Figure 4C) and QGP-1 (Figure 4D) cell lines. Specifically, a decreased survival rate was observed after 48 and 72 hours of treatment with simvastatin (10−5 M; 21.4% and 34.5%, respectively), and after 72 hours of treatment with atorvastatin (10−5 M; 15.2%) in BON-1 cells (Figure 4C). The effect of simvastatin was more pronounced than that of atorvastatin at 72 hours (P < 0.05; Figure 4C). In QGP-1 cells, a reduction in the proliferation rate was observed after 48 and 72 hours of incubation with all the statins tested (Figure 4D). Thus, simvastatin, atorvastatin, lovastatin, and rosuvastatin (10−5 M) decreased survival rates by a range of 14.7% to 17.2% after 72 hours.

Based on their antiproliferative effects, phenformin and simvastatin were chosen as representative compounds of these two classes of drugs to perform further functional experiments (i.e., migration, apoptosis, and serotonin secretion). Moreover, metformin was also included in these analyses because of its relevance in clinical practice.

Migration Capacity in PNET Cells in Response to Metformin, Phenformin, and Simvastatin Treatment

Metformin and simvastatin (after 24 hours of incubation) significantly decreased the migration capacity of BON-1 cells (100% and 38.6%, respectively; representative images are presented in Figure 5A). In contrast, it was not possible to measure the migration capacity in response to phenformin in BON-1 cells, perhaps due to a treatment-related toxicity of this compound (discussed later in Results). As previously reported,[54] it was not feasible to measure QGP-1 cells with this functional assay, because these cells form aggregates or clusters in culture, which do not allow correct measurement of the migration capacity under basal conditions or in response to any given treatment.

Figure 5.

Effect of biguanides and statins on (A) cell migration in BON-1 cells; (B) apoptosis rate in BON-1 and (C) QGP-1 cells; and (D) serotonin secretion in (D) BON-1 and (E) QGP-1 cell lines. Migration and serotonin secretion were assessed after 24 hours of incubation; apoptosis rate was evaluated after 48 hours. Representative images of wound healing after 24 hours of treatment are presented in (A), lower panels. Treatment rates were compared with those in controls by multiple comparison tests. *P< 0.05; **P < 0.01; ***P < 0.001.

Effect of Metformin, Phenformin, and Simvastatin Treatment on Apoptosis

In BON-1 cells, phenformin caused a threefold increase in apoptosis (Figure 5B). However, metformin or simvastatin treatment did not alter apoptosis in BON-1 cells. In QGP-1 cells, a twofold increase in apoptosis was also observed in response to phenformin (Figure 5C). In addition, simvastatin increased the apoptotic rate in QGP-1 cells by 58.1% (Figure 5C). Conversely, metformin treatment did not alter apoptosis in QGP-1 cells.

Effect of Biguanides and Statins on Serotonin Secretion in PNET Cell Lines

In BON-1 cells, phenformin, but not simvastatin, decreased serotonin secretion after 24 hours of incubation (P < 0.05; Figure 5D). Metformin treatment also tended to decrease serotonin release (P = 0.06; Figure 5D). In contrast, none of these treatments altered serotonin secretion from QGP-1 cells (Figure 5E).

Effects of Metformin, Phenformin, and Simvastatin on ERK1/2 and AKT Signaling Pathways

To start exploring the signaling pathways affected by biguanides (i.e., metformin and phenformin) and simvastatin to induce their functional actions in NET cells, the levels of phosphorylation of AKT and ERK were evaluated. In BON-1 cells, both biguanides and simvastatin similarly decreased phosphorylation levels of AKT and ERK compared with controls (Figure 6A). In marked contrast, in QGP-1 cells, only phenformin and simvastatin decreased phosphorylation levels of ERK without altering those of AKT (Figure 6B).

Figure 6.

Effects of biguanides and statins on AKT and ERK phosphorylation in (A) BON-1 and (B) QGP-1 cells. Phosphorylation levels compared with those in controls was assessed by multiple comparison tests. *P < 0.05; **P < 0.01; ***P < 0.001.

Effect of Metformin, Phenformin, and Simvastatin in the Expression of key Genes in PNET Pathophysiology

In BON-1 cells, metformin and phenformin severely decreased the mRNA levels of INSR (P < 0.001; Figure 7A). Also, a trend toward an increase in the expression GLUT4was observed in response to phenformin (Figure 6A). In QGP-1 cells, GLUT4 expression was increased in response to both biguanides and simvastatin, but this difference only reached statistical significance in the case of phenformin (Figure 7B). No significant changes were observed in the expression of INSR in QGP-1 cells in response to these compounds (Figure 7B). Finally, metformin treatment did not significantly alter the expression of SSTRs in BON-1 and QGP-1 cells (data not shown).

Figure 7.

Effects of biguanides and statins on mRNA expression in (A) BON-1 and (B) QGP-1 cells. mRNA expression compared with that in controls was assessed by multiple comparison tests. *P < 0.05; ***P< 0.001.

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