EMT reversal in human cancer cells after IR knockdown in hyperinsulinemic mice

Abstract

Type 2 diabetes (T2D) is associated with increased cancer risk and cancer-related mortality. Data herein show that we generated an immunodeficient hyperinsulinemic mouse by crossing the Rag1(-/-) mice, which have no mature B or T lymphocytes, with the MKR mouse model of T2D to generate the Rag1(-/-) (Rag/WT) and Rag1(-/-)/MKR(+/+) (Rag/MKR) mice. The female Rag/MKR mice are insulin resistant and have significantly higher nonfasting plasma insulin levels compared with the Rag/WT controls. Therefore, we used these Rag/MKR mice to investigate the role of endogenous hyperinsulinemia on human cancer progression. In this study, we show that hyperinsulinemia in the Rag/MKR mice increases the expression of mesenchymal transcription factors, TWIST1 and ZEB1, and increases the expression of the angiogenesis marker, vascular endothelial growth factor A (VEGFA). We also show that silencing the insulin receptor (IR) in the human LCC6 cancer cells leads to decreased tumor growth and metastases, suppression of mesenchymal markers vimentin, SLUG, TWIST1 and ZEB1, suppression of angiogenesis markers, VEGFA and VEGFD, and re-expression of the epithelial marker, E-cadherin. The data in this paper demonstrate that IR knockdown in primary tumors partially reverses the growth-promoting effects of hyperinsulinemia as well as highlighting the importance of the insulin receptor signaling pathway in cancer progression, and more specifically in epithelial-mesenchymal transition.

Keywords: cancer; epithelial–mesenchymal transition; hyperinsulinemia; insulin receptor; type 2 diabetes.

Figure 1

Characterization of an immunodeficient hyperinsulinemic mouse model. (A) Body weights of male and female mice from Rag/WT and Rag/MKR mice, measured weekly from 3 weeks of age to 10 weeks of age. * p<0.05 between Rag/WT male and Rag/MKR male mice. # p<0.05 between Rag/WT female and Rag/MKR female mice. N=5 mice per group, error bars represent SEM. (B) MRI results showing whole body fat in Rag/WT and Rag/MKR mice. N=5 mice per group, error bars represent SEM. (C) Insulin Tolerance Test (ITT) performed on fasted 8 week male and female Rag/WT and Rag/MKR mice. Blood glucose levels were measured at time 0 and 15, 30, and 60 minutes post insulin injection. Mice were injected with 0.75U/kg insulin. * p<0.05 between Rag/WT male and Rag/MKR male mice. # p<0.05 between Rag/WT female and Rag/MKR female mice. N=5 mice per group, error bars represent SEM. (D) Plasma insulin levels of non-fasted male and female Rag/WT and Rag/MKR mice. * p<0.05 between groups as indicated. Graphs represent the mean of each group (n=5 mice per group), error bars are SEM.

Figure 2

Silencing the insulin receptor leads to decreased tumor growth. 8–10 week old Rag/WT control and Rag/MKR hyperinsulinemic mice were injected with either 5×10⁶ LCC6 control (Ctrl) or 5×10⁶ LCC6 insulin receptor knockdown (IRKD) cells, into the 4th mammary fat pad (n=8–10 mice per group). (A) Mammary tumor volume was calculated from tumor measurements taken twice weekly with calipers. * p<0.05 between Rag/MKR Ctrl and Rag/WT Ctrl tumors. ** p<0.05 between Rag/MKR Ctrl and Rag/MKR IRKD. # p<0.05 between Rag/WT Ctrl and Rag/WT IRKD tumors. N=8–10 mice per group, error bars represent SEM. (B) Tumor weight at the end of the study. (* p<0.05 between groups as indicated). N=8–10 mice per group, error bars represent SEM.

Figure 3

Expression of IR and IGF1R in LCC6 Tumors. (A) Primary tumors from Rag/WT and Rag/MKR mice were assessed for the gene expression of the insulin receptor, demonstrating an 83% reduction of IR in the tumors from the LCC6 IRKD cells. (* p<0.05, n=8–10 mice per group, error bars represent SEM.). (B) Primary tumors from Rag/WT and Rag/MKR mice were assessed for the gene expression of the insulin like growth factor-1 receptor IGF1R (* p<0.05 n=8–10 mice per group, error bars are SEM.)

Figure 4

Reduction of insulin signaling pathway in LCC6 IRKD tumors. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for phospho-Akt (pAKT) and total AKT expression. B-Actin antibody used as loading control. Densitometry of Western blot (* p<0.05, graphs represent mean per group (n=8–10 mice per group) and error bars represent SEM). (B) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for phospho-S6 ribosomal protein (pS6RP) and total S6RP expression. B-Actin antibody used as loading control. Densitometry of Western blot (* p<0.05, graphs represent mean per group (n=8–10 mice per group) and error bars represent SEM). (C) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for c-MYC expression. B-Actin antibody was used as the loading control. Densitometry of Western blot of c-MYC/B-Actin (* p<0.05, graphs represent mean per group (n=8–10 mice per group) and error bars are SEM).

Figure 5

EMT and angiogenesis in Ctrl and IRKD tumors. RNA extracted from tumor tissue was analyzed for (A) TWIST1, (B) ZEB1, (C) SLUG, and (D) Vascular endothelial growth factor A (VEGFA) gene expression by qRT-PCR. Calculations were made using ribosomal protein L19 (RPL19) as housekeeping gene. Graphs represent mean per group (n=5 per group) and error bars are SEM. (* p<0.05).

Figure 6

Tumors from LCC6 IRKD cells have reversal of Epithelial-Mesenchymal Transition phenotype. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for Vimentin expression. B-Actin antibody was used as the loading control. Densitometry of western blot of Vimentin/B-Actin. (B) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for TWIST1 expression. B-Actin antibody was used as the loading control. Densitometry of western blot TWIST1/B-Actin. (C) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for SLUG expression. B-Actin antibody used as loading control. Densitometry of western blot of SLUG/B-Actin. (D) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for E-cadherin expression. B-Actin antibody used as loading control. Densitometry of western blot of E-cadherin/B-Actin. Graphs represent mean per group (n=8–10 mice) and error bars are SEM. * p<0.05 between groups as indicated.

Figure 7

Insulin Receptor Knockdown inhibited pulmonary metastases in hyperinsulinemic mice. Lungs were fixed, paraffin embedded and sectioned. Representative images of H&E stained lung sections from Rag/WT and Rag/MKR mice injected with LCC6 Ctrl and LCC6 IRKD cells. (A, B) 0/5 (0%) of Rag/WT injected with LCC6 Ctrl or LCC6 IRKD cells had pulmonary metastasis. (C) Representative pulmonary micrometastases seen in 2/5 (40%) Rag/MKR injected with LCC6 control cells. (D) 0/5 (0%) of Rag/MKR mice injected with LCC6 IRKD cells had pulmonary metastases. 10× magnification with scale bar of 100um in length.