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Inhibiting PI3K reduces mammary tumor growth and induces hyperglycemia in a mouse model of insulin resistance and hyperinsulinemia.

Gallagher EJ, Fierz Y, Vijayakumar A, Haddad N, Yakar S, LeRoith D - Oncogene (2011)

Bottom Line: NVP-BKM120 treatment decreased phosphorylation of Akt and S6 ribosomal protein (S6rp); no change in Erk1/2 phosphorylation was seen.Therefore, we examined whether the oral PI3K/mTOR inhibitor NVP-BEZ235 augmented the tumor suppressing effects of PI3K inhibition.We also investigated the effect of targeted PI3K/mTOR inhibition on PI3K/Akt/mTOR and Erk1/2 signaling, and the potential effects on glycemia.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Diabetes and Bone Disease, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Women with type 2 diabetes mellitus (T2DM) are at a greater risk of developing and dying from breast cancer than women without T2DM. Insulin resistance and hyperinsulinemia underlie the pathogenesis of T2DM. In the MKR mouse model of insulin resistance, we have previously shown increased activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway in association with accelerated mammary tumor growth. In this study, we demonstrate that inhibiting PI3K with the oral pan-class I PI3K inhibitor, NVP-BKM120 reduced the growth of Met-1 and MCNeuA mammary tumor orthografts in the MKR mouse. NVP-BKM120 treatment decreased phosphorylation of Akt and S6 ribosomal protein (S6rp); no change in Erk1/2 phosphorylation was seen. Hyperglycemia, hypertriglyceridemia and greater hyperinsulinemia developed in the MKR mice treated with NVP-BKM120. We previously reported reduced tumor growth using intraperitoneal rapamycin in the MKR mouse, with the development of hyperglycemia and hypertriglyceridemia. Therefore, we examined whether the oral PI3K/mTOR inhibitor NVP-BEZ235 augmented the tumor suppressing effects of PI3K inhibition. We also investigated the effect of targeted PI3K/mTOR inhibition on PI3K/Akt/mTOR and Erk1/2 signaling, and the potential effects on glycemia. NVP-BEZ235 suppressed the growth of Met-1 and MCNeuA tumor orthografts, and decreased Akt and S6rp phosphorylation, despite increased Erk1/2 phosphorylation in Met-1 orthografts of MKR mice. Less marked hyperglycemia and hyperinsulinemia developed with NVP-BEZ235 than NVP-BKM120. Overall, the results of this study demonstrated that inhibiting PI3K/Akt/mTOR signaling with the oral agents NVP-BKM120 and NVP-BEZ235 decreased mammary tumor growth in the hyperinsulinemic MKR mouse. Inhibiting PI3K alone led to more severe metabolic derangement than inhibiting both PI3K and mTOR. Therefore, PI3K may be an important target for the treatment of breast cancer in women with insulin resistance. Monitoring for hyperglycemia and dyslipidemia should be considered when using these agents in humans, given the metabolic changes detected in this study.

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NVP-BKM120 reduces the increased proliferation of Met-1 cell orthografts in MKR mice. Assessment of proliferation of Met-1 orthografts by BrdU incorporation, after intraperitoneal injection of BrdU (10µl/g body weight) and detection by immunofluorescence (mouse monoclonal antibody against BrdU, secondary antibody AlexaFluor 488-conjugated goat antimouse IgG (green), and nuclear counterstaining with DAPI (blue). 3A: Representative 40X objective images of BrdU positive Met-1 tumors (top row) and composite images of DAPI and BrdU positive Met-1 tumors (bottom row). White arrows point to BrdU positive cells. 3B: Quantitative analysis representing the percent of BrdU positive cells to total number of DAPI positive cells, expressed as means ± SEM. *P<0.05 between MKR vehicle treated group and all other groups (n = 2–3 mice per group, with six 40X fields per mouse). (WT = wild type, V = vehicle treated, BKM = NVP-BKM120 treated).
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Figure 3: NVP-BKM120 reduces the increased proliferation of Met-1 cell orthografts in MKR mice. Assessment of proliferation of Met-1 orthografts by BrdU incorporation, after intraperitoneal injection of BrdU (10µl/g body weight) and detection by immunofluorescence (mouse monoclonal antibody against BrdU, secondary antibody AlexaFluor 488-conjugated goat antimouse IgG (green), and nuclear counterstaining with DAPI (blue). 3A: Representative 40X objective images of BrdU positive Met-1 tumors (top row) and composite images of DAPI and BrdU positive Met-1 tumors (bottom row). White arrows point to BrdU positive cells. 3B: Quantitative analysis representing the percent of BrdU positive cells to total number of DAPI positive cells, expressed as means ± SEM. *P<0.05 between MKR vehicle treated group and all other groups (n = 2–3 mice per group, with six 40X fields per mouse). (WT = wild type, V = vehicle treated, BKM = NVP-BKM120 treated).

Mentions: We have previously demonstrated increased proliferation in the Met-1 tumors in MKR mice by evaluation of protein extracts for proliferating cell nuclear antigen (PCNA) on western blot (Fierz 2010). To examine whether this increase in S phase growth was inhibited with NVP-BKM120, the mice were injected with 5-bromo-2’-deoxyuridine (BrdU) 2 hours prior to tumor tissue collection. The percent of cells that stained positive for BrdU in the Met-1 tumors was evaluated by immunofluorescence. We found a significantly higher percent of BrdU positive cells in the vehicle treated MKR mice (9.9 ±0.76%), compared to vehicle treated WT mice (7.8±0.35%) (Figure 3A). Treatment of the MKR mice with NVP-BKM120 led to a significantly lower percent of BrdU positive cells (5.8±1.13%), compared to vehicle treated MKR mice (P=0.001). The percent of BrdU positive cells in the Met-1 tumors from the MKR mice treated with NVP-BKM120 was comparable to the percent of BrdU positive cells in the WT NVP-BKM120 treated group (5.8±0.63%). Therefore, in the MKR mice, inhibiting PI3K with NVP-BKM120 attenuated the increased cellular proliferation in Met-1 tumors from the MKR mice (Figure 3B). Western blot analysis of apoptosis, using the antibody to the anti-apoptotic protein Bcl-2 revealed decreased levels of Bcl-2 when normalized to β actin, in the MKR vehicle treated group, compared to the MKR NVP-BKM120 treated group (data not shown).


Inhibiting PI3K reduces mammary tumor growth and induces hyperglycemia in a mouse model of insulin resistance and hyperinsulinemia.

Gallagher EJ, Fierz Y, Vijayakumar A, Haddad N, Yakar S, LeRoith D - Oncogene (2011)

NVP-BKM120 reduces the increased proliferation of Met-1 cell orthografts in MKR mice. Assessment of proliferation of Met-1 orthografts by BrdU incorporation, after intraperitoneal injection of BrdU (10µl/g body weight) and detection by immunofluorescence (mouse monoclonal antibody against BrdU, secondary antibody AlexaFluor 488-conjugated goat antimouse IgG (green), and nuclear counterstaining with DAPI (blue). 3A: Representative 40X objective images of BrdU positive Met-1 tumors (top row) and composite images of DAPI and BrdU positive Met-1 tumors (bottom row). White arrows point to BrdU positive cells. 3B: Quantitative analysis representing the percent of BrdU positive cells to total number of DAPI positive cells, expressed as means ± SEM. *P<0.05 between MKR vehicle treated group and all other groups (n = 2–3 mice per group, with six 40X fields per mouse). (WT = wild type, V = vehicle treated, BKM = NVP-BKM120 treated).
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Figure 3: NVP-BKM120 reduces the increased proliferation of Met-1 cell orthografts in MKR mice. Assessment of proliferation of Met-1 orthografts by BrdU incorporation, after intraperitoneal injection of BrdU (10µl/g body weight) and detection by immunofluorescence (mouse monoclonal antibody against BrdU, secondary antibody AlexaFluor 488-conjugated goat antimouse IgG (green), and nuclear counterstaining with DAPI (blue). 3A: Representative 40X objective images of BrdU positive Met-1 tumors (top row) and composite images of DAPI and BrdU positive Met-1 tumors (bottom row). White arrows point to BrdU positive cells. 3B: Quantitative analysis representing the percent of BrdU positive cells to total number of DAPI positive cells, expressed as means ± SEM. *P<0.05 between MKR vehicle treated group and all other groups (n = 2–3 mice per group, with six 40X fields per mouse). (WT = wild type, V = vehicle treated, BKM = NVP-BKM120 treated).
Mentions: We have previously demonstrated increased proliferation in the Met-1 tumors in MKR mice by evaluation of protein extracts for proliferating cell nuclear antigen (PCNA) on western blot (Fierz 2010). To examine whether this increase in S phase growth was inhibited with NVP-BKM120, the mice were injected with 5-bromo-2’-deoxyuridine (BrdU) 2 hours prior to tumor tissue collection. The percent of cells that stained positive for BrdU in the Met-1 tumors was evaluated by immunofluorescence. We found a significantly higher percent of BrdU positive cells in the vehicle treated MKR mice (9.9 ±0.76%), compared to vehicle treated WT mice (7.8±0.35%) (Figure 3A). Treatment of the MKR mice with NVP-BKM120 led to a significantly lower percent of BrdU positive cells (5.8±1.13%), compared to vehicle treated MKR mice (P=0.001). The percent of BrdU positive cells in the Met-1 tumors from the MKR mice treated with NVP-BKM120 was comparable to the percent of BrdU positive cells in the WT NVP-BKM120 treated group (5.8±0.63%). Therefore, in the MKR mice, inhibiting PI3K with NVP-BKM120 attenuated the increased cellular proliferation in Met-1 tumors from the MKR mice (Figure 3B). Western blot analysis of apoptosis, using the antibody to the anti-apoptotic protein Bcl-2 revealed decreased levels of Bcl-2 when normalized to β actin, in the MKR vehicle treated group, compared to the MKR NVP-BKM120 treated group (data not shown).

Bottom Line: NVP-BKM120 treatment decreased phosphorylation of Akt and S6 ribosomal protein (S6rp); no change in Erk1/2 phosphorylation was seen.Therefore, we examined whether the oral PI3K/mTOR inhibitor NVP-BEZ235 augmented the tumor suppressing effects of PI3K inhibition.We also investigated the effect of targeted PI3K/mTOR inhibition on PI3K/Akt/mTOR and Erk1/2 signaling, and the potential effects on glycemia.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Diabetes and Bone Disease, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Women with type 2 diabetes mellitus (T2DM) are at a greater risk of developing and dying from breast cancer than women without T2DM. Insulin resistance and hyperinsulinemia underlie the pathogenesis of T2DM. In the MKR mouse model of insulin resistance, we have previously shown increased activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway in association with accelerated mammary tumor growth. In this study, we demonstrate that inhibiting PI3K with the oral pan-class I PI3K inhibitor, NVP-BKM120 reduced the growth of Met-1 and MCNeuA mammary tumor orthografts in the MKR mouse. NVP-BKM120 treatment decreased phosphorylation of Akt and S6 ribosomal protein (S6rp); no change in Erk1/2 phosphorylation was seen. Hyperglycemia, hypertriglyceridemia and greater hyperinsulinemia developed in the MKR mice treated with NVP-BKM120. We previously reported reduced tumor growth using intraperitoneal rapamycin in the MKR mouse, with the development of hyperglycemia and hypertriglyceridemia. Therefore, we examined whether the oral PI3K/mTOR inhibitor NVP-BEZ235 augmented the tumor suppressing effects of PI3K inhibition. We also investigated the effect of targeted PI3K/mTOR inhibition on PI3K/Akt/mTOR and Erk1/2 signaling, and the potential effects on glycemia. NVP-BEZ235 suppressed the growth of Met-1 and MCNeuA tumor orthografts, and decreased Akt and S6rp phosphorylation, despite increased Erk1/2 phosphorylation in Met-1 orthografts of MKR mice. Less marked hyperglycemia and hyperinsulinemia developed with NVP-BEZ235 than NVP-BKM120. Overall, the results of this study demonstrated that inhibiting PI3K/Akt/mTOR signaling with the oral agents NVP-BKM120 and NVP-BEZ235 decreased mammary tumor growth in the hyperinsulinemic MKR mouse. Inhibiting PI3K alone led to more severe metabolic derangement than inhibiting both PI3K and mTOR. Therefore, PI3K may be an important target for the treatment of breast cancer in women with insulin resistance. Monitoring for hyperglycemia and dyslipidemia should be considered when using these agents in humans, given the metabolic changes detected in this study.

Show MeSH
Related in: MedlinePlus