Modulation of insulin/IGFs pathways by sirtuin-7 inhibition in drug-induced chemoreistance

Abstract

Background: Insulin and insulin-like growth factors (IGFs) are key regulators of metabolism and growth. Recent evidences suggest a key role of these pathways in non-classical tissues and the metabolic pathways by which these hormones exert their effects in neoplasia is unclear.

Aims: To study insulin/IGFs pathways in drug sensitive and resistant cancer cells representing breast cancer (MCF-7), osteosarcoma (SaOS-2), and ovarian cancer (A2780) and to examine the effect of Sirtuin-7 (Sirt7) inhibition on insulin/IGFs pathways in MCF-7 cell line.

Methods: Drug resistant cells were generated by continuous incubation of parental cell lines with stepwise increases in Doxorubicin or Cisplatin over a period of 3 to 6 months. MCF-7 cells were transfected with cloned hairpin siRNA template for Sirt7 using the Amaxa GmbH transfection system. mRNA expression of Sirt7, INSR, IRS-1, IRS-2, IRS-4, IGF-1, IGF-2, MDR-1, MRP-1, BCRP was measured by qPCR and Sirt7 by standard Western blotting. FITC-insulin uptake was imaged with Leica Confocal Microscope.

Results: Insulin receptor (INSR), insulin receptor substrate-1 (IRS-1) were inhibited in drug-induced resistance, whereas IRS-2 was significantly induced in all the chemoresistant cells tested when compared to their parental counterparts. IGF-1 and IGF-2 were also upregulated in all the drug resistant cells tested. Sirt7 was significantly reduced in all chemoresistant cells tested. Knockdown of Sirt7 expression in human breast MCF-7 cell line by siRNA induced premature senescence-like phenotype and multi-drug resistance, suggesting that this gene may play an active role in regulating cancer cell response to stress. Suppression of Sirt7 selectively inhibited INSR and IRS-1, whereas it had minimal effect on that of IRS-2. Sirt7 suppression in MCF-7 also inhibited insulin uptake. Additionally, Sirt7 inhibition upregulated IGF-1, IGF-2 and IGFR expression.

Conclusion: Our data demonstrate that stress-induced Sirt7 inhibition significantly increases stress resistance and modulates insulin/IGF-1 signaling pathways. More importantly, this study links Sir2 family proteins to insulin/IGF signaling in drug-induced stress resistance in neoplasia.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1135426681234493.

Figure 1

mRNA expression of mdr1, MRP1 and BCRP. mRNA expression measured by real time PCR of A) mdr1, B) MRP1 and C) BCRP in drug resistant cells and the respective parental cells normalized to GAPDH mRNA expression. mRNA expression of mdr1 is significantly higher in all Doxorubicin resistant cells (MCF-7 and SaOS2). No significant increase in mdr1 expression by Cisplatin resistant A2780 was observed.

Figure 2

mRNA and protein expression of Sirt7. A) mRNA expression of Sirt7 normalized to GAPDH mRNA expression by RT-PCR, where the expression of Sirt7 is reduced in all chemoresistant cell lines tested (MCF-7, SaOS2, and A2780); B) A representative Western blot for Sirt7 protein expression showing a significant reduction of Sirt7 expression in all drug-resistant cells (MCF-7, SaOS2, and A2780); C) Densitometric quantitation of Sirt7/β-actin protein expression ratio.

Figure 3

mRNA and protein expression of INSR in drug-resistant cell lines. A) mRNA expression of INSR normalized to GAPDH mRNA expression by RT-PCR; B) A representative Western blot for INSR protein expression showing a significant reduction of INSR expression in drug-resistant cell lines (MCF-7, SaOS2, and A2780); C) Densitometric quantitation of INSR/GAPDH protein expression ratio.

Figure 4

mRNA expression levels of IRS-1, IRS-2 and IRS-4. mRNA expression of A) IRS-1; B) IRS-2; and C) IRS-4 in drug resistant and their corresponding parental cells (MCF-7, SaOS2, and A2780).

Figure 5

mRNA expression levels of IGF-1 and IGF-2 in drug resistant and their corresponding parental cells. mRNA expression of A) IGF-1 and B) IGF-2 in drug resistant and their corresponding parental cells (MCF-7, SaOS2, and A2780).

Figure 6

Protein expression of Sirt7 and mRNA expression of insulin/IGF transduction pathway protein following inhibition of Sirt7. A) Protein expression of Sirt7 following inhibition of Sirt7 in MCF-7 by siRNA; B) mRNA expression of Sirt7, INSR, IRS-1, IRS-2, IRS-4, IGF1, IGF2, GAPDH and β-actin in MCF-7 following inhibition of Sirt7 by siRNA.

Figure 7

FITC-conjugated Insulin uptake by MCF-7 following Sirt7 inhibition. A) Representative image for FITC-conjugated Insulin uptake by MCF-7 transfected with either mock or Sirt7 siRNA vectors. Stably transfectcted MCF-7 cells were cultured in chamber slides in 0.1% FBS containing medium overnight. Cells were then incubated with FITC-conjugated Insulin (0 and 1000 nM) for 2 hours and then washed, fixed and analyzed by Leica confocal microscope. Nucleus was counterstained with DAPI (blue) while cytoplasm was counterstained with Rhodamine Phalloidin (red); B) Insulin uptake by MCF-7 Mock transfected and incubated with different FITC-labeled insulin for different time intervals; and C) Insulin uptake by MCF-7 cells transfected with Sirt7 siRNA.