Sphingosine kinase (SphK) 1 and SphK2 play equivalent roles in mediating insulin's mitogenic action - PubMed (original) (raw)

Sphingosine kinase (SphK) 1 and SphK2 play equivalent roles in mediating insulin's mitogenic action

Lan Dai et al. Mol Endocrinol. 2014 Feb.

Abstract

Insulin, an established mitogen that promotes breast cancer cell growth, has been implicated in the link between obesity and an increased risk of breast cancer. However, the current understanding of signaling pathways that mediate the mitogenic action of insulin remains incomplete. Here we provide the first evidence that insulin is capable of activating both sphingosine kinase (SphK) 1 and SphK 2, two isoenzymes that often exhibit opposing effects in the regulation of cell survival and growth. Insulin stimulates the phosphorylation of both SphK1 and SphK2 in a similar time- and dose-dependent manner. Interestingly, both isoenzymes are responsible equally for insulin-induced cell cycle progression and proliferation of MCF7 breast cancer cells, although SphK1 and SphK2 display different roles in mediating insulin-induced ERK1/2 and Akt activation. Moreover, the sphingosine 1-phosphate receptor 3, a key component of the SphK signaling system, is important for insulin-mediated mitogenic action in breast cancer cells. Furthermore, insulin receptor and type 1 IGF receptor (IGF1R) are responsible for the insulin-promoted mitogenic action on MCF7 breast cancer cells. Notably, IGF1R mediates insulin-stimulated phosphorylation of both SphK1 and SphK2, whereas insulin receptor is involved only in SphK1, but not SphK2, activation. Collectively the current study illustrates a new signaling system controlling the mitogenic action of insulin in breast cancer cells, suggesting a new strategy that pharmaceutically targets both isoenzymes of SphK for the management of breast cancer.

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Figures

Figure 1.

Figure 1.

Inhibition of SphK by SKI-II suppresses insulin-induced breast cancer cell proliferation. MCF7 (A), MDA-MB-231 (B), and MCF10A cells (C) were serum starved and treated with insulin (10 nM) in the presence or absence of SKI-II (2.5 μM) for 72 hours. Cell growth was measured using the MTS assay. D, Serum-starved cells were pretreated with 2.5 μM SKI-II for 2 hours, followed by stimulation with or without 10 nM insulin for 24 hours. The percentage of cells in synthesis phase (S phase) was measured by flow cytometry. Ctrl, Control. E, MCF7 cells were plated at 1000 cells/well in six-well plates and serum starved for 24 hours. Cells were then incubated with insulin (10 nM) with or without SKI-II (1 μM) in DMEM medium supplemented with 1% fetal bovine serum. Colonies were counted after 14 days. Data are mean ± SD. *, P < .05 vs control; #, P < .05 vs insulin alone.

Figure 2.

Figure 2.

Insulin activates SphK1 and SphK2 in MCF7 breast cancer cells. A, Total SphK activity was determined in MCF7 cells treated with 10 nM insulin for 15 minutes. B, Serum-starved MCF7 cells were treated with 10 nM insulin for the indicated time. Activation of SphK1 and SphK2 was determined by immunoblotting with specific antiphosphorylated SphK1 (pSphK1) or pSphK2 antibodies. *, Nonspecific bands. Densitometric analysis of pSphK1 and pSphK2 (normalized to total SphK1 or SphK2) are shown in bottom. C, Serum-starved MCF7 cells were treated with increasing doses of insulin as indicated. Levels of pSphK1 and pSphK2 were determined after 15 minutes and 10 minutes of stimulation, respectively. Bottom panel shows densitometric analysis of pSphK1 and pSphK2 (normalized to total SphK1 or SphK2). Data are mean ± SD. *, P < .05 vs control.

Figure 3.

Figure 3.

Insulin-induced phosphorylation of SphK1 and SphK2 is ERK dependent. MCF7 cells were serum starved for 24 hours, washed, and pretreated with 5 μM U0126 for 2 hours and then stimulated without or with 10 nM insulin for 15 minutes. Levels of phosphorylated (pSphK1) (A) and pSphK2 (B) were then determined by Western blot assays. *, Nonspecific bands. Bottom panel shows densitometric analysis of pSphK1 and pSphK2 (normalized to SphK1 and SphK2, respectively) corresponding to the bands shown in the Western blots. Data are mean ± SD. *, P < .05.

Figure 4.

Figure 4.

The effect of SphK1 and SphK2 on insulin-induced cell proliferation. MCF7 cells were transfected with siRNAs targeting SphK1 and/or SphK2 and control siRNA as indicated. A, Levels of SphK1 and SphK2 mRNA were determined by quantitative RT-PCR after 24 hours of transfection and normalized to GAPDH mRNA levels. Data are expressed as the change with respect to control siRNA. The right panel shows PCR products separated on agarose gels. B, SphK activity was measured in the siRNA transfected MCF7 cells treated with insulin (10 nM) for 15 minutes. C and D, The siRNA transfected MCF7 cells were labeled with [3H]sphingosine (1.5 μM, 0.4 μCi) in serum-free medium for 10 minutes and washed extensively. [3H]S1P formation in cells (C) and released into the medium (D) was measured in the radioactively labeled cells treated with or without insulin (10 nM) for 30 minutes. E, MCF7 cells were transfected with the indicated siRNAs, followed by stimulation with or without insulin (10 nM) for 24 hours. The percentage of cells in the S phase was measured by flow cytometry. F, Cell growth was measured using MTS assays in the siRNA-transfected cells exposed to insulin (10 nM) for 72 hours. G, Phosphorylated ERK (pERK) and pAkt were determined by immunoblot assays in the siRNA-transfected MCF7 cells treated with insulin (10 nM) for 30 minutes. Right panels show densitometric analysis of pERK and pAkt (normalized to total ERK or Akt) corresponding to the bands shown in the Western blots. Data are mean ± SD. *, P < .05 vs control/−insulin; #, P < .05 vs control/+insulin.

Figure 5.

Figure 5.

The effect of S1P3 on insulin-induced cell proliferation. A, MCF7 cells were transfected with control or S1P3-siRNA as indicated. Levels of mRNA were determined by quantitative RT-PCR after 24 hours of transfection. Top panels, the PCR products were separated on an agarose gel. Bottom panels, S1P3 mRNA levels were normalized to GAPDH mRNA. Data are expressed as the change with respect to control siRNA. Ctrl, Control. B, MCF7 cells were transfected with the indicated siRNAs, followed by stimulation with or without insulin (10 nM) for 24 hours. Percentage of cells in the S phase was measured by flow cytometry. C, The siRNA-transfected cells were incubated with insulin (10 nM) for 72 hours. Cell growth was measured using MTS assays. D, Left panels, Cells were transfected with the indicated siRNAs and stimulated with or without insulin (10 nM) for 30 minutes. Levels of phosphorylated Akt and ERK were determined by immunoblotting. Right panels, Densitometric analysis of pERK and pAkt (normalized to ERK or Akt) corresponding to the bands shown in the Western blots. Data are mean ± SD. *, P < .05 vs control/−insulin; #, P < .05 vs control/+insulin.

Figure 6.

Figure 6.

The effects of IGF1R and IR on insulin-induced SphK activation. A, MCF7 cells were transfected with control, IGF1R, or IR-siRNA as indicated. Top panels, Total cell lysates were assayed for the expression of the indicated proteins by Western blotting. Bottom panel, Densitometric analysis of the blots (normalized to actin expression levels) from at least three independent experiments. Ctrl, Control. Levels of pSphK1 (B) and pSphK2 (C) were determined by Western blot assays in MCF7 cells transfected with IGF1R, IR, or control siRNA, followed by stimulation without or with 10 nM insulin. *, Nonspecific bands. Bottom panels show densitometric analysis of pSphK1 and pSphK2 (normalized to total SphK1 and SphK2, respectively). Data are mean ± SD. *, P < .05.

Figure 7.

Figure 7.

The role of IR and IGF1R in cell proliferation induced by insulin. A, MCF7 cells were transfected with the indicated siRNAs, followed by stimulation without or with 10 nM insulin for 24 hours. Percentage of cells in the S phase was measured by flow cytometry. Ctrl, Control. B, The siRNA-transfected cells were incubated with insulin (10 nM) for 72 hours, and then cell growth was measured using MTS assays. C, Cells were transfected with the indicated siRNAs and stimulated without or with 10 nM insulin for 30 minutes. Levels of phosphorylation of Akt (pAkt) and ERK1/2 (pERK1/2) were determined by immunoblotting. Densitometric analysis of pERK and pAkt (normalized to ERK or Akt) are shown in bottom. Data are mean ± SD. *, P < .05.

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