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. 2022 Dec 6;41(10):111759.
doi: 10.1016/j.celrep.2022.111759.

The insulin and IGF signaling pathway sustains breast cancer stem cells by IRS2/PI3K-mediated regulation of MYC

Ji-Sun Lee  1 , Michael W Lero  1 , Jose Mercado-Matos  1 , Sha Zhu  1 , Minjeong Jo  1 , Claire E Tocheny  1 , Jennifer S Morgan  1 , Leslie M Shaw  2
Affiliations

The insulin and IGF signaling pathway sustains breast cancer stem cells by IRS2/PI3K-mediated regulation of MYC

Ji-Sun Lee et al. Cell Rep. .

Abstract

Despite the strong association of the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway with tumor initiation, recurrence, and metastasis, the mechanism by which this pathway regulates cancer progression is not well understood. Here, we report that IIS supports breast cancer stem cell (CSC) self-renewal in an IRS2-phosphatidylinositol 3-kinase (PI3K)-dependent manner that involves the activation and stabilization of MYC. IRS2-PI3K signaling enhances MYC expression through the inhibition of GSK3β activity and suppression of MYC phosphorylation on threonine 58, thus reducing proteasome-mediated degradation of MYC and sustaining active pS62-MYC function. A stable T58A-Myc mutant rescues CSC function in Irs2-/- cells, supporting the role of this MYC stabilization in IRS2-dependent CSC regulation. These findings establish a mechanistic connection between the IIS pathway and MYC and highlight a role for IRS2-dependent signaling in breast cancer progression.

Keywords: CP: Cancer; CSC; IGF; IRS2; MYC; PI3K; breast cancer; cancer stem cell; insulin; insulin receptor substrate; insulin-like growth factor.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. IIS pathway activation increases breast cancer stemness
(A) Mammosphere assays were performed with SUM-159 cells in the presence of B27 supplement containing insulin (B27+) or without insulin (B27−). Scale bar, 200 μm. (B and C) Mammosphere assays (B) and in vitro limiting-dilution assays (C) were performed with SUM-159 cells in B27+, B27−, or B27− medium supplemented with individual ligands (50 ng/mL). (D–F) SUM-159 cells treated with non-targeting guide RNA (sgNT), IR knockout (KO) cells (IR−/−), or IGF1R KO cells (IGF1R−/−) were assayed for mammospheres in B27+ supplement (D), ALDH activity (E), and tumor initiation by in vivo limiting-dilution assay (F). For (E), the ALDH inhibitor DEAB was included as a negative control. For (F), cells were injected into the mammary fat pads of NCG mice in 10-fold serial dilutions at the concentrations indicated. For (C) and (F), data are presented as a log-log plot, and the frequency of stem cells is calculated by extreme limiting-dilution analysis. The mammosphere data shown represent the mean ± SD of a representative experiment performed three times independently. The ALDH data shown represent the mean ± SD of six independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.
Figure 2.
Figure 2.. IRS2 supports self-renewal of CSC in a PI3K-dependent manner
(A and B) IRS1−/−, IRS2−/− SUM-159 cells (A) and Irs1−/−, Irs2−/− PyMT cells (B) expressing empty vector (EV), IRS1, or IRS2 were assayed for mammospheres in B27+ supplement. (C) Irs1−/−, Irs2−/− PyMT cells expressing EV or Irs2 were assayed for mammospheres with B27+ or B27− supplement. (D and E) IRS2−/− SUM-159 cells (D) or Irs2−/− PyMT cells (E) were assayed for mammospheres in B27+ supplement. (F and G) ALDH activity was examined in SUM-159 cells (F) and PyMT cells (G). The ALDH inhibitor DEAB was included as a negative control. (H) Irs2fl/fl PyMT and Irs2−/− PyMT cells were injected into the mammary fat pads of NOD/SCID mice in 10-fold serial dilutions. (I and J) IRS1−/−, IRS2−/− SUM-159 cells (I) and Irs1−/−, Irs2−/− PyMT cells (J) expressing EV, Irs2-WT (WT), or Irs2-Y5F (Y5F) were assayed for mammospheres in B27+ supplement. (K and L) IRS1−/−, IRS2−/− SUM-159 cells (K) and Irs1−/−, Irs2−/− PyMT cells (L) expressing EV, Irs2-WT, or Irs2-Y5F were assayed for ALDH activity. (M) Irs1−/−, Irs2−/− PyMT cells expressing EV, Irs2-WT, or Irs2-Y5F were injected into the mammary fat pads of NOD/SCID mice in 10-fold serial dilutions. For (H) and (M), data are presented as a log-log plot, and the frequency of stem cells is calculated by extreme limiting-dilution analysis. The mammosphere data shown represent the mean ± SD of a representative experiment performed three times independently. The ALDH data shown represent the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figures S2 and S3.
Figure 3.
Figure 3.. IRS2/PI3K/ GSK3β axis sustains MYC expression
(A and B) Relative expression of MYC protein in IRS2−/− SUM-159 cells (A) or IRS2−/− SUM-159 cells with restored IRS2 expression (B). (C and D) Relative levels of MYC expression and pS62-MYC phosphorylation in MG132-treated IRS2−/− SUM-159 cells (C) or IRS1−/−, IRS2−/− SUM-159 cells with restored IRS2 expression (D). (E) IRS1−/−, IRS2−/− SUM-159 cells expressing IRS2 were serum starved and stimulated with each ligand (50 ng/mL) for the time periods indicated. (F) Irs1−/−, Irs2−/− PyMT cells expressing Irs2-WT were stimulated with insulin (500 ng/mL) for the time periods indicated. (G) Irs1−/−, Irs2−/− PyMT cells expressing Irs2-WT or Irs2-Y5F were stimulated with IGF1 (50 ng/mL) for the time periods indicated. The data shown in all graphs represent the mean ± SD of three or more independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.
Figure 4.
Figure 4.. MYC-mediated IRS2 regulation of breast cancer stemness
(A) ALDH activity of IRS1−/−, IRS2−/− SUM-159 cells expressing EV or IRS2 after treatment with the Myc inhibitor 10074-G5. The ALDH inhibitor DEAB was included as a negative control. (B) IRS1−/−, IRS2−/− SUM-159 cells expressing EV or IRS2 were assayed for mammospheres in the absence or presence of 10074-G5 in B27+ supplement. (C) Irs2−/− PyMT cells expressing EV, Myc-WT, and Myc-T58A were analyzed for mammospheres in B27+ supplement. (D) In vitro limiting-dilution assay of Irs2−/− PyMT cells expressing EV, Myc-WT, and Myc-T58A. (E) Model for IRS2/PI3K-dependent regulation of breast cancer stemness. IRS2/PI3K mediates IIS-induced inactivation of GSK3β through phosphorylation on serine 9. Inactive pS9-GSK3β lacks the ability to phosphorylate threonine 58 of MYC, thus blocking proteasome-mediated degradation of MYC and allowing active pS62-MYC to accumulate. Created with BioRender.com. The mammosphere data shown in all graphs represent the mean ± SD of a representative experiment performed three times independently. The ALDH data shown represent the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.
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