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

Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

“…Introduction of an auxin inducible degradation (AID) sequence at the N-terminus of the IRS2 gene provided rapid and reversible control of IRS2 protein degradation to investigate the impact of inducible reduction of IRS2 expression on cellular function. Our studies demonstrate that acute loss of IRS2 expression reduces CSC numbers, confirming the role of IRS2 in the regulation of CSC function that was observed upon chronic knockout or knockdown of IRS2 (12). Knock-in of mNeon-green at the N-terminus facilitated the analysis of endogenous IRS2 expression and localization.…”

“…3-IAA treatment induced the degradation of IRS2 in both SUM-159-DFN and MDA-MB-231-DFN cells in a time dependent manner, with IRS2 levels significantly diminished after 6 hours and reaching a plateau by 24 hours (Fig. 3A A role for IRS2 in cancer stem cell (CSC) regulation was established in our previous studies using cells chronically lacking IRS2 expression through Cre-lox recombination or CRISPR/Cas9-mediated gene knockout (12). To evaluate the impact of acute reduction of IRS2 expression on CSC function, in vitro limiting dilution assays were performed to quantify CSCs in the population after exposure to auxin to induce IRS2 degradation (12).…”

“…3A A role for IRS2 in cancer stem cell (CSC) regulation was established in our previous studies using cells chronically lacking IRS2 expression through Cre-lox recombination or CRISPR/Cas9-mediated gene knockout (12). To evaluate the impact of acute reduction of IRS2 expression on CSC function, in vitro limiting dilution assays were performed to quantify CSCs in the population after exposure to auxin to induce IRS2 degradation (12). Parental and DFN-IRS2 SUM-159 cells were treated with 3-IAA for 24 hrs prior to performing in vitro limiting dilution assays.…”

“…In contrast, IRS2 expression is elevated in more aggressive subtypes such as Basal/Triple Negative Breast Cancer (TNBC) and the localized expression of IRS2 is associated with reduced overall survival of breast cancer patients (8,9). Irs2 contributes to both initiation of primary tumor growth and establishment of secondary metastases through regulation of CSC function and invasion in response to IIS (10)(11)(12)(13). In contrast, loss of Irs1 expression enhances metastatic rates (14).…”

“…Much of the understanding about how the IRS proteins impact tumor cell functions has come from studies of cell lines in which IRS expression has been stably knocked down by shRNA or knocked out by CRISPR/Cas9 mediated gene targeting and restoration of exogenously expressed WT and mutant proteins (11)(12)(13). These studies have provided important information about these proteins, but a rigorous understanding of endogenous protein localization in living cells and the functional impact of acute loss of protein expression is lacking.…”