Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST - PubMed (original) (raw)

Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

Kassondra Meyer et al. Mol Cell Biol. 2015.

Abstract

Loss of repressor element 1 silencing transcription factor (REST) occurs in 20% of breast cancers and correlates with a poor patient prognosis. However, the molecular basis for enhanced malignancy in tumors lacking REST (RESTless) is only partially understood. We used multiplatform array data from the Cancer Genome Atlas to identify consistent changes in key signaling pathways. Of the proteins screened in the reverse-phase protein array, we found that insulin receptor substrate 1 (IRS1) is the most highly upregulated protein in RESTless breast tumors. Analysis of breast tumor cell lines showed that REST directly represses IRS1, and cells lacking REST have increased levels of IRS1 mRNA and protein. We find that the upregulation of IRS1 function is both necessary and sufficient for enhanced signaling and growth in breast cancer cells lacking REST. IRS1 overexpression is sufficient to phenocopy the enhanced activation of the signaling hubs AKT and mitogen-activated protein kinase (MAPK) of MCF7 cells lacking REST. Loss of REST renders MCF7 and MDA-MB-231 breast tumor cells dependent on IRS1 activity for colony formation in soft agar. Inhibition of the type 1 insulin-like growth factor receptor (IGF1R) reduces the enhanced signaling, growth, and migration in breast tumor cells that occur upon REST loss. We show that loss of REST induces a pathogenic program that works through the IGF1R/IRS1 pathway.

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Figures

FIG 1

Protein expression in tumors lacking REST. (A) ER+ tumors were subjected to unsupervised hierarchical clustering using Euclidean distance with the 24-gene RESTless gene signature. The heat map shows a group of tumors clustered together that exhibited high expression levels of the majority of RESTless signature genes (labeled). (B) GSEA on the above-mentioned tumors in TCGA demonstrates a robust enrichment of REST target genes in those samples labeled “RESTless” (P < 10−3; false discovery rate [_q_] < 10−3; enrichment score [ES] = 0.466; normalized ES [NES] = 2.258). (C) Interaction network of upregulated proteins (displayed by EMBL STRING as gene symbols). The confidence view (left) shows stronger associations by thicker lines. Interactions in the interaction view (right) are indicated as follows: red lines, inhibitory; blue lines, binding; yellow lines, coexpressed; yellow dots, directionality of interaction with unknown action. (D) Schematic of MEK/ERK and PI3K/AKT pathways downstream of IRS1.

FIG 2

Loss of REST leads to increased IRS1 protein levels. (A) Western blot images showing REST and IRS1 expression in MCF7 (ER+), MDA-MB-231 (ER−), and ZR751 (ER+) cells. (B) Quantifications of relative protein expression depicted in a graph (for shRNA set 1), expressed as fold changes compared to levels in RESTnorm cells (n = 3).

FIG 3

REST directly represses IRS1. (A) Primer sites flanking the RE-1 site positioned 12.4 kb upstream of the IRS1 promoter were used for ChIP and RE-1 reporter assays. (B and C) ChIP of REST at GAPDH, BDNF, and IRS1 in MCF7 (B) and MDA-MB-231 (C) cells (n = 3). (D) Luciferase reporter assay with pGL3Pro-IRS1RE1 (RE-1 site) or pGL3Pro (empty) transfected in MCF7 cells. A two-way ANOVA was used for statistical analysis (n = 3) (***, P = 0.0008; n.s., not significant). (E to G) IRS1 mRNA levels are higher in RESTlow MCF7 (n = 15) (E), MDA-MB-231 (n = 3) (F), and ZR751 (n = 6) (G) breast cancer cells. Data were analyzed with an unpaired, two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Induced REST expression represses IRS1 mRNA in MDA-MB-231 cells but not in MCF7 cells. (A) REST and IRS1 mRNAs extracted from MDA-MB-231 cells expressing Tet-inducible REST. Significance was determined by using a t test (n = 3). (B) Western blotting of MDA-MB-231 cells expressing Tet-REST treated with 100 μg/ml doxycycline for 24 h. (C) ChIP of REST or IgG to determine REST occupancy at IRS1 in Tet-induced REST MDA-MB-231 cells treated with doxycycline (n = 3). (D) Same as panel A but with MCF7 cells (n = 3). (E) Western blot of MCF7 cells expressing Tet-REST treated with 100 μg/ml doxycycline for 24 h. (F) Same as panel C but with MCF7 cells (n = 3).

FIG 5

Cells lacking REST have enhanced signaling in response to IGF. Shown are Western blot images from lysates of RESTnorm and RESTlow cells treated with IGF for 10 min for MCF7 cells (A) or for 2 h for MDA-MB-231 cells (B) (n = 3).

FIG 6

Heightened signaling of PI3K/AKT and MAPK/ERK is required for enhanced growth of RESTlow cells. (A) Inhibitors of the IGF1R/IRS1 pathway. (B and C) Soft-agar growth with inhibitor treatment in RESTnorm and RESTlow MCF7 (n = 27) (B) or MDA-MB-231 (n = 18) (C) cells. A two-way ANOVA was used to determine significance. Relative colony numbers are expressed as the proportion of colonies compared to that of DMSO-treated RESTnorm cells. Significance is annotated relative to DMSO treatment. *, P < 0.05; **, P < 0.005; ***, P < 0.001; †, P < 0.0001. (D) Representative pictures of colonies from panels B and C. (E and F) Western blotting of MCF7 cells (n = 2) (E) or MDA-MB-231 cells (n = 3) (F) treated with OSI-906 for 30 min prior to stimulation with IGF. (G) Quantitation of pERK levels from OSI-906 and IGF treatments of MDA-MB-231 cells (n = 3).

FIG 7

Overexpression of IRS1 is sufficient to enhance signaling and growth in soft agar. (A) Western blot showing overexpression of IRS1-HA in MCF7 cells (pLNCX-IRS1-HA) alongside an empty vector control (pLNCX2-control). (B) Western blot of MCF7-IRS1 cells treated with IGF. (C) Soft-agar assay with MCF7-IRS1 cells and RESTlow MCF7 cells with OSI-906 treatment. Significance was determined by two-way ANOVA (n = 3). Relative colony numbers are expressed as the proportion of colonies compared to that of DMSO-treated RESTnorm cells (***, P < 0.001; **, P < 0.01; *, P < 0.05).

FIG 8

IRS1 activity is necessary for enhanced signaling and growth in MCF7 and MDA-MB-231 cells. (A) Schematic of protein domains in the IRS1-DN construct compared to full-length IRS1. The domains conserved in the IRS1-DN are the pleckstrin homology (PH) domain and the phosphotyrosine binding (PTB) domain. (B) Western blot showing IRS1-DN with a HA tag or the empty vector (control) stably expressed in MCF7 and MDA-MB-231 cells with or without REST. (C and D) Western blot showing lysates from control or IRS1-DN MCF7 (C) and MDA-MB-231 (D) cells in the presence (+) or absence (−) of REST that were treated with IGF (n = 3). (E and F) Relative colony numbers of RESTnorm or RESTlow MCF7 (n = 6) (E) or MDA-MB-231 (n = 9) (F) cells expressing IRS1-DN with OSI-906 treatment, expressed as the proportion of colonies compared to that of DMSO-treated RESTnorm control cells. Significance was determined by two-way ANOVA. (G and H) siRNA directed to IRS1 or nontargeting siRNA was transfected into cells prior to stimulation with IGF to measure downstream signaling in MCF7 (G) or MDA-MB-231 (H) cells.

FIG 9

Loss of REST confers cell-type-specific phenotypes. (A and B) Relative absorbance of crystal violet stain representing cell numbers for MCF7 (n = 24) (A) and MDA-MB-231 (n = 18) (B) cells, expressed as a signal proportional to the RESTnorm, untreated signal. Significance was determined by two-way ANOVA. ***, P < 0.001; **, P < 0.01; *, P < 0.05. (C and D) Migration of MCF7 (n = 17) (C) and MDA-MB-231 (n = 12) (D) cells with IGF treatment in chemotaxis chambers. A two-way ANOVA was used for statistical analysis. (E) MTT signal in the presence of IGF in RESTnorm or RESTlow cells expressing IRS1-DN (n = 12). Relative absorbance is presented as the signal relative to that with zero treatment of RESTnorm control cells. A two-way ANOVA was used to determine significance (†, P < 0.0001). (F) Migration of MDA-MB-231 cells using 10% FBS as a chemoattractant with or without 20 μM OSI-906 treatment in the upper chamber (n = 6). A two-way ANOVA was used to determine significance. (G) Western blot showing expression of receptors in MCF7 and MDA-MB-231 cells. The IGF1R (long) blot for MDA-MB-231 cells was imaged separately with a longer exposure to observe expression differences. (H) Data from blots from 4 different experiments were compiled and quantified.

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Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST - PubMed (original) (raw)