KRAS and YAP1 converge to regulate EMT and tumor survival

Abstract

Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

Figure 1. Systematic identification of genes that rescue loss of viability induced by KRAS suppression

(A) Schematic diagram of an arrayed format screen to identify ORFs that rescue loss of cell viability induced by suppression of KRAS in KRAS-dependent cells. (B) Suppression of KRAS in HCTtetK cells, and rescue by KRAS ORF. Data represent mean ± SD normalized to cell viability in untreated conditions. (C) Distribution of scores for all screened genes averaged across 3 replicates. KRAS rescue score indicates SD from mean of negative control wells. Red line, 3 SD. Blue, gene ‘hits.’ (D) Characterization of 147 hits by in-cell western of ERK and S6 phosphorylation. Each point represents average of duplicate wells. Lines indicate 2SD above mean of negative controls. Gray, negative controls. See also Figure S1 and Table S1 and Table S2.

Figure 2. YAP1 rescues KRAS mutant cancer cells in vitro

(A) Morphology of HCTtetK cells expressing the indicated vectors at 20x magnification. The indicated ORFs were expressed and cells were treated with doxycycline (KRAS suppressed). (B) Viability of HCTtetK cells upon KRAS suppression in cells expressing the indicated genes, normalized to cell viability in media condition. (C) Consequences of expressing YAP1 in KRAS-mutant cell lines after KRAS suppression. Viability of shKRAS normalized to shLuciferase in the presence of each indicated ORF. (D) Response of HCT116 cells to MYC suppression in cells that express the indicated ORFs. (E) Effect of YAP1 suppression on anchorage independent growth of HA1E transformed with KRAS^G13D or YAP1 ORF. (F) Effect of doxycycline-induced KRAS suppression on activation of ERK, AKT, and S6 in HCTtetK cells expressing LacZ, KRAS, or YAP1. (G) Effect of a PI3K inhibitor (PI3Ki; GDC-0941) or a MEK inhibitor (MEKi; AZD-6244) on the ability of YAP1 to rescue KRAS suppression. Cells were treated with 1uM of GDC-0941, 1uM of AZD-6244, both, or DMSO. Data normalized to viability of cells without KRAS suppression (media) with DMSO treatment. (B–E, G) Mean ± SD of at least 3 replicates in a representative experiment shown. See also Figure S2.

Figure 3. Structure-function analysis of YAP1

(A) YAP1 domain structure and YAP1 mutants. (B) Effects of expressing YAP1 TEAD-defective mutants on the activity of a TEAD reporter in 293T cells. (C) Viability after KRAS suppression in HCTtetK cells expressing YAP1 mutants defective in TEAD activation. (D) Viability, in arbitrary luminescence units (ALU), after KRAS suppression in HCTtetK cells expressing a constitutively active TEAD2-VP16 fusion. (E) Effects of expressing the YAP1 mutants defective in transcriptional activation or nuclear localization in HCTtetK cells after KRAS suppression. **P-value <0.01. (B–E) Mean ± SD of 6 replicates of a representative experiment shown. (C–E) Viability of doxycycline treated relative to untreated samples displayed. See also Figure S3.

Figure 4. YAP1 and KRAS converge to regulate the AP-1 transcription factor FOS

(A) Genes rescued by YAP1, KRAS, or both, in the context of KRAS suppression. (B) Categories of transcription factor motifs enriched amongst genes rescued by both KRAS and YAP1. (C) AP-1 reporter activity in 293T cells expressing YAP1. Arbitrary luminescence units normalized to LacZ condition. (D) Expression of FOS rescues suppression of KRAS in HCTtetK cells. Viability of doxycycline treated relative to untreated samples displayed. (E) Effects of suppressing FOS on YAP1-mediated cell transformation of HA1E cells. (C–E) Mean ± SD of at least 3 replicates in a representative experiment shown. See also Figure S4 and Table S3.

Figure 5. YAP1 regulates the epithelial-mesenchymal transition (EMT)

(A) Enriched gene sets rescued by both YAP1 and KRAS. (B) qRT-PCR validation of EMT regulation by KRAS and YAP1 in HCT116 cells. Data represent mean +/− SD of four replicates relative to LacZ control. (C) Viability of doxycycline treated relative to untreated HCTtetK cells expressing Slug and Snail. (D) Effects of suppressing MYC after expressing FOS, Slug, and Snail in HCT116 cells. Viability of shMYC normalized to shLuciferase control in the presence of each indicated ORF. (E) Effect of Slug on the ability of YAP1 to rescue KRAS suppression. Viability of doxycycline-treated HCTtetK cells expressing YAP1 after expression of each indicated shRNA, normalized to media-treated shLuciferase control. (B–E) Mean ± SD of at least 3 replicates in a representative experiment shown. See also Figure S5.

Figure 6. YAP1 and FOS interact at promoter regions to regulate EMT

(A) Co-immunoprecipitation using control antibody or target-specific antibody for YAP1 and FOS in lysates from HCT116 cells expressing YAP1 and V5-tagged FOS. Binding of the reciprocal protein was assessed by immunoblotting. YAP1 indicated by arrowhead. *, IgG heavy and light chains. MW, molecular weight in kDa. (B) mRNA expression of Vimentin (VIM) and Slug after FOS suppression. Mean ± SD of four replicates relative to shLuciferase control in HCT116 cells shown. (C) Chromatin immunoprecipitation in HCT116 to assess YAP1 DNA binding at promoter regions of SLUG (SNAI2) and Vimentin (VIM). (D) Chromatin immunoprecipitation in HCT116 to assess YAP1 binding at SLUG promoter after FOS suppression. (C,D) Bars represent enrichment of promoter compared to 3′ region of each gene. Mean ± SD of 3 replicates shown. *P-value <0.05. See also Figure S6 and Table S4.

Figure 7. Yap1 activity is required for endogenously acquired KRAS resistance in vivo

(A) Schematic of mouse transplant model of KRAS-driven lung cancer. Kras^G12D;p53^fl/fl lung adenocarcinoma cells were infected with retroviral vectors expressing rtTA3, luciferase and a tet-on shKras. Cells were transplanted into recipient mice by tail vein injection. 7 d later, mice were fed a doxycycline diet to induce shKras in tumor cells (D0). (B) Time course of tumor regression and relapse after Kras suppression. Mean ± SD shown. N=3 off dox and N=10 on dox. (C) Suppression of Kras in tumor tissue. Kras mRNA was measured by qRT-PCR in microdissected lung tumors after the indicated days of doxycycline treatment. (D) Enrichment of a published YAP1 signature (Dupont et al., 2011) after 21 d doxycycline treatment versus untreated cells. (E) Enrichment of a published EMT signature (Taube et al., 2010) after 21 d doxycycline treatment versus untreated cells. (F) Yap1 localization in tumors that escape Kras suppression. Immunohistochemistry was performed with Yap1 antibody on frozen tissue sections from tumors which developed after Kras suppression (dox on) for 21 d and tumors which formed with continued Kras expression (dox off). (G) Tumor response to suppression of Kras in combination with Yap1 or control suppression. Mean ± SD shown. See also Figure S7 and Table S5.