Selective targeting of KRAS-driven lung tumorigenesis via unresolved ER stress

Abstract

Lung cancer with oncogenic KRAS makes up a significant proportion of lung cancers and is accompanied by a poor prognosis. Recent advances in understanding the molecular pathogenesis of lung cancer with oncogenic KRAS have enabled the development of drugs, yet mutated KRAS remains undruggable. We performed small-molecule library screening and identified verteporfin, a yes-associated protein 1 (YAP1) inhibitor; verteporfin treatment markedly reduced cell viability in KRAS-mutant lung cancer cells in vitro and suppressed KRAS-driven lung tumorigenesis in vivo. Comparative functional analysis of verteporfin treatment and YAP1 knockdown with siRNA revealed that the cytotoxic effect of verteporfin was at least partially independent of YAP1 inhibition. A whole-transcriptome approach revealed the distinct expression profiles in KRAS-mutant lung cancer cells between verteporfin treatment and YAP1 knockdown and identified the selective involvement of the ER stress pathway in the effects of verteporfin treatment in KRAS-mutant lung cancer, leading to apoptotic cell death. These data provide novel insight to uncover vulnerabilities in KRAS-driven lung tumorigenesis.

Keywords: Cell Biology; Drug screens; Lung cancer; Oncogenes; Oncology.

Figure 1. Verteporfin inhibits KRAS-mutant lung cancer cell growth.

(A) Scheme of the protocol used to screen 1271 drugs. (B) Scatterplot of the difference in z score between KRAS-mutant cells and WT cells. (C) Quantification of cell viability in KRAS-mutant and WT cells after treatment with verteporfin. The values are the mean ± SD (8 KRAS-mutant cell lines and 8 WT cell lines, each n = 4). Statistical significance was determined using an unpaired 2-tailed Student’s t test. ***, P < 0.001.

Figure 2. Verteporfin exerts apoptotic effects in KRAS-mutant cells in vivo.

(A) Schematic timeline of the animal study. (B) Representative pictures of tumors harvested from KRAS-mutant xenograft mice and WT xenograft mice. Tumor weight was measured at the time of sample collection. The values are the mean ± SD (n = 6). Statistical significance was determined using an unpaired 2-tailed Student’s t test. *, P < 0.05. (C) Representative histology of H&E-stained sections, Ki67-stained sections, and TUNEL-stained sections of tumors from KRAS-mutant xenograft mice and WT mice. Quantification of the percentage of Ki67-positive (D) and percentage of TUNEL-positive (E) cells. The values are the mean ± SD (n = 3). Statistical significance was determined using an unpaired 2-tailed Student’s t test. **, P < 0.01.

Figure 3. Verteporfin treatment and YAP1 knockdown have different effects on cell proliferation and YAP1 expression.

(A) Effects of YAP1 siRNAs on the proliferation of A-549 cells. The values are the mean ± SD. Statistical significance was determined using Dunnett’s multiple-comparison test. *, P < 0.05. (B) Validation experiments using 8 KRAS-mutant cell lines and 8 WT cell lines in which YAP1 was knocked down by siRNAs. The values are the mean ± SD (8 KRAS-mutant cell lines and 8 WT cell lines, each n = 4). Statistical significance was determined using an unpaired 2-tailed Student’s t test. (C) Effects of combination treatment of verteporfin and YAP siRNA knockdown in KRAS-mutant and WT cells. The values are the mean ± SD (each, n = 4). (D) Western blots from A-549 cells after treatment with verteporfin. (E) Western blots from A-549 cells after treatment with YAP1 siRNAs.

Figure 4. A whole-transcriptome analysis in KRAS-mutant lung cancer cells between verteporfin treatment and YAP1 knockdown.

(A) Principal component analysis (PCA) of A-549 cells treated with YAP1 siRNA and verteporfin at various concentrations. (B) Heatmap showing the upregulated and downregulated genes following treatment with YAP1 siRNAs and verteporfin at various concentrations. (C) GSEA of the A-549 cells treated with verteporfin at various concentrations. NES, normalized enrichment score. The P values in the graphs were calculated by GSEA. (D) GSEA of the A-549 cells treated with YAP1 siRNAs. The P values in the graphs were calculated by GSEA. (E) Normalized enrichment scores from GSEA of all hallmark gene sets by following verteporfin treatment. EMT, epithelial-mesenchymal transition. (F) Normalized enrichment scores from GSEA of all hallmark gene sets by YAP1-knockdown experiments.

Figure 5. Verteporfin treatment is associated with the ER stress pathway.

(A) Venn diagram showing upregulated genes and downregulated genes following treatment with YAP1 siRNAs and verteporfin at various concentrations. (B) Correlation of the normalized enrichment scores following verteporfin treatment and YAP1 knockdown from GSEA of all hallmark gene sets. (C) Related pathways and functional annotation of genes specifically altered by verteporfin treatment analyzed by ingenuity pathway analysis (QIAGEN). (D) GSEA to assess apoptotic signaling in response to ER stress in A-549 cells treated with various concentrations of verteporfin. GO, Gene Ontology. The P values in the graphs were calculated by GSEA. (E) Heatmap of leading-edge genes in the ER stress pathway.

Figure 6. Characterization of unresolved ER stress mechanism in KRAS-mutant lung cancer cells by verteporfin treatment.

(A) Relative expression analysis of leading genes in ER stress pathways in KRAS-mutant cells treated with verteporfin or YAP1 knockdown by RNA-Seq. (B) Relative expression analysis of leading genes in ER stress pathways in KRAS-mutant and WT cells treated with verteporfin (control and 10 μM) determined by quantitative reverse transcription PCR. The values are the mean ± SD (3 KRAS-mutant cell lines [A-549, H-23 and H-1573] and 3 WT cell lines [H-1650, H-522 and Calu-3], each n = 3). Statistical significance was determined using an unpaired 2-tailed Student’s t test. *, P < 0.05. (C) Characterization of unspliced XBP1 (uXBP1) and spliced XBP1 (sXBP1) in KRAS-mutant and WT cells treated by verteporfin in various concentrations. The normalization control was performed by total XBP1 (tXBP1). The values are the mean ± SD (n = 3). (D) Effects of combination therapy of verteporfin and TUDCA (ER stress inhibitor) in KRAS-mutant cells. The results of combination therapy of eeyarestatin (ERAD inhibitor) and TUDCA are shown as a control. E, eeyarestatin. The values are the mean ± SD (n = 3). Statistical significance was determined using an unpaired 2-tailed Student’s t test. **, P < 0.01 and ***, P < 0.001. FPKM, fragments per kilobase per million mapped reads.