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Scribble mis-localization induces adaptive resistance to KRAS G12C inhibitors through feedback activation of MAPK signaling mediated by YAP-induced MRAS

Nature Cancer volume 4, pages 829–843 (2023)Cite this article

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Abstract

Tumor cells evade targeted drugs by rewiring their genetic and epigenetic networks. Here, we identified that inhibition of MAPK signaling rapidly induces an epithelial-to-mesenchymal transition program by promoting re-localization of an apical-basal polarity protein, Scribble, in oncogene-addicted lung cancer models. Mis-localization of Scribble suppressed Hippo-YAP signaling, leading to YAP nuclear translocation. Furthermore, we discovered that a RAS superfamily protein MRAS is a direct target of YAP. Treatment with KRAS G12C inhibitors induced MRAS expression, which formed a complex with SHOC2, precipitating feedback activation of MAPK signaling. Abrogation of YAP activation or MRAS induction enhanced the efficacy of KRAS G12C inhibitor treatment in vivo. These results highlight a role for protein localization in the induction of a non-genetic mechanism of resistance to targeted therapies in lung cancer. Furthermore, we demonstrate that induced MRAS expression is a key mechanism of adaptive resistance following KRAS G12C inhibitor treatment.

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Fig. 1: Scrib mis-localization following KRAS G12C inhibition is a cause of resistance to KRAS G12C inhibitors.
Fig. 2: Inhibition of the MAPK pathway induces Scrib mis-localization.
Fig. 3: ZDHHC7 is involved in the regulation of Scrib localization.
Fig. 4: Scrib mis-localization induces nuclear translocation of YAP, leading to adaptive resistance to KRAS G12C inhibitors.
Fig. 5: MRAS is transcriptionally regulated by YAP.
Fig. 6: KRAS G12C inhibition rewires signaling from a Scrib–SHOC2 complex to an MRAS–SHOC2 complex, leading to reactivation of MAPK signaling.
Fig. 7: Membrane-localized MRAS is activated by receptor tyrosine kinases.
Fig. 8: MRAS knockout enhances sotorasib efficacy in vivo.

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Data availability

RNA-sequencing data generated in this study have been deposited in the BioProject database at DNA Data Bank of Japan under accession code DRA014731. Source data for Figs. 3e,f, 4c,h, 5b,f–h, 6e, 7a,b and 8a and Extended Data Figs. 1e,f,h–j, 3b,e,k,l, 6a,c,d, 8a–c and 9a–c,g have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This study is supported by the Fund for the Promotion of Joint International Research from JSPS (15KK0303), P-CREATE from the AMED under grant no. 20cm0106513h0005, Princess Takamatsu Cancer Research Fund and Takeda Science Foundation, Aichi Cancer Center Joint Research Project on Priority Areas (to H.E.) and The Hori Sciences and Arts Foundation (to Y.A.). The super-computing resource was provided by Human Genome Center, Institute of Medical Science, The University of Tokyo (http://sc.hgc.jp/shirokane.html).

Author information

Authors and Affiliations

  1. Division of Molecular Therapeutics, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan

    Yuta Adachi, Ryo Kimura, Kentaro Hirade, Shogo Yanase, Yuki Nishioka, Natsumi Kasuga & Hiromichi Ebi

  2. Division of Cancer Systems Biology, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan

    Rui Yamaguchi

  3. Division of Cancer Informatics, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan

    Rui Yamaguchi

  4. Division of Advanced Cancer Therapeutics, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan

    Hiromichi Ebi

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Contributions

Y.A. and H.E. conceived and designed the project. Y.A. performed most of the experiments. R.K., K.H., Y.N. and S.Y. helped with biochemistry experiments. N.K. helped with animal experiments. R.Y. analyzed the sequencing data. Y.A. and H.E. wrote the manuscript and all authors contributed to the writing and/or critical review of the manuscript.

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Correspondence to Hiromichi Ebi.

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Nature Cancer thanks Sandra Misale and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Scrib mis-localization following KRAS G12C inhibition.

a. The NCI-H358 KRAS G12C mutant lung cancer cell line was treated with 500 nM adagrasib for 48 hours. Representative immunofluorescence images of E-cadherin are shown. Scale bar, 10 μm. b. NCI-H358 cells were treated with 1 μM sotorasib at the indicated time points. Representative immunofluorescence images of Scrib are merged with 4’,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. c. NCI-H358 and LU65 cells were treated with 1 μM sotorasib at the indicated time points. Whole cell extracts (WCE) from untreated cells and cell membrane fractions prepared from each timepoint were blotted with anti-Scrib antibody. CD71 and GAPDH are membrane and cytoplasmic markers, respectively. Western blot was repeated two independent times with similar results. Representative images are shown. d. LU65 cells were transfected with siRNA targeting KRAS or scramble siRNA and cultured for 72 hours. Cells were stained with anti-Scrib or DAPI (blue). Scale bar, 10μm. e. NCI-H358 and LU65 cells were treated with 1 μM sotorasib for 48 or 72 hours and analyzed by FACS to quantify annexin positive cells. The average ± SD of 3 independent experiments is shown. Also, LU65 cells were treated with 1 μM sotorasib for 72 hours or with 5 μM docetaxel for 24 hours. Cells were stained with anti-Scrib (alexa 568), anti-Cleaved PARP (alexa 647), CFSE, a cell permeant molecule used for the fluorescent intracellular labeling of live cells, or DAPI (blue). Scale bar, 10μm. Docetaxel was used as a positive control for induction of cleaved PARP. f. qPCR analysis of mRNA levels of snail and ZEB1 normalized to ubiquitin in NCI-H358 and LU65 cells treated with sotorasib at 1 μM for 48 hours. Data are mean ± SD, n = 3 independently treated cell cultures. g. NCI-H358 single-cell clones with CRISPR-Cas9 KO of Scrib, and introduction of wild-type, P305L mutant, or CAAX-fused Scrib were generated. Each clone and parental cells were lysed and blotted with anti-Scrib antibody. GAPDH is a loading control. h, i. NCI-H358 and LU65 single-cell clones with CRISPR-Cas9 KO of Scrib, or introduction of wild-type or P305L mutant Scrib, were treated with 1 μM sotorasib for 72 hours. Relative cell growth levels were normalized to DMSO-treated cells. All data are represented as mean ± SD, n = 3, independently treated cell cultures (two-sided Student’s t-test, * p < 0.01). j. NCI-H358 or LU65 cells were treated with 1 μM sotorasib for 72 hours. Half of the cells then continued to receive sotorasib with drug replenishment, while treatment was discontinued for the other half. After 72 hours, these cells were re-challenged with sotorasib for 72 hours. Error bars represent mean ± SD, n = 3, independently treated cell cultures. a, b, d, e. Images of immunofluorescence are representative of two independent experiments.

Source data

Extended Data Fig. 2 Inhibition of the driver protein induces Scrib mis-localization in KRAS G12C mutant colorectal cancer, but this is not observed in PI3K-driven breast cancer.

a. NCI-H358 cells expressing cDNA encoding activated AKT (myristoylated AKT (NCI-H358 Myr-AKT)) or MEK (NCI-H358 MEK-DD) were treated with 1 μM sotorasib for 48 hours. Cell membrane fractions were blotted with anti-Scrib and anti-CD71 antibodies. Western blot was repeated two independent times with similar results. Representative images are shown. b. MCF7 (PIK3CA E545K mutant) and T47D (PIK3CA amplified) PI3K-driven breast cancer cell lines were treated with a pan-PI3K inhibitor GDC-0941 at 250 nM for 48 hours and localization of Scrib was determined. c. SW837 and SNU1411 KRAS G12C mutant colorectal cancer cells were treated with 1 μM sotorasib for 48 hours. Representative immunofluorescence images of Scrib are merged with DAPI. Scale bar, 10μm. b, c. Images of immunofluorescence are representative of two independent experiments.

Source data

Extended Data Fig. 3 ZDHHC7 is involved in the regulation of Scrib localization.

a. LU65 cells were treated with 1 μM sotorasib for 48 hours. Representative immunofluorescence images of ZDHHC7 and GM130, a marker for the Golgi apparatus, are shown. Scale bar, 10 μm. b. NCI-H358 cells were treated with 1 μM sotorasib for 48 hours. ZDHHC7 (green) and GM130 (red) were stained and merged. Scale bar, 10μm. The line indicates the profiles taken for the relative fluorescent intensities for ZDHHC7 and GM130. Peaks of fluorescence were identical between ZDHHC7 and GM130. c. The expression of ZDHHC7 in whole cell extracts (WCE), cytoplasm, and nuclear extracts (nucleus) was detected by western blot analysis. Lamin B1 and GAPDH are controls for nuclear and cytoplasmic protein, respectively. d. NCI-H358 and LU65 cells were treated with 1 μM sotorasib for the indicated time points, and lysates were probed with the indicated antibodies. e. qPCR analysis of mRNA levels of ZDHHC7 normalized to ubiquitin in NCI-H358 and LU65 cells treated with 1 μM sotorasib for the indicated time points. Data are mean ± SD, n = 3 independently treated cell cultures. f. NCI-H358 cells were treated with 250 nM GDC-0941 for 48 hours. Representative immunofluorescence images of ZDHHC7 and GM130 are shown. Scale bar, 10μm. g. LU65 cells expressing Myr-AKT or MEK-DD were treated with 1 μM sotorasib for 48 hours. Note that constitutive MEK introduction upregulated ZDHHC7 expression. h. HCC827 and NCI-H2228 cells were treated with 50 nM trametinib for 48 hours. Representative immunofluorescence images of ZDHHC7 and GM130 are shown. Scale bar, 10μm. i. NCI-H358 cells transduced with either control (VC) or ZDHHC7 expression plasmids (OE) were treated with 1 μM sotorasib for 48 hours. Cell membrane fractions were blotted with anti-Scrib and anti-CD71 antibodies, and WCE were blotted with ZDHHC7 and GAPDH. j. NCI-H358 cells were treated with 1 μM sotorasib, 5 μM ML349, or the combination of these drugs for 24 hours. Cell membrane fractions were blotted with anti-Scrib and anti-CD71 antibodies. k. NCI-H358 and LU65 cells were treated with sotorasib, ML349, or the combination of these drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment. Error bars represent mean ± SD, n = 6, independently treated cell cultures. l. NCI-H358 or LU65 single-cell clones with CRISPR-Cas9 KO of Scrib, and introduction of CAAX-fused Scrib were treated with 1 μM sotorasib with or without 5 μM ML349 for 3 days. Relative cell growth levels were normalized to DMSO-treated cells. All data are represented as mean ± SD n = 3, independently treated cell cultures. a, b, f, h. Images of immunofluorescence are representative of two independent experiments. c, d, g, i, j. Western blot was repeated two independent times with similar results. Representative images are shown.

Extended Data Fig. 4 Scrib mis-localization induces nuclear translocation of YAP.

a, b. NCI-H358 or LU65 single-cell clones with CRISPR-Cas9 KO of Scrib, and introduction of wild-type or P305L mutant Scrib, were stained with Scrib (red), YAP (green), and DAPI, and the images were merged. Fluorescence imaging data are representative of three independent biological experiments. Scale bar, 10 μm. c. NCI-H358 and LU65 cells were treated with 500 nM adagrasib for 48 hours and stained as in a. Images of immunofluorescence are representative of two independent experiments. d. NCI-H358 and LU65 cells were treated with 1 μM sotorasib for the indicated time points, and lysates were probed with the indicated antibodies. e. Lysates from NCI-H358 and LU65 cells transduced with control sgRNA or sgYAP/TAZ were probed with the indicated antibodies. f. g. Optical density readings OD590 nm derived from 10% acetic acid elution of the crystal violet staining from Fig. 4d (for f) and 4e (for g). h. NCI-H358 cells were treated with 1 μM sotorasib, 5 μM ML349, or the combination of these drugs for 48 hours. Cells were stained as in a. Scale bar, 5μm. Images of immunofluorescence are representative of two independent experiments. i. In NCI-H358 and LU65 cell lines, single-cell clones with CRISPR-Cas9 KO of Scrib, and introduction of wild-type or CAAX-fused Scrib were treated with 1 μM sotorasib for the indicated time points and lysates were probed with the indicated antibodies. j. NCI-H358 and LU65 cells were treated with 1 μM sotorasib with or without 5 μM ML349 for the indicated time points and lysates were probed with the indicated antibodies. d, e, i, j. Western blot was repeated two independent times with similar results. Representative images are shown.

Extended Data Fig. 5 Scrib mis-localization induces nuclear translocation of YAP in oncogene-addicted lung cancer models.

a. HCC827 and NCI-H2228 cancer cell lines were treated with 100 nM osimertinib, 500 nM alectinib, or 50 nM trametinib for 72 hours as indicated. Representative immunofluorescence images of Scrib (red) and YAP (green) were merged. Scale bar, 10 μm. Images of immunofluorescence are representative of two independent experiments. b. HCC827 and NCI-H2228 cells were treated with 100 nM osimertinib, or 500 nM alectinib with or without 5 μM ML349 for the indicated time points and lysates were probed with the indicated antibodies. Western blot was repeated two independent times with similar results. Representative images are shown.

Extended Data Fig. 6 The combination of Sotorasib with K-975, a TEAD inhibitor, enhances the efficacy of the KRAS G12C inhibitor.

a. NCI-H358 cells were treated with either DMSO, 1 μM sotorasib, 1μM K-975, or the combination of these drugs for 48 hours. qPCR analysis of mRNA levels of CYR61 and ANKRD1 normalized to ubiquitin following treatment is shown. Data are mean ± SD (n = 3, independently treated cell cultures). b. Optical density readings (OD590 nm) derived from 10% acetic acid elution of the crystal violet staining from Fig. 4g. c. NCI-H358 and LU65 cells were treated with sotorasib, K-975, or the combination of these drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment. Error bars represent mean ± SD, n = 6, independently treated cell cultures. d. Xenograft tumors derived from NCI-H358 cells were treated as shown in Fig. 4h. These xenografted tumors were harvested at the time of last treatment (Day 24 for vehicle, 38 for K-975, and 66 for sotorasib and the combination of these drugs). mRNA was extracted from these tumors, and qPCR analysis of mRNA levels of CTGF and ANKRD1 are shown. Data are mean ± SD of six tumors. Statistical significance was determined by two-sided Mann–Whitney U Tests. * p = 0.026 for CTGF and <0.01 for ANKRD1, respectively.

Extended Data Fig. 7 Pathways enriched in constitutively active YAP expressed or sotorasib-treated cells.

To analyze the enrichment of the genes upregulated in YAP-S6A expressing cells (n = 3874), sotorasib 72 hr treated cells (n = 3863), and their overlap (n = 845) in NCI-H358, the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) (http://www.webgestalt.org/) was used. The enrichment of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Reactome pathway were processed through Overrepresentation Enrichment Analysis (ORA) with a false discovery rate (FDR) less than 0.05 selected as the cut-off value.

Extended Data Fig. 8 MRAS induction following inhibition of driver oncogenes.

a. NCI-H358 single-cell clones with CRISPR-Cas9 KO of Scrib, and introduction of wild-type or CAAX-fused Scrib, were treated with 1 μM sotorasib for 48 hours and mRNA levels of MRAS were determined by qPCR. Data are mean ± SD (n = 3, independently treated cell cultures). b. NCI-H358 cells were treated with 1 μM sotorasib with or without 5 μM ML349 for 48 hours, and mRNA levels of MRAS normalized to ubiquitin were determined by qPCR. Data are mean ± SD (n = 3, independently treated cell cultures). c. qPCR analysis of mRNA levels of RRAS, RRAS2, and MRAS normalized to ubiquitin in NCI-H358 cells treated with sotorasib at 1 μM for the indicated time points. Data are mean ± SD (n = 3, independently treated cell cultures). d. NCI-H358 and LU65 cells transduced with either control (Ctr) or constitutively active YAP mutant (YAP-S6A) were lysed and blotted with the indicated antibodies. e. NCI-H358 and LU65 cells were treated with either 1 μM sotorasib or 500 nM adagrasib for 72 hours. Representative immunofluorescence images of MRAS are shown. Scale bar, 10 μm. Images of immunofluorescence are representative of three independent experiments. f. HCC827 and NCI-H2228 cells were treated with 100 nM osimertinib or 500 nM alectinib for the indicated time points. Cell lysates were blotted with the indicated antibodies. g. Primer design for ChIP–qPCR. Consensus sequence (GGAATG) recognized and bound by YAP through TEAD around the transcription start site (TSS) are shown as gray vertical bars. Exons are represented as blue vertical bars. Arrows are primers used for ChIP–qPCR in Fig. 5f. d, f. Western blot was repeated two independent times with similar results. Representative images are shown.

Extended Data Fig. 9 MRAS is a cause of feedback activation of the MAPK pathway following sotorasib treatment.

a. qPCR analysis of mRNA levels of MRAS, NRAS, and HRAS normalized to ubiquitin in NCI-H358 cells treated with 1 μM sotorasib for the indicated time points. Data are mean ± SD (n = 3 independently treated cell cultures). b. NCI-H358 cells were treated with 1 μM sotorasib for 4 hours. Scrib (red) and CRAF (green) were stained and merged. Scale bar, 10μm. The lines indicate the profiles taken for the relative fluorescent intensities. While peaks of fluorescence were identical between Scribble and CRAF in untreated cells, the peaks were not merged in sotorasib-treated cells. Images of immunofluorescence are representative of two independent experiments. c. Chemiluminescent quantifications of normalized p-ERK levels from western blots as in Fig. 6d are illustrated. d. NCI-H358 single-cell clones with CRISPR-Cas9 KO of Scrib, and introduction of wild-type or CAAX-fused Scrib, were treated with 1 μM sotorasib for the indicated time points, and lysates were probed with the indicated antibodies. e. Control or YAP/TAZ-knockout cells were treated with 1 μM sotorasib for the indicated time points, and lysates were probed with the indicated antibodies. f. NCI-H358 and LU65 cells were treated with 1 μM sotorasib with or without 5 μM ML349 for the indicated time points. Lysates were probed with the indicated antibodies. GAPDH is a loading control. g. NCI-H358 cells were transfected with either scramble (Scr), NRAS (siNRAS), or MRAS (siMRAS) siRNA and cultured for 24 hours. Media was then replaced, and cells were treated with either 1 μM sotorasib or DMSO for an additional 72 hours. Lysates were probed with the indicated antibodies (left), or relative cell survival was determined (right). Data are mean ± SD, n = 3, independently treated cell cultures. d, e, f, g. Western blot was repeated two independent times with similar results. Representative images are shown.

Extended Data Fig. 10 Tight correlation between EMT markers and MRAS expression.

Scatter plot analysis showing the relationship between MRAS, Vimentin, and ZEB1. Expression data of KRAS mutant lung cancer in TCGA dataset was downloaded from cBioportal. p < 0.0001, both by linear regression analysis.

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Unprocessed western blots for all main figures.

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Source Data for main figures.

Source Data Extended Data Fig. 1

Source Data for Extended Data Figs.

Source Data Extended Data Fig. 2

Unprocessed blots for Extended Data Figs.

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Adachi, Y., Kimura, R., Hirade, K. et al. Scribble mis-localization induces adaptive resistance to KRAS G12C inhibitors through feedback activation of MAPK signaling mediated by YAP-induced MRAS. Nat Cancer 4, 829–843 (2023). https://doi.org/10.1038/s43018-023-00575-2

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