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RasGRP1 opposes proliferative EGFR–SOS1–Ras signals and restricts intestinal epithelial cell growth

Nature Cell Biology volume 17, pages 804–815 (2015)Cite this article

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Abstract

The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR–SOS1–Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras–ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR–RasGRP1 and EGFR–SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma.

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Figure 1: RasGRP1 is expressed in CRC cell lines and CRC patient tumour samples.
Figure 2: Rasgrp1 plays a role in intestinal epithelial cell proliferation and goblet cell generation.
Figure 3: Loss of one or two Rasgrp1 alleles exacerbates serrated dysplasia of KRasG12D epithelium.
Figure 4: EGFR connects to both RasGRP1 and SOS1.
Figure 5: Hyperactivation of RAS in EGF-stimulated KRASMUT CRC cells.
Figure 6: EGFR–SOS1 signals promote EGFR–Ras signalling and tumorigenesis of KRASMUT CRC cells.
Figure 7: RasGRP1 feedback restricts EGFR–SOS1–Ras signals.
Figure 8: RasGRP1 limits CRC cell proliferation and in vivo tumour growth.

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Acknowledgements

The authors thank J. Stone, T. Jacks and A. Ma for Rasgrp1-deficient, KRASLSL−G12D and VillinCre mice, A. Karnezis and O. Yilmaz for helpful comments on the intestinal hyperplasia, L. Westerveld for technical support, and A. Balmain, M. McMahon, C. Bonnans, N. Duesbery and O. Klein for critically reading the manuscript. Our research was supported by the Sandler Program in Basic Science (start-up), NIH-NCI Physical Science Oncology Center grant U54CA143874, NIH grant 1P01AI091580-01, a Gabrielle’s Angel Foundation grant, a UCSF ACS grant, and a UCSF Research Allocation Program (RAP) pilot grant (all to J.P.R.), as well as by grants from the NCI (R01 CA057621 to Z.W. and K01CA118425 to K.M.H.), from the Jeannik M. Littlefield foundation (to R.W.), and from the Ministry of Science and Technology, Taiwan (104-2917-I-006-002 to C-Y.W.), and by the KWF (Dutch Cancer Society) (R.A.H.v.d.V., L.M.H.) and Saal van Zwanenberg Foundation (L.M.H.).

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  1. Linda M. Henricks, Robert A. H. van de Ven & Ed Lemmens

    Present address: Present addresses: Department of Molecular Pathology, the Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands (L.M.H.); Department of Pathology, Utrecht University, 3582 BW Utrecht, The Netherlands (R.A.H.v.d.V.); Aduro BioTech, Berkeley, California 94710, USA (E.L.).,

Authors and Affiliations

  1. Department of Anatomy, University of California, San Francisco, San Francisco, California 94143, USA

    Philippe Depeille, Linda M. Henricks, Robert A. H. van de Ven, Ed Lemmens, Chih-Yang Wang, Zena Werb & Jeroen P. Roose

  2. Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan

    Chih-Yang Wang

  3. Department of Surgery, University of California, San Francisco, San Francisco, California 94143, USA

    Mary Matli, David Donner & Robert Warren

  4. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

    Kevin M. Haigis

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Contributions

P.D. performed most experiments. L.M.H., R.A.H.v.d.V. and E.L. assisted with soft-agar colony formation, biochemistry, and tissue culture. M.M., D.D. and R.W. provided RNA of patient specimens. C-Y.W., supported by Z.W., assisted with bioinformatics approaches. R.W., D.D. and K.M.H. provided insights and comments on the manuscript. P.D. and J.P.R. conceived the study, analysed data, and wrote the manuscript.

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Correspondence to Philippe Depeille or Jeroen P. Roose.

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P.D., R.W., and J.P.R. at UCSF filed a provisional patent (81906-937600-220200US) based on this work.

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Supplementary Figure 3 Loss of Rasgrp1 alleles exacerbates the serrated dysplasia and hyperproliferation driven by KRasG12D.

(a) Higher magnifications of the distal colon of the indicated three mouse models depicted in Fig. 3b. Sections have been immunostained for Ki67 expression in brown. Scale bar indicates 100 μm. (b) Quantification of counted Ki67-positive cells in open crypts of the distal colon in mice from the indicated genotypes. Data are mean ± s.e.m. determined from n = 150 crypts that were pooled from three mice for each genotype. ∗∗∗p < 0.0001 (One way Anova, Bonferroni’s multiple comparison test). (c) Aberrant serration in the distal colon in KRasG12DRasgrp1WT/- heterozygous mice. H&E staining of a representative section of the distal colon of KRasG12DRasgrp1WT/- mice. Scale bar indicates 100 μm.

Supplementary Figure 4 Increased branching of villi in the small intestine of KRasG12D mice when Rasgrp1 alleles are deleted.

(a) Large field, low magnification overviews of the small intestine (duodenum) sections stained by H&E from control WT-, KRasG12D-, KRasG12DRasgrp1WT/- -, and KRasG12DRasgrp1-/- - mice. Fields depicted here are distinct from the ones show in Fig. 3 to highlight the penetrance of the phenotypes. Scale bars 100 μm. (b) A selected field with branching villi in the duodenum of KRasG12DRasgrp1WT/- heterozygous mice at higher magnification. Scale bars indicate 100 μm. (c) Quantification of measurements of villus lengths (μm) in the duodenum from mice with the indicated genotype. Data are mean ± s.e.m. determined from n = 150 villi pooled from 3 mice for each specific specific genotype. NS = not significant, ∗∗∗p < 0.0001 (One way Anova, Bonferroni’s multiple comparison test). (d) Representative images of ‘swiss rolls’ of entire intestinal tracts from stomach to rectum for the four indicated genotypes, sectioned transversally and stained with H&E. Scale bars represent 1 mm.

Supplementary Figure 5 Taqman expression analysis of all RasGRP family members in colorectal cancer cell lines.

(a) Real-time PCR of RasGRP1 mRNA level in seven selected colorectal (CRC) cell lines as shown Supplementary Table 1. WiDr was used as an arbitrary reference and set at 1.0. All data are shown as fold difference ± s.e.m. and compared to the value of 1.0 in WiDr and data is plotted from n = 6 independent RNA extractions for each cell type. (b) Same data sets for WiDr and SW403 as in Supplementary Fig. 3a but depicted on a different scale to highlight low expression of RasGRP1 in WiDr and nearly complete absence of RasGRP1 in SW403 CRC cells. WiDr was used as an arbitrary reference and set at 1.0. All data are shown as fold difference ± s.e.m. and compared to the value of 1.0 in WiDr and data is plotted from n = 6 independent RNA extractions for each cell type. (c–e) RasGRP2- (c), RasGRP3- (d) and RasGRP4- (e) mRNA expression levels in the indicated seven CRC cell lines. WiDr was used as an arbitrary reference and set at 1.0. All data are shown as fold difference ± s.e.m. and compared to the value of 1.0 in WiDr and data is plotted from n = 6 independent RNA extractions for each cell type.

Supplementary Figure 6 ERK activation in HCT15 colorectal cancer cell lines.

(a,b) Analysis of ERK phosphorylation and total ERK protein levels in the human HCT15 colorectal cancer cell line stimulated with EGF. a,b are independent experiments confirming the observations in the experiment presented in Fig. 7c. Mean values of phospho-ERK for the three independent experiments with statistical analysis are provided in Fig. 7d. Unstimulated WT HCT15 cells are arbitrarily set at 1.0. Uncropped western blot images are shown in Supplementary Fig. 7.

Supplementary Figure 7 Correlation of RASGRP1 expression in CRC with clinical patient survival.

(a) The GSE12945 dataset comprises 185 RasGRP1 high and 38 RasGRP1 low samples from patients. High and low were defined using a cut-off finder as described. P = 0.0102, Log-rank (Mantel-Cox) test. (b) The GSE14333 dataset comprises 40 RasGRP1 high and 11 RasGRP1 low samples. P = 0.0235, Log-rank (Mantel-Cox) test.

Supplementary Figure 8 Analysis of ApcMin/+ :Rasgrp1-/- mice.

(a) Representative H&E images from colonic tumours from ApcMin/+ and ApcMin/+ :Rasgrp1-/- mice. Top panels (same as in Fig. 8) show the entire tumours and bottom panel show the same tumours at larger magnification. Top panels have a scale of 150 μm, whereas the bottom panels scale is 30 μm. (b) Representative colonic tumour sections from ApcMin/+ and ApcMin/+ :Rasgrp1-/- mice that were stained for BrdU (in brown, 2 h in vivo BrdU labeling). Scale 150 μm. Details within the traced squares are shown at higher magnification. (c) Representative tumour sections stained for cleaved caspase-3 (in brown) to reveal apoptotic cells in ApcMin/+ and ApcMin/+ :Rasgrp1-/- mice with an inset showing rare, positive cells at higher magnification. Arrows indicate the rare cleaved caspase-3 positive cells. Scale 150 μm. Details within the traced squares are shown at higher magnification.

Supplementary Table 1 RasGRP1 expression profile and KRAS mutational status in selected colorectal cancer cell lines.
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Supplementary Table 2 Sensitivity of colorectal cancer cell lines to the Erlotinib EGFR inhibitor.
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Depeille, P., Henricks, L., van de Ven, R. et al. RasGRP1 opposes proliferative EGFR–SOS1–Ras signals and restricts intestinal epithelial cell growth. Nat Cell Biol 17, 804–815 (2015). https://doi.org/10.1038/ncb3175

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