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ROCK-mediated selective activation of PERK signalling causes fibroblast reprogramming and tumour progression through a CRELD2-dependent mechanism

Nature Cell Biology volume 22, pages 882–895 (2020)Cite this article

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Abstract

It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer–microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK–PERK–ATF4–CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.

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Fig. 1: ROCK activation enhances tumour burden, fibroblast numbers and ECM production.
Fig. 2: ROCK education of mammary fibroblasts yields a tumour-promoting fibroblast phenotype.
Fig. 3: ROCK education of mammary fibroblasts yields a tumour-promoting fibroblast phenotype through paracrine Creld2.
Fig. 4: Perk is selectively activated downstream of ROCK in mammary cancer.
Fig. 5: Perk signalling is required for the ROCK-induced, tumour-promoting education of mammary tumour fibroblasts.
Fig. 6: ROCK activation in mammary cancers induces Atf4-regulated Creld2 transcription downstream of actin stress.
Fig. 7: High CRELD2 expression is associated with progressive human breast cancer.

Data availability

MS data have been deposited at ProteomeXchange with the primary accession code PXD018253 (http://www.ebi.ac.uk/pride). Source data for Figs. 1–7 and Extended Data Figs. 1–7 are provided with the paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank J. Visvader for mouse mammary cancer tissue; P. Thomas for providing CRISPR vector pDG461; and M. White for assistance in designing CRISPR guides. The PyMT mammary cancer cell line was provided by K. Blyth through SEARCHBreast (https://searchbreast.org/). This research was supported by the NHMRC (to M.S.S., P.T., S.M.P., A.F.L. and M.K.), ARC (to M.S.S. and P.T.), the Cancer Councils of South Australia (M.S.S.) and New South Wales (P.T.), the RAH Research Fund (M.S.S.) and the Fay Fuller Foundation (S.M.P). S.T.B. was supported by a RAH Research Committee Early Career Fellowship. We thank donors to the Health Services Charitable Gifts Board of SA and the Australian Cancer Research Foundation (Cancer Discovery Accelerator) for funding the imaging equipment used.

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Author notes

  1. Alexander Charles Lewis

    Present address: Translational Haematology Program, Peter McCallum Cancer Centre, Melbourne, Victoria, Australia

Authors and Affiliations

  1. Centre for Cancer Biology, SA Pathology and the University of South Australia, Adelaide, South Australia, Australia

    Sarah Theresa Boyle, Valentina Poltavets, Jasreen Kular, Natasha Theresa Pyne, Alexander Charles Lewis, Natasha Kolesnikoff, Paul Andre Bartholomew Moretti, Melinda Nay Tea, Vinay Tergaonkar, Stuart Maxwell Pitson, Angel Francisco Lopez, Marina Kochetkova & Michael Susithiran Samuel

  2. Division of Systems Biology and Personalised Medicine, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Jarrod John Sandow & Andrew Ian Webb

  3. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

    Jarrod John Sandow & Andrew Ian Webb

  4. The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Cancer Division, St Vincent’s Clinical School, University of NSW, Sydney, New South Wales, Australia

    Kendelle Joan Murphy & Paul Timpson

  5. Institute of Molecular and Cell Biology, A*STAR and Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Vinay Tergaonkar

  6. Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia, Australia

    Stuart Maxwell Pitson, Angel Francisco Lopez & Michael Susithiran Samuel

  7. Breast, Endocrine and Surgical Oncology Unit, Royal Adelaide Hospital, Adelaide, South Australia, Australia

    Robert John Whitfield

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Contributions

S.T.B. designed and performed much of the experimental work, provided substantial intellectual input and wrote the manuscript. V.P. and J.K. performed biochemical analyses. N.T.P. performed histological analyses. J.J.S. and A.I.W. performed proteomic MS analyses. N.K. generated the Creld2 CRISPR plasmids. A.C.L performed Creld2 promoter ChIP analyses, P.A.B.M. generated the Creld2 promoter luciferase reporter constructs and M.N.T. performed Creld2 promoter luciferase analyses with intellectual input and supervision from S.M.P. K.J.M. performed CDM experiments with intellectual input and supervision from P.T. A.F.L., V.T. and R.J.W. provided intellectual input. M.K. and M.S.S. conceived, supervised and were intellectual drivers of the study, designed and performed experiments, and wrote the manuscript.

Corresponding authors

Correspondence to Marina Kochetkova or Michael Susithiran Samuel.

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Extended data

Extended Data Fig. 1 ROCK activation enhances tumor burden, fibroblast numbers and ECM production; Related to Fig. 1.

ROCK a, and pMYPT1 b, in normal (n = 12 normal/hyperplastic) and cancerous (n = 108 IDC) human breast. Scales: 50 µm. Each TMA was immunolabeled once with each antibody. c, Rock1/2 mRNA in advanced PyMT cells transfected with non-targeting control (NTC) or Rock1 and Rock 2 siRNA (n = 5/group). Mean+SEM, two-sided unpaired t-test. d, pMlc2 protein following Rock1/2 knockdown in tumor cells. e, pMlc2 (greyscale, green) and pMypt1 (greyscale, magenta) in tumor cells following Rock1/2 knockdown. Counter-labels: Phalloidin (F-actin, red); DAPI (nuclei, blue). Scales: 50 µm. (d,e) Repeated twice with similar results. f, ROCK (mean ± SEM; n = 5 NTC, 3 siRNA; two-sided unpaired t-test) and pMlc2 (mean; n = 5 NTC, 2 siRNA) (greyscale and green) in tumors one week after engraftment of tumor cells with Rock1/2 knockdown. Counter-labels: E-cadherin (red); PyMT (blue). Scales: 50 µm. g, Collagen (SHG: magenta, transmission: grey) and fibronectin, periostin and tenascin C (green, counter-labels E-Cadherin (red); DAPI (blue)) in R-PyMT and K-PyMT tumors. Scales: 100 µm. Experiment repeated at least twice and pooled data analyzed. h, Fsp1/S100a4 (n = 6 veh, 5 fas) and Pdgfrβ (n = 10/group) (both green) in tumors from mice engrafted with tumor cells and treated with fasudil or vehicle. Counter-labels: E-Cadherin (red); DAPI (blue). Scales: 100 µm. Median±IQR, two-sided Mann-Whitney. i, Fsp1/S100a4 (n = 7/group) and Pdgfrβ (n = 10 NTC, 7 siRNA) (both green) in tumors in mice engrafted with Rock1/2-depleted tumor cells. Counter-labels: E-Cadherin (red); DAPI (blue). Scales: 100 µm. Median±IQR, two-sided Mann-Whitney. j, Epithelial cells (CK8/18/14, green), fibroblasts (Pdgfrβ, red), immune cells (CD45, green) and endothelial cells (CD31) in total cell suspensions from mammary tissue digest (top), and cultured fibroblasts (bottom). Counter-label: DAPI. Scales: 100 µm. Pie charts show cell type proportions. k, Ex vivo-cultured mammary epithelial cells and fibroblasts. Scales: 200 µm. j,k, Repeated at least twice with similar results. See also Statistics Source Data for Extended Data Fig. 1 and Unprocessed blots for Extended Data Fig. 1.

Source data

Extended Data Fig. 2 ROCK-education of mammary fibroblasts yields a tumor-promoting fibroblast phenotype; Related to Fig. 2.

a, Schematic of the inverse invasion assay assessing fibroblast motility ex vivo. b, Confocal z-stack images (7 µm intervals) through a Matrigel plug following migration by fibroblasts from ROCK-activated R-PyMT and K-PyMT mammary tumors. Repeated more than 3 times with similar results. c, Schematic of ex vivo cell-derived matrix (CDM) establishment to study cancer cell anisotropy. d, Images of picrosirius red-stained CDM formed by fibroblasts from ROCK-activated R-PyMT and K-PyMT mammary tumors. Scales: 100 µm. e, Streaming images of PyMT tumor cells seeded onto CDM generated from ROCK- or KD-educated fibroblasts. Arrows demonstrate streaming direction (insets of boxes shown below). d,e, Repeated 3 times. f, Schematic of co-engraftment strategy to examine educated fibroblast function in vivo. g, ECM proteins fibronectin, periostin and tenascin C (green) in tumors arising in mice that were co-engrafted with PyMT tumor cells and either ROCK- or KD-educated fibroblasts. Counter-label: DAPI (blue). Scales: 100 µm. Dot plots show the area coverage of respective ECM component IF signals. Median±IQR, n = 5 tumors/group, two-sided Mann-Whitney tests. h, Left: Western analysis of pMlc2 in ex vivo-cultured K-PyMT and R-PyMT tumor cells following 4−hydroxy tamoxifen (4HT) treatment. Graphical representations of starved and 4HT-treated densitometries are shown below. Right: pMlc2 (green) in ex vivo-cultured K-PyMT and R-PyMT tumor cells treated with 4HT. Counter-labels: Phalloidin (F-actin, red) and DAPI (blue). Scales: 100 µm. Repeated at least twice with similar results. i, Schematic of co-engraftment strategy to examine the effect of paracrine signaling between ROCK-activated tumor cells and fibroblasts on tumor promotion in vivo. See also Statistics Source Data for Extended Data Fig. 2 and Unprocessed blots for Extended Data Fig. 2.

Source data

Extended Data Fig. 3 ROCK-education of mammary fibroblasts yields a tumor-promoting fibroblast phenotype via paracrine Creld2; Related to Fig. 3.

a, Creld2 protein in 4HT-treated K-PyMT and R-PyMT ex vivo-cultured tumor cells. Experiment repeated twice with similar results. b, Creld2 (greyscale and green) in tumors one week following engraftment of tumor cells transfected with non-targeting control (NTC) or Rock1/2 siRNA. Counter-labels: E-cadherin (red); PyMT (blue). Scales: 50 µm. Mean±SEM (n = 5 NTC, 3 siRNA), two-sided unpaired t-test. c, Creld2 (green) in advanced tumor cells treated with fasudil and Y-27632. Counter-labels: Phalloidin (F-actin, red) and DAPI (blue). Scales: 50 µm. Mean±SEM (n = 10/group), one-way ANOVA. FOV = Field of view. d, Creld2 mRNA in R-PyMT cells following Creld2 knockdown (n = 3/group). Mean+SEM, one-way ANOVA. e, Creld2 (greyscale and green) in immortalized PyMT cell line following Creld2-CRISPR, clone E4. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 50 µm. Mean±SEM (n = 13 Parental, 10 E4), two-sided unpaired t-test. f, Mean distance migrated by fibroblasts exposed to CM from PyMT cells with Creld2-CRISPR (n = 3/group) in inverse invasion assay. Mean±SEM, two-sided unpaired t-test. g, Co-engraftment strategies to examine effect of Creld2 on fibroblast function in vivo. h, Fibronectin (n = 5 KD + NTC,6 ROCK + NTC,6 ROCK + siRNA) and periostin (n = 6 KD + NTC,6 ROCK + NTC,5 ROCK + siRNA) (green) in tumors following co-engraftment of tumor cells and fibroblasts exposed to tumor cell CM following Creld2 knockdown. Counter-labels: E-Cadherin (red); DAPI (blue). Scales: 100 µm. Mean±SEM, one-way ANOVA. i, Progressive tumor volume following co-engraftment of tumor cells with fibroblasts exposed to CM from K-PyMT cells following Creld2 knockdown (n = 8 tumors/group). Mean&SEM, 2-way ANOVA. j, Final weights of tumors from i, two-sided Mann-Whitney test. Box plot: midline=median; box=25-75th percentile; whisker=minima to maxima. k, Fibronectin and periostin (green) in tumors following co-engraftment of tumor cells and fibroblasts treated with recombinant Creld2. Counter-labels: E-Cadherin (red); DAPI (blue). Scales: 100 µm. Median±IQR (n = 8 tumors untreated, 9 Creld2), two-sided Mann-Whitney. See also Statistics Source Data for Extended Data Fig. 3 and Unprocessed blots for Extended Data Fig. 3.

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Extended Data Fig. 4 Perk is selectively activated downstream of ROCK in mammary cancer; Related to Fig. 4.

a, Western analysis of Atf4 in K-PyMT and R-PyMT ex vivo-cultured mammary tumor cells following serum-starvation and 4HT-stimulation. Experiment repeated twice with similar results. b, Atf4 (greyscale and green) in 4HT-treated K-PyMT and R-PyMT tumor cells. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 100 µm. Higher magnification images from an independent experiment shown in Fig. 4d. Experiment repeated 3 times with similar results. c, Phosphorylated Perk (green) in R-PyMT and K-PyMT tumor cells cultured ex vivo. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 50 µm. Mean+SEM (n = 5 K-PyMT, 6 R-PyMT), two-sided unpaired t-tests. d, Phosphorylated Mlc2 and Perk (both green) in advanced PyMT tumor cells cultured ex vivo, serum-starved and serum-treated following starvation, in the presence of ROCK pharmacological inhibitors fasudil and Y27632. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 50 µm. Mean+SEM (n = 8 untreated, 10 Y27632, 10 fasudil) relative to untreated, one-way ANOVA. See also Statistics Source Data for Extended Data Fig. 4 and Unprocessed blots for Extended Data Fig. 4.

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Extended Data Fig. 5 Perk signaling is required for ROCK-induced production of Creld2; Related to Fig. 5.

a, mRNA expression of indicated genes in R-PyMT tumor cells following transfection with non-targeting control (NTC) or siRNAs against Atf6 (Atf6 UPR pathway), Ern1 (Ire1α UPR pathway), Eif2ak2 (Pkr ISR pathway), Eif2ak4 (Gcn2 ISR pathway) and Eif2ak3 (Perk pathway) (n = 2/group). Mean values shown. ND = not detected. Representative data from more than 3 independent experiments. b, Bip (Atf6 pathway), Xbp1−spliced (Ire1 pathway) and Atf4 (Perk pathway) (green) in R-PyMT and K-PyMT tumor cells, transfected with either NTC or siRNAs to separately knockdown UPR pathways as indicated. Counter-labels: Phalloidin (F-Actin, red); DAPI (blue). Scales: 50 µm. Repeated twice with similar results. c, Mean distance migrated by PyMT fibroblasts exposed to CM from 4HT-treated K-PyMT tumor cells transfected with NTC or siRNAs against Ern1, Atf6 and Eif2ak3 (n = 6 KD + NTC, 4 KD + Ern1, 4 KD + Atf6, 5 KD + Eif2ak3) in inverse invasion assay. Mean±SEM, one-way ANOVA. d, Atf4 in K-PyMT and R-PyMT tumor cells transfected with NTC or siRNA targeting Perk (Eif2ak3). Mean±SEM (n = 8 KD + NTC, 11 ROCK + NTC, 9 ROCK + siRNA) relative to KD, one-way ANOVA. Representative images shown in e. e, Atf4 (green) in R-PyMT and K-PyMT tumor cells, transfected with NTC or siRNAs to knockdown the ISR mediators, Eif2ak2 (Pkr), Eif2ak4 (Gcn2) and Eif2ak3 (Perk). Counter-labels: Phalloidin (red) and DAPI (blue). Scales: 50 µm. f, Creld2 (green) in R-PyMT tumor cells treated with ISR inhibitors against Pkr and Gcn2. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 50 µm. Mean±SEM (n = 4/group) relative to untreated, one-way ANOVA. See also Statistics Source Data for Extended Data Fig. 5.

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Extended Data Fig. 6 ROCK activation in mammary cancers induces Atf4-regulated Creld2 transcription downstream of actin stress; Related to Fig. 6.

a, Atf4, Bip and Xbp1-spliced (green), indicating induction of Perk, Atf6 and Ire1 UPR pathways respectively in PyMT tumor cells treated with tunicamycin (10 µg/ml) to induce ER stress. Counter-labels: Phalloidin (F-actin, red); DAPI (blue). Scales: 50 µm. Experiment repeated 3 times with similar results. b, mRNA levels of Creld2 in PyMT tumor cells treated with tunicamycin (10 µg/ml), mean ± SEM (n = 3/group) relative to untreated, two-sided unpaired t-test. c, Creld2 (green) in PyMT tumor cells treated with tunicamycin. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 50 µm. Mean±SEM (n = 25 untreated, 26 tunicamycin), two-sided unpaired t-test. d-f, Immunofluorescence analysis of pPerk (n = 6 KD, 5 ROCK, 5 ROCK + BMS5) d, Atf4 (n = 11/group) e, and Creld2 (n = 17 KD, 16 ROCK, 16 ROCK + BMS5) f, (all green) in K-PyMT and R-PyMT tumor cells treated with BMS5. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 50 µm. Mean±SEM relative to KD, one-way ANOVA tests. See also Statistics Source Data for Extended Data Fig. 6.

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Extended Data Fig. 7 High CRELD2 expression is associated with human breast cancer; Related to Fig. 7.

a, Representative images of immunohistochemical analyses of FSP1 in human normal/hyperplastic mammary specimens and invasive ductal carcinoma. Scales: 100 µm. Graph shows positive pixel analysis of FSP1 on a human breast cancer TMA of 14 normal (N)/hyperplasia (HP)/ductal carcinomas in situ (DCIS) and 50 invasive ductal carcinoma (IDC) specimens, analyzed by two-sided Mann-Whitney test. Box plot: midline=median; box=25−75th percentile; whisker=minima to maxima. b, CRELD2 expression in human patient samples based on breast cancer grade 1 (n = 170), 2 (n = 775) and 3 (n = 957), analyzed using the publicly available METABRIC dataset consisting of 2,000 primary breast cancers (see Methods). Median±IQR, Kruskal-Wallis test. c, Representative images of immunohistochemical analyses of CRELD2 in human normal/hyperplastic mammary specimens and invasive ductal carcinoma. Insets of boxes shown in Fig. 7f. Scales: 200 µm. d, Top: IF images of pMlc2 and Creld2 in normal mouse mammary tissue, ROCK-activated R−PyMT mouse mammary tumor tissue, and various mouse models of breast cancer (MMTV-PyMT, MMTV-Neu, MMTV-Wnt-1 and p53-null) as indicated. Merged images shown in Fig. 7h. Scales: 50 µm. Bottom: Histograms show area coverage analysis of pMlc2 and Creld2 signal, relative to signal in normal gland. Mean+SEM, two-sided unpaired t-tests. pMlc2: n = 5 normal, 5 R−PyMT+TAM, 4 MMTV-PyMT, 5 MMTV-Neu, 6 MMTV-Wnt-1, 6 p53-Null. Creld2: n = 10 normal, 9 R-PyMT+TAM, 8 MMTV-PyMT, 9 MMTV-Neu, 9 MMTV-Wnt-1, 10 p53-Null. See also Statistics Source Data for Extended Data Fig. 7.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: patient details. Supplementary Table 2: experimental conditions for immunofluorescence, immunohistochemistry and western analyses.

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Boyle, S.T., Poltavets, V., Kular, J. et al. ROCK-mediated selective activation of PERK signalling causes fibroblast reprogramming and tumour progression through a CRELD2-dependent mechanism. Nat Cell Biol 22, 882–895 (2020). https://doi.org/10.1038/s41556-020-0523-y

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