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Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to promote an ECM-enriched niche and support tumour initiation

Abstract

Defining drivers of tumour initiation can provide opportunities to control cancer progression. Here we report that lysophosphatidic acid receptor 4 (LPAR4) becomes transiently upregulated on pancreatic cancer cells exposed to environmental stress or chemotherapy where it promotes stress tolerance, drug resistance, self-renewal and tumour initiation. Pancreatic cancer cells gain LPAR4 expression in response to stress by downregulating a tumour suppressor, miR-139-5p. Even in the absence of exogenous lysophosphatidic acid, LPAR4-expressing tumour cells display an enrichment of extracellular matrix genes that are established drivers of cancer stemness. Mechanistically, upregulation of fibronectin via an LPAR4/AKT/CREB axis is indispensable for LPAR4-induced tumour initiation and stress tolerance. Moreover, ligation of this fibronectin-containing matrix via integrins α5β1 or αVβ3 can transfer stress tolerance to LPAR4-negative cells. Therefore, stress- or drug-induced LPAR4 enhances cell-autonomous production of a fibronectin-rich extracellular matrix, allowing cells to survive ‘isolation stress’ and compensate for the absence of stromal-derived factors by creating their own tumour-initiating niche.

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Fig. 1: Pancreatic cancer cells selectively upregulate LPAR4 in response to isolation stress.
Fig. 2: LPAR4 expression in patients with pancreatic cancer and its link to tumour initiation.
Fig. 3: Stress suppresses miR-139-5p to release the brake on LPAR4 expression.
Fig. 4: DEGs common to LPAR4-expressing cells and patient tumours include ECM-related genes.
Fig. 5: LPAR4 expression promotes the cell-autonomous production of FN1.
Fig. 6: FN1, induced by the LPAR4/AKT/CREB signalling, is indispensable for LPAR4-induced TIC properties.
Fig. 7: ECM deposited by LPAR4-positive cells endows stemness features to LPAR4-negative cells in an FN1-dependent manner.

Data availability

RNA-seq data that support the findings of this study have been deposited in the Gene Expressing Omnibus (GEO) under accession code GSE198002.

The human PAAD data were derived from the TCGA Research Network (http://cancergenome.nih.gov/ and http://www.cbioportal.org/). The TNMplot public dataset that supports the findings of this study is available at https://tnmplot.com/analysis/ or in 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

We thank A. Reiss, M. Morgan and M. Advani for their technical support. This study was funded by the University of California Tobacco-Related Disease Research Program (C.W., T29FT0343), and from grants awarded by the NIH, including T32CA009523 (T.R.), T32OD017863 (H.I.W.), T32HL086344 (H.I.W.), K01OD030513 (H.I.W.), R01CA155620 (A.M.L.), R01CA045726 (D.A.C.) and R35CA220512 (D.A.C.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

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Contributions

C.W., S.M.W. and D.A.C. conceived, designed and wrote the manuscript. C.W. performed all the experiments with the exception of: some of the in vitro experiments performed by T.R., T.S., S.J. and J.T.; the in vivo experiments and data analysis assisted by H.I.W., Z.Y. and T.R.; and PDX cells were generated by E.M., B.G.C. and A.M.L. A.C. assisted with the RNA-seq data analysis.

Corresponding author

Correspondence to David A. Cheresh.

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Nature Cell Biology thanks Mara Sherman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 LPAR4 is a stress- inducible gene.

a, Graph showing relative mRNA expression of six LPARs (normalized to housekeeping gene RPL37A) for cells grown in 10% serum and 2D. b, Graph comparing LPARs expression on cells grown in stem-like culture conditions (that is, no serum/3D) for 72 h with respect to cells grown in 10% serum on 2D for 72 h. c, Graph comparing LPARs expression on cells grown in hypoxia condition (1% O2) for 72 h with respect to cells grown in normoxia for 72 h. d, e, Graphs comparing LPARs expression on cells grown in 3D condition treated with varying doses of gemcitabine or paclitaxel for 24 h. Bars represent median value per cell line (ac). Data were presented as mean ± s.d. for n = 3 independent experiments. Statistical analyses were performed using two tailed unpaired one sample t-test (b-e). Source numerical data are available in source data.

Source data

Extended Data Fig. 2 LPAR4 does not impact cell growth in the absence of stress.

a, Immunohistochemistry staining of LPAR4 in a PDAC tissue array consisted of 15 PDAC samples and 4 samples of normal pancreas. Scale bar is 50 μM. Representative images showing normal pancreas (n = 4 biological samples), low-LPAR4 PDAC (n = 9 biological samples), and high-LPAR4 PDAC (n = 6 biological samples). Bar graph shows the relative percentage of LPAR4-low and LPAR4-high patients in the PDAC tissue array. b, Quantitative RT-PCR confirming the ectopic expression and stable knockdown of LPAR4 in 2 pancreatic cancer cell lines (Colo-357, MIA PaCa-2) and 2 patient-derived cancer cells (79E, 34E). Data were shown as mean ± s.d. (n = 4 independent experiments for Colo-357 and 79E cells with LPAR4 stable knockdown, n = 3 for Colo-357, 79E, and MiaPaCa2 cells with LPAR4 ectopic expression, n = 3 for 34E cells with LPAR4 stable knockdown, and n = 4 for 34E with LPAR4 ectopic expression). c, Non-invasive Bioluminescence images showing tumors formed at day 10 for Colo-357+sh-CTRL+ luciferase or Colo-357+sh-R4.1+luciferase of various number implanted in the pancreas of nu/nu mice. The right panel showing the luminescence intensity in a blue-to-red spectrum. d, f, Trypan blue exclusion assay showing the relative viable cell number of cells with or without LPAR4 expression manipulation grown in 10% serum and 2D at day 4 and day 7. e, Tumor growth rates for 1 million Colo-357 cells with or without LPAR4 expression manipulation in a subcutaneous tumor model. Data were presented as mean ± s.d. for n = 8 independent samples for each group. g, Quantitative RT-PCR confirming the knockdown of LPAR1 in Colo-357 cells. h, Effects of LPAR1 knockdown using siRNA on 2D or 3D (methylcellulose sphere forming) cell growth. Data were presented as mean ± s.d. for n = 3 independent experiments (d, f, g, and h). Statistical analyses were performed using two tailed unpaired one sample t-test (b, g, and h) and one-way ANOVA (df). Source numerical data are available in source data.

Source data

Extended Data Fig. 3 miR-139-5p downregulates LPAR4 expression in pancreatic cancer cells.

a, Graphs showing the log2-FC of miR-139-5p expression level in cells with or without LPAR4 expression manipulation. b, Graph shows the log2-FC of mRNA level of LPARs in Colo-357 cells treated with anti-miR-139-5p, normalized to cells treated with scrambled control miRNA. Data were presented as mean ± s.d. for n = 6 independent experiments for LPAR4 and n = 5 for other LPARs. c, Construct map for LPAR4-3’UTR luciferase reporter vector or control vector. d, Immunoblots showing representative of three independent experiments for LPAR4 protein level in Colo-357-sh-CTRL or sh-R4.1 cells treated with scrambled control miRNA vs. anti-miR-139-5p. e, Graphs showing the log2-FC of mRNA level of LPAR4 and various predicated miR-139-5p targets in Colo-357 or 34E cells treated with miR-139-5p mimic, normalized to cells treated with scrambled control miRNA. Data were presented as mean ± s.d. for n = 3 independent experiments (a, b, and e). Statistical analyses were performed using two tailed unpaired one sample t-test (a, b, and e). Source numerical data are available in source data.

Source data

Extended Data Fig. 4 LPAR4 upregulates ECM genes and cancer stemness-related genes in the absence of exogenous LPA.

a, Quantitative RT-PCR confirmation of LPAR4 regulated genes associated with ECM in two additional pairs of +EV vs. +R4 cells, grown in charcoal stripped FBS containing media. All mRNA level was normalized to +EV cells. b, Graphs showing the relative number of viable 79E + EV or 79E + R4 cells treated with various doses of H2O2 or gemcitabine in serum-free media for 72 h, evaluated by the CellTiter-Glo assay. c, Graph showing the number of tumorspheres formed by Colo-34+EV or Colo-357 + R4 cells grown in 3D suspension with serum-free media at day 10. The right panel shows representative images from three biological experiments for spheres formed by +EV or +R4 cells at day 10. d, Relative gene expression of CSC markers and antioxidant genes in +EV vs. +R4 cells grown in charcoal stripped FBS containing media. All mRNA level was normalized to +EV cells. Data were presented as mean ± s.d. for n = 3 independent experiments (a-d). Statistical analyses were performed using two tailed unpaired one sample t-test (a, c, and d). Source numerical data are available in source data.

Source data

Extended Data Fig. 5 LPAR4 induces the expression of FN1 isoforms containing EDA and EDB domains.

a, TNMplot showing FN1 gene expression is significantly higher in pancreatic adenocarcinoma (PAAD) than in normal pancreas (P < 0.0001). The unpaired two-tailed t test was used for statistical analysis. Bars represent median values for each group. b, Graphs showing the relative mRNA level of total FN1, FN1 containing EDA domain (FN1-EDA), and FN1 containing EDB (FN1-EDB) in +EV vs. +R4 cells as indicated. All mRNA expression was normalized to +EV cells. Expression of FN1-EDA or FN1-EDB is quantitated independently by using two different sets of primers. c, Graphs showing the relative LPAR4 mRNA level in cells stably transfected with scrambled shRNA (sh-CTRL) or two different LPAR4 shRNAs (sh-R4.1 and sh-R4.2), treated with hypoxia and no serum for 72 h. All were normalized to LPAR4 mRNA level in normoxia and no serum condition. Data were presented as mean ± s.d. for n = 3 biological experiments (b and c). P-value was calculated using two tailed unpaired one sample t-test. Source numerical data are available in source data.

Source data

Extended Data Fig. 6 FN1 is a critical mediator of LPAR4-induced cancer stemness.

a, Gene Set Enrichment Analysis (GESA) for LPAR4-induced gene expression suggests that AKT signaling is upregulated in 79E + R4 cells in the absence of LPA. b, Immunoblot showing representative of three biological experiments for the protein levels of p-AKT-S473, AKT, p-GSK-3β-S9, p-CREB-S133, CREB, and vinculin in Colo-357+EV and Colo-357 + R4 cells treated with Ipatasertib of a range of doses for 2 h, or with 1 μM Ipatasertib in a time-course experiment. c, Relative mRNA level of ECM-related genes among LPAR4 gene signature in +R4 cells transfected with si-CREB as compared to cells transfected with si-CTRL. d, Graphs showing the cell viability of cells grown on 2D with serum free or with 10% charcoal stripped FBS containing media. Cell viability was assessed by the CellTiter-Glo assay, and all numbers were normalized to EV cells transfected with si-CTRL. e, Quantitative RT-PCR confirmation of FN1 knockdown by using siRNA in three pairs of +EV and +R4 cells as indicated. f, Representative histograms of three biological experiments showing MitoSOX signaling in +EV vs. +R4 cells treated with scrambled siRNA or si-FN1. All cells were cultured in serum free media for 48 h prior to MitoSOX staining. Data were presented as mean ± s.d. for n = 3 biological experiments (ce). P-value was calculated using two tailed unpaired one sample t-test. Source numerical data are available in source data.

Source data

Extended Data Fig. 7 ECM deposited by LPAR4 + cells endows LPAR4-negative cells with growth advantage in the presence of stress.

Representative images for three biological experiments for Colo-357 cells grown on uncoated plate or on plate coated with extracellular matrix deposited by 79E + EV or 79E + R4 cells in serum-free media at day 1, day 2, day 7, and day 12. Scale bar = 50 µM. Yellow color circled areas show representative cell colony.

Extended Data Fig. 8 Validation of LPAR4 antibody for immunohistochemistry application.

Representative LPAR4 immunohistochemistry staining for orthotopic xenograft tumors from 79E + EV (n = 3 biologically independent samples) and 79E + R4 (n = 3 biologically independent samples).

Supplementary information

Supplementary Information

Supplementary figure demonstrating flow cytometry gating strategy for mitoSOX staining.

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Four tables for the main text and the other two tables for information on vectors, siRNA, shRNA, qPCR primers and so on.

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Wu, C., Rakhshandehroo, T., Wettersten, H.I. et al. Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to promote an ECM-enriched niche and support tumour initiation. Nat Cell Biol (2023). https://doi.org/10.1038/s41556-022-01055-y

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