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Colon tumour cell death causes mTOR dependence by paracrine P2X4 stimulation

Nature volume 612, pages 347–353 (2022)Cite this article

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Abstract

Solid cancers exhibit a dynamic balance between cell death and proliferation ensuring continuous tumour maintenance and growth1,2. Increasing evidence links enhanced cancer cell apoptosis to paracrine activation of cells in the tumour microenvironment initiating tissue repair programs that support tumour growth3,4, yet the direct effects of dying cancer cells on neighbouring tumour epithelia and how this paracrine effect potentially contributes to therapy resistance are unclear. Here we demonstrate that chemotherapy-induced tumour cell death in patient-derived colorectal tumour organoids causes ATP release triggering P2X4 (also known as P2RX4) to mediate an mTOR-dependent pro-survival program in neighbouring cancer cells, which renders surviving tumour epithelia sensitive to mTOR inhibition. The induced mTOR addiction in persisting epithelial cells is due to elevated production of reactive oxygen species and subsequent increased DNA damage in response to the death of neighbouring cells. Accordingly, inhibition of the P2X4 receptor or direct mTOR blockade prevents induction of S6 phosphorylation and synergizes with chemotherapy to cause massive cell death induced by reactive oxygen species and marked tumour regression that is not seen when individually applied. Conversely, scavenging of reactive oxygen species prevents cancer cells from becoming reliant on mTOR activation. Collectively, our findings show that dying cancer cells establish a new dependency on anti-apoptotic programs in their surviving neighbours, thereby creating an opportunity for combination therapy in P2X4-expressing epithelial tumours.

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Fig. 1: Resistance of hCRC PDTOs to 5-FU depends on mTOR activation.
Fig. 2: Dying tumour cells induce mTOR survival signalling in a paracrine manner.
Fig. 3: Dying tumour cells release ATP to trigger mTORC1 through P2X4.
Fig. 4: ROS scavenging prevents induced mTOR addiction.

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Data availability

All data generated and/or analysed during this study are included in the Article and its Supplementary Information. Original scans of the kinase activation assay shown in Fig. 1c and the immunoblot experiments shown in Figs. 1d,e, 2a,g, 3a,c,e–g,j,l and 4f and Extended Data Figs. 1a,i, 2b and 3c–i are provided in the Supplementary Information. Tumour measurements for Figs. 1j, 2k, 3p and 4i are included in the Source Data. The FACS gating strategy for Fig. 4e is shown in the Supplementary Information. All oligonucleotide sequences, guide sequences for shRNAs and siRNA sequences are described in Supplementary Tables 1–3. Source data are provided with this paper.

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Acknowledgements

We thank H. Kunkel, S. Bösser, K. Mohs, P. Gupta, E. Rudolf and C. Danneil for expert technical assistance as well as the staff at the Animal Facility, the Histology Core Facility and the Flow Cytometry Core Facility at Georg-Speyer-Haus. Work in the laboratory of F.R.G. is supported by institutional funds from Georg-Speyer-Haus, and by the LOEWE Center Frankfurt Cancer Institute financed by the Hessen State Ministry for Higher Education, Research and the Arts (III L 5 - 519/03/03.001 - (0015)), Deutsche Forschungsgemeinschaft (FOR2438: Gr1916/11-1; SFB1292-Project ID: 318346496-TP16; SFB1479-Project ID: 441891347-P02; GRK2336) and the ERC (Advanced Grant PLASTICAN-101021078). The Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus is financed jointly by the German Federal Ministry of Health and the Ministry of Higher Education, Research and the Arts of the State of Hessen.

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Author notes

  1. Mark Schmitt

    Present address: Institute of Pharmacology, University of Marburg, Marburg, Germany

  2. Jalaj Gupta

    Present address: Stem Cell Research Center, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India

  3. These authors contributed equally: Fatih Ceteci, Jalaj Gupta

Authors and Affiliations

  1. Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany

    Mark Schmitt, Fatih Ceteci, Jalaj Gupta, Marina Pesic, Tim W. Böttger, Adele M. Nicolas, Kilian B. Kennel, Esther Engel, Matthias Schewe, Asude Callak Kirisözü, Valentina Petrocelli, Yasamin Dabiri, Julia Varga, Mallika Ramakrishnan, Madina Karimova, Melek C. Arkan & Florian R. Greten

  2. Frankfurt Cancer Institute, Goethe University Frankfurt, Frankfurt am Main, Germany

    Mark Schmitt, Fatih Ceteci, Jalaj Gupta, Marina Pesic, Tim W. Böttger, Adele M. Nicolas, Kilian B. Kennel, Esther Engel, Matthias Schewe, Asude Callak Kirisözü, Valentina Petrocelli, Yasamin Dabiri, Julia Varga, Madina Karimova, Melek C. Arkan & Florian R. Greten

  3. Global Health Institute, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland

    Andrea Ablasser

  4. Department of Organoid Medicine, Keio University School of Medicine, Tokyo, Japan

    Toshiro Sato

  5. Molecular Oncology, Genentech, South San Francisco, CA, USA

    Frederic J. de Sauvage

  6. German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Florian R. Greten

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Contributions

M. Schmitt, F.C., J.G. and F.R.G. designed the experiments. M. Schmitt, F.C., J.G., E.E., K.B.K., Y.D. and M. Schewe performed and analysed animal and organoid experiments. A.M.N. performed immunostaining and RT–PCR. M.P. generated mutant organoids and shRNA plasmids and contributed to animal experiments. A.C.K. performed and M.C.A. analysed Seahorse experiments. J.V. initiated the project. M.R. helped with FACS experiments. M.K. helped in generation of Sting-knockout organoids. T.W.B. performed siRNA experiments. V.P. performed ROS analysis and ATP measurements. A.A., T.S., M.C.A. and F.J.d.S. provided essential material. F.R.G. conceptualized and conceived the project. M. Schmitt, J.G. and F.R.G. wrote the manuscript. All of the authors commented on the manuscript draft.

Corresponding author

Correspondence to Florian R. Greten.

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Competing interests

M. Schmitt, J.G. and F.R.G. have filed a patent regarding the use of P2X4 inhibitors in combination with cytotoxic compounds. F.J.d.S. is an employee of Genentech and owns Roche shares. F.R.G. is a consultant for Amazentis, which is a company not related to this study. All other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 (Related to Figure 1): CRC resistance to 5-FU depends on mTORC1 but not mTORC2.

a, Immunoblot analysis of two different human tumor organoid lines derived from sporadic CRC treated as indicated (n = 3). b, Reseeding capacity of hCRC organoid lines treated with 5-FU, rapamycin or 5-FU/rapamycin (n = 3, one of three biological replicates). c, Reseeding capacity of doxycycline-inducible hCRCshRAPTORr#2 tumor organoids treated as indicated (n = 4, one of three biological replicates). d, Raptor qRT-PCR analysis of doxycycline treated hCRCshRAPTOR#2 tumor organoids (n = 3 biological replicates). e, Reseeding capacity of doxycycline-inducible hCRCshRICTOR tumor organoids treated as indicated (n = 4, one of three biological replicates). f, Rictor qRT-PCR analysis of doxycycline treated hCRCshRictor#1 tumor organoids (n=3 biological replicates). g, Reseeding capacity of doxycycline inducible hCRCshRICTOR#2 tumor organoids treated as indicated (n = 4, one of three biological replicates). h, Rictor qRT-PCR analysis of doxycycline treated hCRCshRictor#2 tumor organoids (n=3 biological replicates). i, Immunoblot analysis of Lgr5EGFP-DTR tumor organoids treated as indicated (n = 3). j) Profiles for Extracellular Acidification Rate (ECAR) of AOM/DSSATKN tumor organoids treated as indicated for 24h (n=4 for rapamycin treated organoids in all other conditions n=5 biological replicates of one experiment). k) Profiles for Oxygen Consumption Rate (OCR) of AOM/DSSATKN tumor organoids described in (j) (n = 4 for rapamycin treated organoids in all other conditions n = 5 biological replicates of one experiment). l) Percentage of metabolic potential over baseline in ECAR in organoids described in (j) (n = 4 for rapamycin treated organoids in all other conditions n = 5 biological replicates of one experiment). m) Percentage of metabolic potential over baseline in OCR in organoids treated described in (j) (n = 4 for rapamycin treated organoids in all other conditions n = 5 biological replicates of one experiment). All data are mean ±SD (except for j,k where data are mean ±SEM) and analysed by two-tailed Student’s t-test (d,f,h) or 1-way ANOVA with Bonferroni’s multiple comparison (b, c,e,g,l,m).

Extended Data Fig. 2 (related to Figure 2): Dying tumor cells activate mTORC1 in surrounding tumor cells in vivo.

a, Immunofluorescence analysis of p-S6 (red) and EGFP (Lgr5, green) expression in tumor tissues of vehicle or DT treated AOM/DSS mice 24h after injection. Nuclei were counterstained with DAPI. Representative images are shown (n = 3 biological replicates, scale bar = 50 µm). b, Immunoblot analysis of AOM/DSSATKN tumor organoids treated as indicated. For p-γH2AX and the corresponding α-tubulin loading samples were run on a separate gel (see SI). Representative results are shown (n = 3).

Extended Data Fig. 3 (related to Figure 3): Dying tumor cells activate mTORC1 via ATP/P2X4 in human CRC and PDAC.

a, Relative mRNA expression levels of the indicated genes in the colon tumors from Lgr5EGFP-DTR(+) mice 24h after injection of DT injection or vehicle determined by qRT-PCR. (n = 10 tumors for controls and n = 9 tumors for DT). b, Relative mRNA expression levels of the indicated genes in Lgr5EGFP-DTR(+) tumor organoids at 24h after DT treatment determined by qRT-PCR. Expression of vehicle treated organoids was set to 100 arbitrary units (n = 4 biological replicates). c, Immunoblot analysis of Lgr5EGFP-DTR(+) tumor organoids treated as indicated for 30 min (n = 3). d, Immunoblot analysis of Lgr5EGFP-DTR(+) tumor organoids treated as indicated for 16h (n = 3). e, Immunoblot analysis of Sting WT and Sting KO Lgr5EGFP-DTR(+) tumor organoids treated as indicated (n = 4). f, Immunoblot analysis of mouse (CMT93 and CT26, left panel) and human CRC cells (HCT116 and RKO, right panel) treated as indicated (n = 2). g, Immunoblot analysis of Lgr5EGFP-DTR(+) tumor organoids treated as indicated for 12h (n = 3). h, Left: Immunoblot analysis of ATP (100 µM) treated CT26 cells transfected with control or Rheb siRNA (n = 3). Right: Relative mRNA expression levels of Rheb in siRNA transfected cells determined by qRT-PCR (n = 3 biological replicates). i, Left: Immunoblot analysis of ATP (100 µM) treated CT26 cells transfected with control or a combination of RagA and RagB siRNA (n = 3). Right: Relative mRNA expression levels RagA and RagB in siRNA transfected cells determined by qRT-PCR (n = 3 biological replicates). j, Relative mRNA expression levels of P2x and P2y receptors in mouse colon tumor organoids tissues determined by qRT-PCR (n = 3 biological replicates). k, Immunohistochemistry analysis of P2X4 expression in healthy mouse colon or colon tumors of AOM/DSS treated mice. Images from one representative staining (n = 3, scale bar = 300 µm). l, Reseeding capacity of hCRC tumor organoid line 2 treated as indicated (n = 3, one of three biological replicates). m, Reseeding capacity of doxycycline-inducible hCRCshP2X4#2 tumor organoids treated as indicated (n = 4, one of three biological replicates). n, P24 qRT-PCR analysis of doxycycline treated hCRCshP2X4#2 tumor organoids (n = 3 biological replicates). o, Reseeding capacity of human pancreatic tumor organoids treated as indicated (n = 3, one of three biological replicates). p, Reseeding capacity of human pancreatic tumor organoids treated as indicated (n = 3, one of three biological replicates) All data are mean ±SD and analysed by two-tailed Student’s t-test (a,b,h,i,n,o,p) or by 1-way ANOVA with Bonferroni’s multiple comparison (l,m).

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Schmitt, M., Ceteci, F., Gupta, J. et al. Colon tumour cell death causes mTOR dependence by paracrine P2X4 stimulation. Nature 612, 347–353 (2022). https://doi.org/10.1038/s41586-022-05426-1

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