PLCγ1 suppression promotes the adaptation of KRAS-mutant lung adenocarcinomas to hypoxia

Abstract

Mutant KRAS modulates the metabolic plasticity of cancer cells to confer a growth advantage during hypoxia, but the molecular underpinnings are largely unknown. Using a lipidomic screen, we found that PLCγ1 is suppressed during hypoxia in KRAS-mutant human lung adenocarcinoma cancer cell lines. Suppression of PLCγ1 in hypoxia promotes a less oxidative cancer cell metabolism state, reduces the formation of mitochondrial reactive oxygen species and switches tumour bioenergetics towards glycolysis by impairing Ca2+ entry into the mitochondria. This event prevents lipid peroxidation, antagonizes apoptosis and increases cancer cell proliferation. Accordingly, loss of function of Plcg1 in a mouse model of KrasG12D-driven lung adenocarcinoma increased the expression of glycolytic genes, boosted tumour growth and reduced survival. In patients with KRAS-mutant lung adenocarcinomas, low PLCγ1 expression correlates with increased expression of hypoxia markers and predicts poor patient survival. Thus, our work reveals a mechanism of cancer cell adaptation to hypoxia with potential therapeutic value.

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Fig. 1: Hypoxia alters phosphoinositide fatty acid composition and suppresses PLCγ1.
Fig. 2: PLCγ1 suppression promotes cancer cell survival during hypoxia.
Fig. 3: PLCγ1 suppression decreases mitochondrial respiration and enhances cancer cell glycolytic capacity.
Fig. 4: PLCγ1 suppression depletes mitochondrial ROS through impairment of Ca2+ entry into mitochondria.
Fig. 5: Ca2+ entry into mitochondria prevents the glycolytic shift and impairs the survival of cancer cells to hypoxia.
Fig. 6: PLCγ1 suppression reduces hypoxia-induced lipid peroxidation.
Fig. 7: Plcg1 deletion accelerates Kras-driven lung tumorigenesis and results in poor survival.
Fig. 8: Low PLCγ1 levels in human lung adenocarcinomas correlate with poor patient survival.

Data availability

All supporting data are included in this published article and its Supplementary Information. Requests to use the PLCγ1 floxed mice will be redirected to F.H.H. and T.M.S. The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD) dataset was retrieved from the Genomic Data Commons Portal: http://cancergenome.nih.gov. The data were downloaded with the help of the web graphic user interface Xena browser https://xenabrowser.net. Source data are provided with this paper.

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Acknowledgements

We wish to thank J. Sondek for providing the rat PLCγ1 plasmid (UNC Center for Structural Biology, Chapel Hill, USA). The NSCLC cell lines were provided by J. D. Minna (UTSW medical center, USA). We thank A. Traynor-Kaplan (University of Washington, USA) for help with mass spectrometry analysis. We thank G. Ramadori (University of Geneva, Switzerland) for critically reading the manuscript. This study was supported by the German Research Council (DFG; HE6233/4-1 to F.H.H. and SCHN15561-1 to T.M.S.), the Thuringian state program ProExzellenz (RegenerAging-FSU-I-03/14) of the Thuringian Ministry for Research (TMWWDG; to F.H.H.), local funds from the University of Ferrara (FIR-2017), the Italian Ministry of Health (GR-2016-02364602) and the Italian Ministry of Education, University and Research (PRIN Grant 2017XA5J5N) (to A.R.), the Italian Association for Cancer Research (AIRC, IG-18624), Telethon (GGP11139B), the Italian Ministry of Education, University and Research (PRIN Grant 2017E5L5P3), local funds from the University of Ferrara (to P.P.), and the Swiss National Science Foundation (#PP00P3_163929) Professorship (to G.K.).

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Contributions

G.K. and M.S. conceived and designed the experiments. M.S., M.R.S., C.P. and G.K. performed and analysed the data. F.H.H. and T.M.S. generated and provided the Plcg1fl/fl mice. S.S.P., L.B. and S.A.B. (pathologists) and R.S. (surgeon) provided the human lung tissue samples, and S.A.B. performed the PLCγ1 staining grading. A.R. and P.P. performed the Ca2+ measurements. J.B. and S.F. performed the Seahorse experiments. G.K. supervised the study. G.K. and M.S. wrote the manuscript.

Corresponding author

Correspondence to Georgia Konstantinidou.

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Extended data

Extended Data Fig. 1 Hypoxia suppresses PLCγ1 and alters the fatty acid composition of phosphoinositides.

a-d, Total PI (a), PI4P (b), PI(4,5)P2 (c) and PI(3,4,5)P3 (d) peak areas of A549 cells in normoxia and upon shift to hypoxia (1% O2) for 48h measured by ultra-performance liquid chromatography - tandem mass spectrometry; n = 4/group. e, Schematic representation of phosphoinositol signalling and the metabolic fate of PI(4,5)P2. PM: plasma membrane. PI: phosphatidylinositol, PI4P: phosphatidylinositol-4-phosphate, PtdIns(4,5)P2: phosphatidylinositol-4,5-biphosphate, PI(3,4,5)P3: phosphatidylinositol-3,4,5-trisphosphate. f, HIF1a mRNA expression level in A549 cells (relative to normoxia control at 24h). Cells were transduced with either an empty vector (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against HIF1α and incubated in normoxia or hypoxia for the indicated time; n = 3. g, Heatmap display of PI(4,5)P2 lipid alterations (fold change). A549 cells were transduced with either an empty vector control (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1, treated with doxycycline for 48h and treated as in (a); n = 4/group. h, Peak areas of the indicated PI(4,5)P2 lipid species in A549 cells from (g); n = 4/group. i, Total PI(4,5)P2 peak areas in A549 cells treated as in (g); n = 4/group.All graphical data are presented as mean ± SD. Statistical analyses were done using two-tailed unpaired Student’s t test or one-way ANOVA; n, number of biologically independent samples. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical source data are provided in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 PLCγ1 suppression promotes cancer cell proliferation during hypoxia.

a, Immunoblot analysis of the indicated targets in H358 and A427 cells transduced with either an empty vector control (Tet-pLKO-puro, shControl) or 2 doxycycline-inducible shRNAs against PLCγ1. Cells were incubated in the presence of doxycycline for 72h in normoxia before performing the immunoblot analysis. b,c, Relative cell number of A549, H358 and A427 cells transduced with either an empty vector control (Tet-pLKO-puro, shControl), or a doxycycline-inducible shRNA against PLCγ1 (as indicated). After this, cells were plated and incubated for the indicated time in normoxia or hypoxia; n = 3. d, Immunoblot analysis of the indicated targets in A549 cells transduced as in (b). Cells were then incubated in the presence of doxycycline and transfected with either pcDNA3.1 empty vector or pcDNA3.1-rPLCγ1 (shRNA-resistant PLCγ1) and incubated for 72h in normoxia or hypoxia. e, Immunoblot analysis of the indicated targets in A549 cells transduced as in (a). f,g, Immunoblot analysis of the indicated targets (f) and cell proliferation assay (g) of A549 cells treated as indicated in normoxia or hypoxia; n = 3.All graphical data are presented as mean ± SD. Statistical analyses were done using one-way ANOVA; n, number of biologically independent samples. **** p < 0.0001. Statistical source data and unprocessed immunoblots are provided in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 PLCγ1 suppression promotes cancer cell survival during hypoxia.

a, Representative panels of Annexin V-Atto 633/Propidium iodide (PI) flow cytometry analysis (left) and relative quantification of Annexin V (AV) + Annexin V/PI (AV/PI)-positive cells (right) in A549 cells transduced with either an empty vector control (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1, incubated without/with doxycycline for 48h and moved in normoxia or hypoxia for 72h; n = 3. b, Caspase-3 activity assay in A549 cells transduced as in (a). Cells were then transfected with either pcDNA3.1 empty vector or pcDNA3.1-rPLCγ1 (shRNA-resistant PLCγ1), and incubated in normoxia or hypoxia for 48h. Staurosporine (STA, 100nM) is positive control of caspase-dependent death; n = 3. c, Quantification of Annexin V (AV) + Annexin V/PI (AV/PI)-positive cells by flow cytometry in A549 cells transduced as in (a). Cells were then treated with Q-VD-OPH (QVD), a pan-caspase inhibitor for 24h; n = 3. d, Representative panels of Annexin V-Atto 633/Propidium iodide (PI) flow cytometry analysis (left) and relative quantification of Annexin V (AV) + Annexin V/PI (AV/PI)-positive cells (right) in A549 cells transduced with either pcDNA3.1 empty vector or pcDNA3.1-PLCγ1 and incubated in hypoxia for 72h; n = 3. e, Immunoblot analysis of the indicated targets in MEF cells with the indicated genotype in normoxia or hypoxia. The samples derive from the same experiment and the gel/blot were processed in parallel. f, Representative panels of Annexin V-Atto 633/Propidium iodide (PI) flow cytometry analysis of MEF cells with the indicated genotype. Graphical data are mean ± SD. Statistical analyses were done using two-tailed unpaired Student’s t test or one-way ANOVA; n, number of biologically independent samples. **** p < 0.0001. Statistical source data and unprocessed immunoblots are provided in Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 PLCγ1 suppression decreases mitochondrial respiration and enhances cancer cell glycolytic capacity in human NSCLC cells.

a, Immunoblot analysis of the indicated targets in A549 cells transduced with either an empty vector control (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1, incubated without/with doxycycline for 48h and further incubated for 48h in normoxia or hypoxia (3% O2). b-e, Graphs showing oxygen consumption rate (OCR, b and c) and extracellular acidification rate (ECAR, d and e) of A549 cells transduced and treated as in (a); n replicates/group: (b) = 8, (c) = 7 normoxia/8 hypoxia, (d) = 9 normoxia/ 8 hypoxia, (e) = 8. Panels (c and e) are treated without/with doxycycline and serve as a control for the doxycycline effect on cells. f, Bar graph showing ECAR parameters in A549 cells transduced as in (a). Cells were transfected with either pcDNA3.1 empty vector or pcDNA3.1-rPLCγ1 (shRNA-resistant PLCγ1) to rescue the shPLCγ1 #2 and incubated for 48h in normoxia or hypoxia; n = 8. g-h, Oxygen consumption rate (g) and extracellular acidification rate (h) parameters of A549 cells transfected with either pcDNA3.1 empty vector or pcDNA3.1-PLCγ1 and incubated for 48h in normoxia or hypoxia; (g) n = 8 for normoxia and 6 for hypoxia groups, (h) n = 7.OCR was determined during sequential treatments with oligomycin, FCCP and rotenone/antimycin (AA). ECAR was determined during sequential treatments with glucose (Glc), oligomycin and 2 deoxyglucose (2DG).Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n, number of biologically independent samples. **** p < 0.0001. Statistical source data and unprocessed immunoblots are provided in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 PLCγ1 suppression decreases mitochondrial respiration and enhances cancer cell glycolytic capacity in MEFs.

a, Immunoblot analysis of the indicated targets in MEF cells with the indicated genotype transfected with either pcDNA3.1-HA-LIC empty vector or pcDNA3.1-HA-LIC-PLCγ1. 24h later, cells were moved in hypoxia (3% O2) and incubated for additional 48h. b, Oxygen consumption rate (OCR) graph of MEF cells of the indicated genotypes. Cells were incubated for 48h in normoxia or hypoxia (3% O2) before seahorse experiment. OCR was determined during sequential treatments (indicated with arrows) with oligomycin, FCCP and rotenone/antimycin (AA) at the indicated time; n = 3 for the normoxia groups and n = 6 for the hypoxia groups. c, Graph showing extracellular acidification rate (ECAR) of MEF cells with the indicated genotype transduced as in (a). ECAR was determined during sequential treatments (indicated with arrows) with glucose (Glc), oligomycin and 2 deoxyglucose (2DG) at the indicated time; n = 6. d, Flow cytometry panels showing mean fluorescent intensity of A549 cells from experiment reported in main Fig. 3i. A549 cells were previously transduced with either an empty vector control (Tet-pLKO-puro, shControl) or 2 different doxycycline-inducible shRNAs against PLCγ1 (shPLCγ1 #1, shPLCγ1 #2), incubated in the presence of doxycycline for 48h, incubated for additional 48h in normoxia or hypoxia (1% O2), stained with LipidTOX and analyzed by flow cytometry. Sh#1: shPLCγ1 #1, Sh#2: shPLCγ1 #2; n = 3. Graphical data are mean ± SD; n, number of biologically independent samples. Statistical source data and unprocessed immunoblots are provided in Source Data Extended Data Fig. 5. Source data

Extended Data Fig. 6 PLCγ1 suppression depletes mitochondrial ROS through impairment of Ca2+ entry into the mitochondria.

a, Flow cytometry histograms (left) and quantification of DHR (green, right) of A549 cells transduced with an empty vector (Tet-pLKO-puro, shControl) and incubated without/with doxycycline (DOX) for 48h, incubated for additional 48h in normoxia or hypoxia and stained with DHR for flow-cytometry. H2O2: positive control. DHR: Dihydrorhodamine. Normo: normoxia. Hypo: hypoxia. n = 3. b, MCU mRNA levels in A549 cells transduced with an empty vector (Tet-pLKO-puro, shControl) or 2 doxycycline-inducible shRNAs against MCU and incubated with doxycycline for 72h; n = 3. c, Immunoblot analysis of the indicated targets in A549 cells transduced with an empty vector (Tet-pLKO-puro, shControl) or 2 doxycycline-inducible shRNA against PLCγ1, transfected with either pcDNA3.1 empty vector or pcDNA3.1-MCU-flag plasmid and incubated in normoxia or hypoxia for 48h. d, Representative flow cytometry histograms of DHR (green) mean fluorescent intensity of A549 cells from experiment reported in main Fig. 4f. Sh#2: shPLCγ1 #2. e, Representative flow cytometry histograms (left) and quantification of DHR (green) of A549 cells transduced with an empty vector (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1. Cells were then treated with doxycycline for 24h, transfected with either pcDNA3.1 empty vector or pcDNA3.1-MCU-flag plasmid and moved to hypoxia for additional 48h before staining cells with DHR for analysis. DHR: Dihydrorhodamine. Sh#1: shPLCγ1 #1; n = 3. f, Cytosolic (left) and mitochondrial (right) Ca2+ response of A549 cells transduced as in (c), transfected with appropriate targeted-aequorin and histamine-induced Ca2+ was measured 48h after incubation in normoxia or hypoxia; n/group = cytosolic normoxia 7, 8, 7, 7, 8, 7/cytosolic hypoxia 8, 6, 7, 8, 7, 8. n/group = mitochondrial normoxia 11, 7, 9, 10, 8, 6/mitochondrial hypoxia 7, 10, 9, 9, 9, 11.Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n, number of biologically independent samples. **** p < 0.0001. Statistical source data and unprocessed immunoblots are provided in Source Data Extended Data Fig. 6. Source data

Extended Data Fig. 7 PLCγ1 suppression enhances cell proliferation and decreases cell death in hypoxia through impairment of Ca2+ entry into the mitochondria.

a, Immunoblot analysis of the indicated targets in MEFs generated from a LSL-KrasG12D/WT;p53flox/flox;Plcg1wt/wt and LSL-KrasG12D/WT;p53flox/flox;Plcg1flox/flox mouse model, stably transduced with Cre recombinase (pMSCV-hygro-Cre) and transfected with either pcDNA3.1 empty vector or pcDNA3.1- MCU-flag plasmid and moved to hypoxia (1% O2) for additional 48h. b,c, Oxygen consumption rate (b) and extracellular acidification rate (c) of A549 cells transduced with either an empty vector (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1, treated with doxycycline for 24h, transfected with either pcDNA3.1 empty vector or pcDNA3.1-MCU-flag plasmid and incubated for 48h in hypoxia (3% O2). OCR was determined during sequential treatments (indicated with arrows) with oligomycin, FCCP and rotenone/antimycin (AA) at the indicated time. ECAR was determined during sequential treatments (indicated with arrows) with glucose (Glc), oligomycin and 2 deoxyglucose (2DG) at the indicated time; n = 5. d, Fatty acid β-oxidation of A549 cells transduced and treated as in (b); n = 4. e, Relative cell number of A549 cells transduced and treated as in (b); n = 3. Normoxia: * p = 0.03, hypoxia: * p = 0.014, *** p = 0.0005. f, Representative Annexin V-Atto 633/Propidium iodide (PI) flow cytometry analysis panels of A549 cells from experiment reported in main Fig. 5e; n = 3. g, Representative Annexin V-Atto 633/Propidium iodide (PI) flow cytometry analysis panels of MEF cells from experiment reported in main Fig. 5f; n = 3. Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n, number of biologically independent samples. **** p < 0.0001. Statistical source data and unprocessed immunoblots are provided in Source Data Extended Data Fig. 7. Source data

Extended Data Fig. 8 PLCγ1 suppression reduces hypoxia-induced lipid peroxidation.

a, Confocal microscopy of A549 cells transduced with either an empty vector (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1 (shPLCγ1 #1) and incubated in the presence of doxycycline for 48h. Cells were then incubated for 48h in normoxia or hypoxia (1% O2), stained with BODIPY 581/591 C11 (green: oxidized lipids / red: non-oxidized lipids), LipidTOX (grey) to mark lipid droplets and Hoechst (nuclei). White squares represent magnified areas on the right. Scale bars: 20 μm. b, Quantification of lipid peroxidation expressed as percent of oxidized lipids from (a); n/group = 13, 13, 12, 14. c, Quantification of oxidized lipids, expressed as percent, localized in lipid droplets from (a); n/group = 13, 13, 13, 14. d, Relative cell number of A549 (left) and H358 (right) cells transduced with either an empty vector (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1 (shPLCγ1 #1), incubated with doxycycline for 48h and treated as indicated with the DGAT1 inhibitor (T863). The graphs represent day 6 of the cell proliferation assay; n = 3. e, Lipid droplet quantification (relative to control) of A549 and H358 cells treated as in (d), stained with LipidTOX (far red) and analyzed by flow cytometry; n = 3.Graphical data are mean ± SD except the panels b and c that are mean ± SEM. Statistical analyses were done using one-way ANOVA; n, number of biologically independent samples. **** p < 0.0001. Statistical source data are provided in Source Data Extended Data Fig. 8. Source data

Extended Data Fig. 9 PLCγ1 deletion accelerates lung tumorigenesis in mice.

a, Representative hematoxylin & eosin (H&E) staining of LSL-KrasG12D/WT;p53flox/flox;Plcg1wt/wt, LSL-KrasG12D/WT;p53flox/flox;Plcg1wt/flox and LSL-KrasG12D/WT;p53flox/flox;Plcg1flox/flox mouse lung sections, 10 weeks after Cre induction. Scale bar: 5000μm. b, Representative immunohistochemistry images showing Ki67-positive cell staining in mouse lung tumors from LSL-KrasG12D/WT;p53flox/flox;Plcg1wt/wt, KrasG12D/WT;p53flox/flox;Plcg1wt/flox and LSL-KrasG12D/WT;p53flox/flox;Plcg1flox/flox mice 10 weeks after Cre induction. This is related to main Fig. 7e.

Extended Data Fig. 10 Low PLCγ1 levels in human lung adenocarcinomas correlate with poor patient survival.

a, Antibody validation for IHC against PLCγ1 on human tumor tissue microarray. A549 cells previously transduced with either an empty vector (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1 (shPLCγ1 #1) and incubated in the presence of doxycycline for 48h before paraffin embedding. Subsequently IHC was performed as for TMA staining (described in materials and methods). The arrow heads indicate PLCγ1-positive cells shown at higher magnification in the inset b, Representative images of the human lung adenocarcinoma TMA showing the staining intensity with the matched scoring by the pathologist upon immunohistochemistry against PLCγ1. Score 0-I was considered low staining intensity, II was considered moderate and score III was considered high staining intensity.

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Supplementary Fig. 1.

Reporting Summary

Supplementary Tables 1 and 2

Supplementary Table 1: detailed reagents and tools information. Supplementary Table 2: list of the patient age, gender and corresponding PLC 0-3 score (used in the TMA).

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Saliakoura, M., Rossi Sebastiano, M., Pozzato, C. et al. PLCγ1 suppression promotes the adaptation of KRAS-mutant lung adenocarcinomas to hypoxia. Nat Cell Biol 22, 1382–1395 (2020). https://doi.org/10.1038/s41556-020-00592-8

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