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PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia

Abstract

Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2+ xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2+ BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2+ BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2+ BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2+ BC, and possibly other malignancies, in the hypoxic tumour microenvironment.

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Figure 1: Increased hypoxia and necrosis in PTP1B-deficient HER2+ breast tumours.
Figure 2: PTP1B deficiency sensitizes HER2+ BC cells to hypoxia in vitro.
Figure 3: PTP1B-deficient BC cells die due to increased NMOC.
Figure 4: PTP1B deficiency activates multiple α-KG dioxygenases.
Figure 5: Generic α-KGDD inhibitors normalize hypoxia hypersensitivity and NMOC in PTP1B-deficient BC cells.
Figure 6: RNF213 is a novel PTP1B substrate that regulates oxygen consumption and survival of HER2+ BC cells in hypoxia.
Figure 7: PTP1B deficiency, via RNF213, alters the ubiquitylome in HER2+ BC cells.

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Acknowledgements

We thank G. Keller and T. W. Mak (Princess Margaret Cancer Center) for helpful comments on the manuscript. This work was funded by NIH grant R37 CA49152 and Canadian Institutes of Health Research (CIHR) grant 120593 (to B.G.N.), CIHR grant 62975 (to J.W.D.), CIHR grant 136956 (to S.S.S.), CIHR grant 133615 (to T.K.), Terry Fox New Frontiers Research Program PPG09-02005 (to B.G.W.), Cancer Research-UK and the Wellcome Trust (to S.E.W. and C.J.S), NIH grant GM96745 (to S.P.G.) and Kiban Kenkyu grant A-25253047 to A.K. Work in the Neel and Wouters laboratories was partially supported by the Princess Margaret Cancer Foundation and the Ontario Ministry of Health and Long Term Care. B.G.N. and J.W.D. are Canada Research Chairs, Tier 1, and B.G.W. is a Senior Investigator of the Ontario Institute for Cancer Research. T.K. is supported by the Canada Research Chair programme (Tier 2). R.M. was partially supported by a Post-doctoral Fellowship Grant, and R.S.B. by a Doctoral Fellowship Grant, both from the Canadian Breast Cancer Foundation. W.Z. was supported by a CIHR Post-doctoral Fellowship Grant. D.C. was supported by an Ontario Graduate Scholarship. A.A.R. was supported by a MITACS-Accelerate internship. A.S. was supported by the Medical Biophysics Excellence Award and the Kirsti Piia Callum Memorial Fellowship.

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Authors

Contributions

R.S.B. designed and performed most of the experiments, analysed and interpreted the data and wrote the manuscript. C.I. performed and analysed in vitro cell growth and tumour growth experiments. Y.X. performed LC–MS/MS to identify PTP1B-interacting proteins and substrates. R.M. provided conceptual advice and helped to design experiments. D.C. helped set up oxygen consumption measurements. A.A.R., J.P. and J.W.D. prepared, performed and helped to analyse the metabolomics experiments. W.Z. and S.S.S. assisted with the auto-ubiquitylation assays. A.S. performed LC–MS/MS to identify ubiquitylated proteins from HA–Ub pulldowns. C.M.R. and M.I. performed anti-diGly IP–MS to identify endogenous ubiquitylated peptides. S.Z. generated the RNF213-KO line and assisted with some of the ubiquitylation experiments. R.W. performed dot blots with anti-5-meC antibodies. C.V. helped with the bioinformatic analyses. T.Hitomi, T.Habu and A.K. provided reagents for RNF213 detection and expression. S.E.W. and C.J.S. provided conceptual advice and reagents for α-KGDDs. B.G.W. provided conceptual advice on hypoxia and metabolism experiments. B.G.N. conceived and supervised the project, helped to interpret the data and wrote the manuscript. All authors critically analysed data, and edited and approved the manuscript.

Corresponding author

Correspondence to Benjamin G. Neel.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PTP1B-knockdown (1B-KD) in HER2+ breast cancer (BC) cells does not affect proliferation in vitro.

(a) Immunoblot showing PTP1B-KD efficiency and expression of WT mPTP1B WT and a catalytically impaired mutant (RM) in HER2+ BC cells. (b) Growth curves of Control and 1B-KD HER2+ BC cells in normal media (DMEM or RMPI + 10%FBS, as indicated; see Methods). Data points represent mean values from three independent experiments done on different days; see Supplementary Table 8 for raw data. (c) Growth curves of the indicated cells maintained in low serum (1% FBS), low glucose (0.5 g l−1) or no glutamine conditions. Shown are data from three (low serum, low glucose) or two (low glutamine) independent experiments; see Supplementary Table 8 for raw data. (d) Immunoblot showing EGF-induced signaling events in Control and BT474 1B-KD cells. A similar lack of effect of PTP1B-deficiency on downstream signaling components was seen using the other 1B-KD lines, as well as in response to multiple other agonists. (e) Representative images (4× magnification) and quantification of soft agar colonies formed by Control and 1B-KD HER2+ BC cells. Data from two independent experiments are shown. (f) Representative images (10x magnification) of colonies formed by control and 1B-KD HER2+ BC cells in Matrigel. Scale bars represent 250 μM. Results represent mean ± s.e.m. Note that PTP1B deficiency has no consistent effect on HER2+ breast cancer cell proliferation in 2D- or 3D-cultures.

Supplementary Figure 2 Ptpn1−/−; MMTV-NeuNT mice exhibit more hypoxic hyperplastic lesions and delayed tumourigenesis.

(a) Kaplan-Meier curves showing percent survival of Ptpn1+/+; MMTV-NeuNT (30) and Ptpn1−/−; MMTV-NeuNT (20) mice in mixed (129/B6/FVB) background. b. Number of hyperplastic lesions per mammary gland in Ptpn1+/+; MMTV-NeuNT (n = 8) and Ptpn1−/−; MMTV-NeuNT mice at 9 months (n = 10). (c) Representative images of hyperplastic lesions from Ptpn1+/+; MMTV-NeuNT and Ptpn1−/−; MMTV-NeuNT mice, stained for H&E, BrdU (proliferation), EF5 (hypoxia) and CD31 (angiogenesis). Scale bars in 10× magnifications represent 250 μm; scale bars in 20× magnification represent 25 μm. Examples of positive staining are indicated by red arrows. (d) Percentage of cells positive for the indicated stain in hyperplastic lesions from Ptpn1+/+; MMTV-NeuNT (n = 30 lesions, obtained from 4 mice) and Ptpn1−/−; MMTV-NeuNT (n = 38 lesions, obtained from 5 mice). Each graph displays individual data points from every mouse (indicated by the color code), as well as the median, interquartile range (IQR) and whisker bars (1.5× IQR). Significance was determined by using a multi-level t-test (a.k.a., mixed effect/hierarchical model) that considers the lesion measurements from each mouse separately before comparing mouse level aggregate measurements (P < 0.05, precise values in figures). Note that PTP1B deficiency does not affect the number of hyperplastic lesions, but is associated with increased hypoxia.

Supplementary Figure 3 Characterization of Control and 1B-KD breast cancer xenografts.

(a) No apparent effect of PTP1B deficiency on receptor tyrosine kinase signaling. Immunoblot shows levels of pAKT (T308 and S473), pMEK (S217/221), pERK (T202/Y204), and pS6 (S240/244 or S235/236) in xenograft lysates; total ERK2 levels serve as loading controls. Each lane is from a separate tumour. (b) Representative images of BrdU, Ki67 and CD31 staining from BT474, BT474 1B-KD and BT474 1B-KD + m1B WT xenografts at 11 weeks post-injection. (c) Representative images of H&E, BrdU, CD31 and Ki67 from JIMT1 and JIMT1 1B-KD xenografts at 8 weeks post-injection. Insets and main images in b and c represent 0.4× and 10× magnifications, respectively. Scale bars from 0.4× magnifications represent 1 mm; scale bars from 10× magnifications represent 250 μm. (d) Quantification of BrdU, CD31 and Ki67 staining from BT474 (n = 6), BT474 1B-KD (n = 5), BT474 1B-KD + mPtpn1 WT (n = 6), JIMT1 (n = 6) and JIMT1 1B-KD (n = 6) tumours (from b and c). Graphs represent mean ± s.e.m., and were compared by two-tailed Student t-test (JIMT1) or one-way ANOVA, followed by Bonferroni post-hoc test (BT474). (e,f) Scatter plot of EF5 staining versus tumour size from BT474, JIMT1, HCC1954 and BT474-inducible PTPN1-knockdown xenografts (from Fig. 1). Note that 1B-KD HER2+ tumours, although smaller, display as much or increased EF5 staining compared with their larger parental counterparts.

Supplementary Figure 4 PTP1B-deficient HER2+ BC cells undergo non-apoptotic cell death in hypoxia (0.1% O2), but activate known hypoxia response pathways normally.

(a) Representative flow cytometric plots of Sytox blue (DNA stain) and Annexin V staining of Control and 1B-KD HER2+ BC cells exposed to 0.1% O2 for the indicated times. (b) Quantification of Annexin V and Sytox Blue populations from a (n = 4 biologically independent samples; also see Supplementary Table 8). Graphs indicate mean percentage of cells ± s.e.m. Statistical significance was evaluated by two-way ANOVA, followed by Bonferroni post-hoc test. (c) No consistent effect on mTOR pathway or autophagy in PTP1B-deficient HER2+ BC cells. Immunoblots show autophagic flux (by LC3 staining) and mTOR-dependent signaling (by pS6 S240/244) in Control and 1B-KD HER2+ BC cells, following exposure to 0.1% O2 for the indicated times. (d) Precocious HIF1α stabilization in PTP1B-deficient HER2+ BC cells. Immunoblots show HIF1α levels and downstream PDH phosphorylation in Control and 1B-KD HER2+ BC cells following exposure to hypoxia (0.1% O2), as indicated; these findings could be explained by the increased oxygen consumption in PTP1B-deficient cells, resulting in early HIF activation. (e) Scatter plot showing level of expression of 84 hypoxia genes (assessed by qPCR array) in the indicated Control and 1B-KD HER2+ BC cells exposed to 0.1% O2 for 8 h (SKBR3 and HCC1954) or 24 h (BT474 and MDA-MB-361). Array was assessed once for each set of cell lines. f,Expression levels of known HIF1α target genes VEGFA, GLUT1, CA9, PDK1 and REDD1 normalized to RPL13A from Control and 1B-KD HER2+ BC cells exposed to 0.1% O2 as indicated. For BT474, each gene was measured in three biologically independent replicate experiments. For the other lines, data from a single experiment are shown. For raw values, see Supplementary Table 8. (g) PTP1B-deficient HER2+ cells show precocious activation of UPR. Immunoblot shows PERK activation, as assessed by eIF2α phosphorylation, in Control and 1B-KD BC cells in response to exposure to hypoxia (0.1% O2), as indicated. ERK2 and eIF2α serves as loading controls.

Supplementary Figure 5 Mitochondrial mass and levels of glutamate metabolism enzymes are unaffected by PTP1B deficiency.

(a) Immunoblots show levels of the indicated mitochondrial proteins in Control and 1B-KD HER2+ BC cells under normoxia or 0.1% O2, as indicated. SOD1 serves as a loading control. (b) Total and active mitochondria in Control and 1B-KD BT474 cells were quantified by staining with Mitotracker green or CMXROS, respectively, and flow cytometric analysis. Graphs represent geometric mean fluorescent intensity ± s.e.m. Data points are from four biologically independent experiments. See Supplementary Fig. 8 for raw data. (c) Quantification of mitochondrial DNA (by qPCR) in Control and 1B-KD SKBR3 cells in normoxia or in 0.1% O2 hypoxia. Data are from one experiment; raw values are in Supplementary Table 8. (d) Immunoblots show levels of glutamate metabolism enzymes that could affect α-KG levels in Control and 1B-KD HER2+ BC cells; ERK2 serves as a loading control.

Supplementary Figure 6 PTP1B deficiency alters the metabolite profile in BT474 and SKBR3 cells.

(a) Heat map and (c) principal components analysis (PCA) showing levels of 139 metabolites (determined by LC-MS/MS; see Methods) in Control, 1B-KD, and 1B-KD + m1B PTP1B WT BT474 cells after 24 h in normoxia (21% O2) or hypoxia (0.1% O2). (b) Heat map and (d) PCA showing levels of 139 metabolites, in Control and 1B-KD SKBR3 cells in normoxia (21% O2) or hypoxia (0.1% O2) for 18 h. Note the PTP1B-dependent decrease in α-KG in both cell lines (red asterisk). (e) Schematic showing ≥2-fold differences in glycolytic and TCA metabolites in Control and 1B-KD BT474 and SKBR3 cells exposed to 0.1% O2 hypoxia; data in normoxia are shown in Fig. 4a. (f) IDH activity was measured in lysates of Control and 1B-KD BT474 and HCC1954 cells transfected with Control or IDH1 siRNAs. Data were derived from a single experiment with three replicate measurements at each time point. Raw data of independent repeats are in Supplementary Table 8. Activity seen after IDH1 knockdown represents IDH2 activity; the difference in activity between total and IDH1 knockdown cells represents IDH1 activity. ERK2 immunoblot serves as a loading control.

Supplementary Figure 7 RNF213 is a putative PTP1B substrate and regulates hypoxia survival and global ubiquitylation.

(a) PTP1B substrate-trapping mutant (CS/DA) identifies a known PTP1B substrate, EGFR, in BT474 cells. Cell lines were starved for 16 hours, then re-stimulated with EGF (50 ng ml−1), as indicated. Flag-mPTP1B WT- and CS/DA-expressing cells were lysed and subjected to anti-Flag immunoprecipitations. Immune complexes and total cell lysates were immunoblotted with anti-EGFR or anti-Flag antibodies. (b) Numbers of peptides (determined by LC-MS/MS) from proteins bound to WT Flag-mPTP1B or Flag-mPTP1B CS/DA, expressed in BT474 1B-KD BC cells in normoxia (21% O2) or hypoxia (0.1% O2). c,ARHGAP12 immunoblot of anti-Flag co-immunoprecipitates from BT474 1B-KD, 1B-KD + Flag-mPTP1B WT- or CS/DA-expressing cells. (d) Effect of ARHGAP12 knockdown on Control and 1B-KD HCC1954 cell survival after 24 h exposure to normoxia or 0.1% O2. Note depletion of ARHGAP12 at 72 hr post-transfection with ARHGAP12 siRNAs. e,f, Immunoblots showing HIF1αP564 hydroxylation in Control and 1B-KD BT474 (n = 3 biologically independent samples) and HCC1954 (n = 6 biologically independent samples) cells, with or without RNF213-KD, in normoxia or 0.1% O2. Graphs (mean ± s.e.m.) were compared two-way ANOVA, followed by Bonferroni post-hoc test (See Supplementary Table 8). (g) Immunoblot of RNF213 from Control or 1B-KD HCC1954 cells expressing RNF213 or control shRNAs. (h) Immunoblot showing RNF213 in HCC1954 xenografts (from Fig. 6g). (i) Scatter plot of EF5 staining versus tumour size from HCC1954 xenografts (from Fig. 6g–i). Immunoblot of HA-ubiquitin (HA-Ub) from BT474 cells treated with or without PTP1B inhibitor for 24 h (j) or Control and 1B-KD BT474 cells transfected with siControl or siRNF213 and empty vector or HA-Ub, exposed to 0.1% O2 for 24 h (k), and treated with or without proteasomal (MG132) and lysosomal (chloroquine) inhibitors for 3 h (from Fig. 7c, d). (l) HA-Ub immunoblots from Control and PTP1B-deficient BT474 cells treated with IOXI for 24 h. ERK2 and eIF2α as loading controls. (m) Venn diagram showing number of proteins with ≥1.5-fold increased ubiquitylation upon PTPN1-KD alone, ≤0.67-fold decreased ubiquitylation upon RNF213-KD alone or that are affected reciprocally by PTPN1- and RNF213-KD, as revealed by HA-Ub IP-MS or DiGly enrichment; see Supplementary Table 3.

Supplementary Figure 8 Unprocessed blots of key figures.

(a) Immunoblots from Fig. 6a showing that RNF213 interacts with PTP1B substrate-trapping mutant (CS/DA). (b) Immunoblots from Fig. 6b showing co-immunoprecipitation of RNF213 with Flag-mPtp1B WT or different substrate-trapping mutants in the absence and presence of vanadate, a general competitive inhibitor of protein-tyrosine phosphatases. (c) Immunoblots from Fig. 6c showing phospho-tyrosine levels of RNF213 immunoprecipitates from Control and 1B-KD BT474, HCC1954 and MDA-MB-361 cells. (d) Immunoblots showing RNF213 levels in Control and 1B-KD HER2+ BC cells transfected with siControl or siRNF213 (from Fig. 6d). e, Immunoblot showing RNF213 autoubiquitylation activity of Flag-RNF213 immunoprecipitates from Control and 1B-KD BT474 cells (from Fig. 7g). Red boxes indicate cropped portions that appear in the main figures.

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Banh, R., Iorio, C., Marcotte, R. et al. PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia. Nat Cell Biol 18, 803–813 (2016). https://doi.org/10.1038/ncb3376

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