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Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth

Nature Cell Biology volume 23, pages 232–242 (2021)Cite this article

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Abstract

Lysosomes must maintain the integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but it is unknown whether dedicated programmes for maintaining the integrity of the lysosome membrane facilitate pancreatic cancer growth. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor Myoferlin as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosomal localization of Myoferlin is necessary and sufficient for the maintenance of lysosome health and provides an early acting protective system against membrane damage that is independent of the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances the pro-oncogenic activities of the lysosome.

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Fig. 1: Organelle proteomics identifies the Ferlin repair factors as PDA-specific lysosome-associated membrane proteins.
Fig. 2: PDA lysosomes are more resistant to lysosome-membrane damage.
Fig. 3: MYOF is essential for lysosome function in PDA cells.
Fig. 4: The N-terminal C2 domains of MYOF are required for membrane protection.
Fig. 5: MYOF is required for PDA tumour growth.
Fig. 6: High MYOF expression levels correlate with aggressive disease.

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Data availability

The mass spectrometry data have been deposited in MassIVE with the accession code MSV000086769. Source data are provided with this 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 all of the members of the Perera and Debnath laboratories for their helpful discussions. R.M.P. is the Nadia’s Gift Foundation Innovator of the Damon Runyon Cancer Research Foundation (grant no. DRR-46-17) and is additionally supported by an NIH Director’s New Innovator Award (grant no. 1DP2CA216364), the Pancreatic Cancer Action Network Career Development Award and the Hirshberg Foundation for Pancreatic Cancer. H.R.S. is supported by an AACR–Amgen fellowship in Clinical/Translational Cancer Research. G.R. is supported by the Belgian American Educational Foundation and the Léon Fredericq Foundation. R.Z. is supported by grants from the NIH (grant nos R01GM127763 and R01GM130995), a Damon Runyon–Rachleff Innovator Award and an Edward Mallinckrodt, Jr Foundation Grant. A.R. is supported by a Human Frontier Science Program Young Investigator Grant (grant no. RGY0076/2009-C), the Swiss National Fund for Research and an European Research Council Consolidator Grant. D.W.D. receives support from the Hirshberg Foundation for Pancreatic Cancer Research. We thank R. Zalpuri at the University of California, Berkeley Electron Microscope Laboratory for advice and assistance with electron-microscopy sample preparation and data collection.

Author information

Authors and Affiliations

  1. Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA

    Suprit Gupta, Julian Yano, Htet Htwe Htwe, Gilles Rademaker, Zeynep Cakir, Thomas Ituarte & Rushika M. Perera

  2. Department of Biochemistry, University of Geneva, Geneva, Switzerland

    Vincent Mercier & Aurélien Roux

  3. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Hijai R. Shin & Roberto Zoncu

  4. Department of Pathology, University of California, San Francisco, San Francisco, CA, USA

    Kwun W. Wen, Grace E. Kim & Rushika M. Perera

  5. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA

    Kwun W. Wen, Grace E. Kim & Rushika M. Perera

  6. Department of Pathology and Laboratory Medicine and Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA, USA

    David W. Dawson

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Contributions

S.G. performed the majority of the experiments and drafted the manuscript. J.Y. developed the FKBP-FRB* assay and conducted surface biotinylation experiments, molecular cloning and data analysis. V.M. conducted FLIM experiments and data analysis. H.H.H. assisted with the mouse experiments and immunohistochemistry. H.R.S. performed the electron microscopy. G.R. performed RT–qPCR and data analysis. Z.C. performed the proteinase K protection assay. T.I. conducted data and pathway analysis. K.W.W. and G.E.K. provided pathology analysis of patient samples. R.Z. provided intellectual feedback and supervised H.R.S. A.R. provided intellectual feedback and supervised V.M. D.W.D provided the PDA tissue microarray and conducted independent pathology analysis and statistical testing. R.M.P. conceived the project, supervised the research, and wrote and edited the manuscript.

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Correspondence to Rushika M. Perera.

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Peer review information Nature Cell Biology thanks Harald Stenmark, Kay Macleod and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 MYOF is a novel lysosomal membrane protein in PDA cells.

a, Co-localization of T192–mRFP-3xHA and endogenous LAMP2 in KP4 cells. b, RT-qPCR analysis of MYOF and DYSF mRNA levels across 10 human PDA cell lines and 3 non-PDA cell lines. Data is representative of n=3 independent biological replicate experiments. c,d, Immunofluorescence staining of MYOF–HA (green) and LAMP2 (red) in PDA cell lines (MiaPaCa, PaTu8902 and Panc0203) (c) and non-PDA (U20S and HPDE) cell lines (d). Arrowheads show examples of co-localization. e, Treatment of KP4, MiaPaCa and PaTu8988T cells with 75nM BafA1 for the indicated times causes an increase in p62 levels but not MYOF or DYSF. Graph shows the quantification of normalized fold change relative to LAMP1, averaged from 3 independent experiments. Data are mean ± s.d. Scale, 20μm for all panels. Statistics source data are provided.

Source data

Extended Data Fig. 2 PDA lysosomes are resistant to multiple membrane perturbing agents.

a, Time course of LysoTracker red staining in 293T and U20S cells following treatment with LLOMe. (293T, n= 86, 88, 88, 87 cells per time point ; U20S n= 69, 63, 69, 65 cells per time point). b, Immunoblot showing the expression of Cathepsin C and Cathepsin B in the indicated cell lines. Asterisk denotes cell lines used throughout the study. c, Immunofluorescence quantification of Magic Red assay-based cathepsin protease activity in n = 65 cells per cell line. d, Time course of LysoTracker red staining in HPDE and KP4 cells following treatment with 0.5M sucrose. e, Normalized fold change of LysoTracker staining. (HPDE, n = 64, 64, 65, 65 cells per time point; KP4, n = 64, 63, 63, 64 cells per time point). f, Time course of LysoTracker red staining in HPDE and KP4 cells following treatment with 100 μg/ml silica. g, Normalized fold change of LysoTracker staining. (HPDE, n = 65 cells per time point; KP4, n = 65 cells per time point). h, Immunoblots for the indicated proteins in MiaPaca cells following a time course of 1mM LLOMe treatment. i, Immunoblots for the indicated proteins in HPDE, KP4 and MiaPaca cells following treatment for 1hr with increasing doses of LLOMe. Scale, 20μm for all panels. For box-and-whisker plots centre lines indicate median values and whiskers represent minimum and maximum values. Data are mean ± s.d. P values determined by unpaired two-tailed Student’s t-tests. Statistics source data are provided. Unmodified blots are provided in Source Data Extended Data Fig. 2. Experiment depicted in b are representative of two independent experiments.

Source data

Extended Data Fig. 3 Recruitment of ESCRT proteins to PDA lysosomes is delayed following acute damage.

a-c, Time course of LLOMe (a), 0.5M sucrose (b), 100 μg/ml silica (c) treatment of HPDE and KP4 cells followed by immunofluorescence staining for ALIX (green) and LAMP1 (red). Graphs show the quantification of percentage co-localization of ALIX (LLOMe, n = 60 cells/condition for HPDE and KP4; sucrose, HPDE n = 13 fields/conditions, KP4 n= 13, 15, 15 fields/condition; silica, HPDE n = 14 fields/condition, KP4 n= 13, 13, 12 fields/condition) with LAMP1-positive lysosomes. d, Time course of LLOMe treatment of HPDE and KP4 cells followed by immunofluorescence staining for CHMP1A (green) and LAMP1 (red). e, Graph shows quantification of percentage co-localization of CHMP1A (n = 40 per cell line) with LAMP1-positive lysosomes. f, Immunoblot for the indicated proteins in lysosome fractions and flow through fractions isolated from HPDE- and KP4-T192–mRFP-3xHA stable cell lines treated with LLOMe for 10min. Scale, 20μm. Data are mean ± s.d. P values determined by unpaired two-tailed Student’s t-tests. Statistics source data are provided. Unmodified blots are provided in Source Data Extended Data Fig. 3. Experiment depicted in f are representative of two independent experiments.

Source data

Extended Data Fig. 4 MYOF loss leads to lysosome dysfunction in PDA cells.

a, Immunoblot of MYOF in KP4 and PaTu8902 cells. b, LAMP2 staining (arrowheads) in MYOF KO PaTu8902 cells. Graph shows lysosome diameter in control (Cas9; n = 252), MYOF KO #1 (n = 255) and MYOF KO #2 (n = 258) cells. c,d, LAMP2 staining (arrowheads) following shRNA-mediated knockdown of MYOF in PaTu8902 (c) and KP4 (d) cells. Graphs show lysosome diameter [PaTu8902 shGFP (n = 258), shMYOF#1 (n = 256), shMYOF#2 (n = 263); KP4 shGFP (n = 254), shMYOF#1 (n = 239), shMYOF#2 (n = 243) cells]. e, LysoTracker staining in KP4 cells following knockdown of MYOF. Graph shows normalized fold change in LysoTracker fluorescence in control (shGFP, n = 70 cells) and KD (shMYOF#1, n = 65; shMYOF#2, n = 77) conditions. f, Recruitment of CHMP1A (green) to LAMP1-positive lysosomes (red) in MYOF KO (#1, n = 60; #2, n = 61) relative to Cas9 control (n = 58) KP4 cells. Graph shows the percentage CHMP1A co-localization with LAMP1. g,h, Representative FLIM images (g) of lysosomes labelled with Lyso Flipper in KP4 cells 72hrs post-transfection with siCTRL (left) or siMYOF (right) and lifetime (Tau 1) measurements (h) from siCTRL and siMYOF transfected cells (n = 4 experiments). Scale, 10 µm. i, Lyso Flipper lifetime measurements from KP4 Cas9 and MYOF KO cells (n = 3 experiments). j, Galectin 3 (GAL3; green) and LAMP1 (red) staining following LLOMe treatment of KP4 Cas9 or MYOF KO cells (n = 60 cells/condition). k, LC3B staining in PaTu8902 cells in shGFP or shRNA MYOF knockdown cells (n = 14 fields per condition). Graph shows LC3B puncta per field. l, Degradation of macropinocytosed DQ-BSA in lysosomes [n = 54 (shGFP), 57 (shMYOF#1), 52 (shMYOF#2), n = 54 (BafA1) fluorescent spots/cell co-localizing with LAMP2-positive lysosomes]. m, Immunoblots from KP4 control (shGFP) or MYOF knockdown cells treated with or without 1mM LLOMe. Data are mean ± s.d. Scale, 20μm unless otherwise indicated. P values determined by unpaired two-tailed Student’s t-tests. Statistics source data are provided. Unmodified blots are provided in Source Data Extended Data Fig. 4. Experiment depicted in m are representative of two independent experiments.

Source data

Extended Data Fig. 5 Autophagy suppression reverses lysosome damage response in MYOF KO cells.

a, Immunoblot confirming shRNA-mediated knockdown of ATG3 and autophagy blockade in KP4 MYOF KO cells. b,c, Recruitment of CHMP1A (green) (b) or CHMP3 (green) (c) to lysosomes (LAMP1; red) in KP4 cells in the presence (WT) and absence (KO) of MYOF. shRNA-mediated knockdown of ATG3 to suppress autophagy causes a decrease in lysosome localization of CHMP1A and CHMP3 in MYOF KO cells (n = 12 fields per condition). Scale, 20μm. Data are mean ± s.d. P values determined by unpaired two-tailed Student’s t-test. Statistics source data are provided. Unmodified blots are provided in Source Data Extended Data Fig. 5. Experiment depicted in a are representative of two independent experiments.

Source data

Extended Data Fig. 6 Lysosomal targeting of MYOF delays onset of membrane damage.

a, U20S cells stably expressing T192-FLAG-FKBP and transiently transfected with MYOF-FRB* were treated with 1mM LLOMe for the indicated time points in the presence or absence of AP, followed by immunostaining for HA (green) and Galectin 3 (GAL3; red). Recruitment of MYOF-FRB* (green) protects against LLOMe induced GAL3 recruitment [n = 40 (control), 39 (15mins), 43 (30 mins), 41 (120mins) cells in the absence of AP and n = 39 (control), 45 (15mins), 41 (30mins) and 39 (120mins) cells in the presence of AP]. b, U20S cells stably expressing T192-FLAG-FKBP and transfected with MYOFΔC2-FRB* variant were treated as in ‘a’, followed by immunostaining for HA (green) and GAL3 (red). Recruitment of MYOFΔC2 (green) does not protect against LLOMe induced GAL3 recruitment [n = 26 cells per condition (-AP and +AP)]. Graphs at right show quantification of GAL3 spots per cell in response to LLOMe. Scale, 20μm for all panels. Data are mean ± s.d. P values determined by unpaired two-tailed Student’s t-tests. n.s. not significant. Statistics source data are provided.

Source data

Extended Data Fig. 7 MYOF is required for growth of mouse PDA tumours.

a, Immunoblot of the indicated proteins in mouse KPC cells (FC1245) following CRISPR-mediated knockout of Myof. b,c, Immunofluorescence staining of LC3B-positive autophagosomes in FC1245 Myof KO cells relative to Cas9 control cells. Graph shows quantification of LC3B puncta from n = 11 fields per condition. Data are mean ± s.d. d,e, Immunofluorescence images of LysoTracker red staining in FC1245 Myof KO cells compared to Cas9 control cells. Graph shows quantification from n = 70 (Cas9 control), 71 (KO#1), 70 (KO#2) cells per condition. Data are mean ± s.d. f, Growth rate of Cas9 control and Myof KO FC1245 allografts following s.c. transplantation in syngeneic C57BL/6 host mice. N=6 (Cas9 ctrl), 5 (KO#1), 5 (KO#2) animals per group. Scale, 20μm for all panels. Data represent mean ± s.e.m. P values determined by unpaired two-tailed Student’s t-tests. Statistics source data are provided. Unmodified blots are provided in Source Data Extended Data Figure 7. Experiment depicted in a are representative of two independent experiments.

Source data

Extended Data Fig. 8 DYSF expression in PDA patient samples.

DYSF transcript levels in human PDA patient specimens from 4 independent datasets as indicated. The number of samples are indicated under each graph in parentheses. Data are mean ± s.d. P values determined by unpaired two-tailed Student’s t-tests. Statistics source data are provided.

Source data

Extended Data Fig. 9 MYOF and DYSF expression in colorectal, NSCLC and breast cancer.

a,c,e, MYOF transcript levels in human colorectal cancer (CRC) (a), Non-small cell lung cancer (NSCLC) (c), and invasive breast cancer (e) patient specimens relative to normal colon, lung and breast from the indicated datasets (4 per tissue type). b,d,f, DYSF transcript levels in CRC (b), NSCLC (d), and invasive breast cancer (f) patient specimens relative to normal colon, lung and breast from the indicated datasets (as in a,c,e). Note MYOF levels are elevated in only 2 datasets while DYSF levels are elevated in 1 dataset. The number of samples are indicated under each graph in parentheses. Data are mean ± s.d. P values determined by unpaired two-tailed student’s t-tests. Statistics source data are provided.

Source data

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. Proteins identified in PDA-lysosome elutes that were >twofold significantly enriched. Supplementary Table 2. Cohort of PDA lysosomal proteins associated with vesicle trafficking and endocytosis that were >twofold significantly enriched. Supplementary Table 3. Clinicopathological characteristics and group membership in ULCA TMA (encompassing resected stage I/II pancreatic cancer). Supplementary Table 4. Cox proportional hazard models for prognostic factors. Supplementary Table 5. Antibodies used.

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Gupta, S., Yano, J., Mercier, V. et al. Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth. Nat Cell Biol 23, 232–242 (2021). https://doi.org/10.1038/s41556-021-00644-7

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