Calpain-6 controls the fate of sarcoma stem cells by promoting autophagy and preventing senescence
Caroline Andrique, Laetitia Morardet, Laetitia K. Linares, Madi Y. Cissé, Candice Merle, Frédéric Chibon, Sylvain Provot, Eric Haÿ, Hang-Korng Ea, Martine Cohen-Solal, Dominique Modrowski
Caroline Andrique, Laetitia Morardet, Laetitia K. Linares, Madi Y. Cissé, Candice Merle, Frédéric Chibon, Sylvain Provot, Eric Haÿ, Hang-Korng Ea, Martine Cohen-Solal, Dominique Modrowski
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Research Article Oncology

Calpain-6 controls the fate of sarcoma stem cells by promoting autophagy and preventing senescence

Abstract

Sarcomas are still unsolved therapeutic challenges. Cancer stem cells are believed to contribute to sarcoma development, but lack of specific markers prevents their characterization and targeting. Here, we show that calpain-6 expression is associated with cancer stem cell features. In mouse models of bone sarcoma, calpain-6–expressing cells have unique tumor-initiating and metastatic capacities. Calpain-6 levels are especially high in tumors that have been successfully propagated in mouse to establish patient-derived xenografts. We found that calpain-6 levels are increased by hypoxia in vitro and calpain-6 is detected within hypoxic areas in tumors. Furthermore, calpain-6 expression depends on the stem cell transcription network that involves Oct4, Nanog, and Sox2 and is activated by hypoxia. Calpain-6 knockdown blocks tumor development in mouse and induces depletion of the cancer stem cell population. Data from transcriptomic analyses reveal that calpain-6 expression in sarcomas inversely correlates with senescence markers. Calpain-6 knockdown suppresses hypoxia-dependent prevention of senescence entry and also promotion of autophagic flux. Together, our results demonstrate that calpain-6 identifies sarcoma cells with stem-like properties and is a mediator of hypoxia to prevent senescence, promote autophagy, and maintain the tumor-initiating cell population. These findings open what we believe is a novel therapeutic avenue for targeting sarcoma stem cells.

Authors

Caroline Andrique, Laetitia Morardet, Laetitia K. Linares, Madi Y. Cissé, Candice Merle, Frédéric Chibon, Sylvain Provot, Eric Haÿ, Hang-Korng Ea, Martine Cohen-Solal, Dominique Modrowski

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Figure 1

Calpain-6–expressing cells are tumor-initiating cells.

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Calpain-6–expressing cells are tumor-initiating cells. (A) Fluorescence ...
(A) Fluorescence microscopy of Calp6-P-GFP K7M2 cells. (B) Flow cytometry of GFP in Calp6-P-GFP K7M2 cells. (C) RT-qPCR analysis of calpain-6 mRNA expression in sorted Calp6-P-GFP– and -GFP+ K7M2 cells. Data are the mean ± SD from 3 independent RNA extracts compared by 2-tailed Student’s t test. (D) Immunofluorescence of luciferase-expressing cells (tumor cells) in sections of bone injected with unsorted Luc-Calp6-P-GFP or sorted Luc-Calp6-P-GFP+ or -GFP– K7M2 cells. The bones were collected at 6 weeks after implantation. Bone tissues from noninjected mice served as control. Scale bars: 100 μm. (E) Quantification of bioluminescent signals in tibias of Luc-Calp6-P-GFP K7M2–implanted mice during tumor growth. n = 6 mice/group. Data are medians, box edges are the interquartile range, and whiskers are the range. Outliers were identified by Grubbs’ test (α = 0.05). Data were compared by 2-way ANOVA. (F) Typical images of the bioluminescent signals in tibias of K7M2-implanted mice. (G) Representative H&E-stained lung sections from mice implanted for 6 weeks with unsorted Calp6-P-GFP or sorted Calp6-P-GFP– or -GFP+ K7M2 cells. Scale bars: 100 μm. (H) Metastasis area. n = 7 mice (unsorted Calp6-P-GFP K7M2 cells), n = 7–8 mice (sorted Calp6-P-GFP– or -GFP+ K7M2 cells). Data are the mean ± SD compared by 1-way ANOVA. (I) Crystal violet staining of K7M2 cells in lung cell cultures. Each well was seeded with lung cells from a K7M2 bone–implanted mouse. Arrows indicate cell clones.