Calpain 2 Isoform-Specific Cleavage of Filamin A Enhances HIF1α Nuclear Translocation, Promoting Metastasis in Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) remains a challenge due to its aggressive nature and limited therapeutic options. Calpain 2, a member of the calcium-dependent cysteine protease family, is particularly associated with poor prognosis in TNBC. This study explores the isoform-specific role of calpain 2 in TNBC, examining its correlation with prognosis and its mechanistic impact on metastasis. Bioinformatic analyses, including Kaplan–Meier survival plots, univariate Cox proportional analysis, and gene set enrichment analysis (GSEA), assessed CAPN2 expression and its association with mesenchymal genes in TNBC. Results of cell-based experiments with CAPN2 knockdown or overexpression indicate that elevated CAPN2 expression correlates with poor clinical outcomes and enhanced metastatic potential in TNBC. CAPN2 knockdown inhibited the epithelial–mesenchymal transition (EMT), reducing cancer cell proliferation, migration, and invasion. Calpain 2 downregulation reversed the EMT by reducing isoform-specific cleavage of filamin A, HIF1α nuclear localization and TWIST1 transcription. CNa 29, a calpain 2-specific inhibitor, reduced cancer cell proliferation, decreased filamin A cleavage, downregulated TWIST1 expression, and significantly retarded metastasis,. In conclusion, calpain 2 plays a critical role in TNBC progression by modulating HIF1α and TWIST1, to promote the EMT and metastasis. Isoform-selective inhibition of calpain 2 with CNa 29 presents a promising therapeutic strategy for managing TNBC.

Keywords

calpain 2 / calpain inhibitor / epithelial–mesenchymal transition / metastasis / triple-negative breast cancer

References

[1] A. C. Garrido-Castro, N. U. Lin, and K. Polyak, “Insights Into Molecular Classifications of Triple-Negative Breast Cancer: Improving Patient Selection for Treatment,” Cancer Discovery 9 (2019): 176-198.

[2] G. Bianchini, C. De Angelis, L. Licata, and L. Gianni, “Treatment Landscape of Triple-Negative Breast Cancer—Expanded Options, Evolving Needs,” Nature Reviews Clinical Oncology 19 (2022): 91-113.

[3] Y. Li, H. Zhang, Y. Merkher, et al., “Recent Advances in Therapeutic Strategies for Triple-Negative Breast Cancer,” Journal of Hematology & Oncology 15 (2022): 121.

[4] O. Ginsburg, F. Bray, M. P. Coleman, et al., “The Global Burden of Women's Cancers: A Grand Challenge in Global Health,” Lancet 389 (2017): 847-860.

[5] B. D. Lehmann, J. A. Bauer, X. Chen, et al., “Identification of Human Triple-Negative Breast Cancer Subtypes and Preclinical Models for Selection of Targeted Therapies,” Journal of Clinical Investigation 121 (2011): 2750-2767.

[6] S. Y. Hwang, S. Park, and Y. Kwon, “Recent Therapeutic Trends and Promising Targets in Triple Negative Breast Cancer,” Pharmacology & Therapeutics 199 (2019): 30-57.

[7] X. Guan, “Cancer Metastases: Challenges and Opportunities,” Acta Pharmaceutica Sinica B 5 (2015): 402-418.

[8] S. Liu and M. S. Wicha, “Targeting Breast Cancer Stem Cells,” Journal of Clinical Oncology 28 (2010): 4006-4012.

[9] B. Zhang, R. Zhao, Q. Wang, et al., “An EMT-Related Gene Signature to Predict the Prognosis of Triple-Negative Breast Cancer,” Advances in Therapy 40 (2023): 4339-4357.

[10] Y. Qi, X. J. Wu, J. B. Shi, et al., “Sanhuang Xiexin Decoction Ameliorates TNBC by Modulating JAK2-STAT3 and Lipid Metabolism,” Chinese Journal of Integrative Medicine 30, no. 12 (2023): 1080-1089.

[11] K. Ganesan, C. Xu, J. Wu, et al., “Ononin Inhibits Triple-Negative Breast Cancer Lung Metastasis by Targeting the EGFR-Mediated PI3K/Akt/mTOR Pathway,” Science China Life Sciences 67 (2024): 1849-1866.

[12] T. Guan, M. Li, Y. Song, et al., “Phosphorylation of USP29 by CDK1 Governs TWIST1 Stability and Oncogenic Functions,” Advanced Science 10 (2023): e2205873.

[13] H. Sorimachi, S. Ishiura, and K. Suzuki, “Structure and Physiological Function of Calpains,” Biochemical Journal 328, no. Pt 3 (1997): 721-732.

[14] A. Lakshmikuttyamma, P. Selvakumar, R. Kanthan, S. C. Kanthan, and R. K. Sharma, “Overexpression of M-Calpain in Human Colorectal Adenocarcinomas,” Cancer Epidemiology and Prevention Biomarkers 13 (2004): 1604-1609.

[15] A. Mamoune, J. H. Luo, D. A. Lauffenburger, and A. Wells, “Calpain-2 as a Target for Limiting Prostate Cancer Invasion,” Cancer Research 63 (2003): 4632-4640.

[16] C. Miao, C. Liang, Y. Tian, et al., “Overexpression of CAPN2 Promotes Cell Metastasis and Proliferation via AKT/mTOR Signaling in Renal Cell Carcinoma,” Oncotarget 8 (2017): 97811-97821.

[17] H. S. Jang, S. Lal, and J. A. Greenwood, “Calpain 2 Is Required for Glioblastoma Cell Invasion: Regulation of Matrix Metalloproteinase 2,” Neurochemical Research 35 (2010): 1796-1804.

[18] S. J. Storr, K. W. Lee, C. M. Woolston, et al., “Calpain System Protein Expression in Basal-Like and Triple-Negative Invasive Breast Cancer,” Annals of Oncology 23 (2012): 2289-2296.

[19] N. Afifi and C. A. Barrero, “Understanding Breast Cancer Aggressiveness and Its Implications in Diagnosis and Treatment,” Journal of Clinical Medicine 12 (2023): 1375.

[20] Z. Deng, H. Wang, J. Liu, Y. Deng, and N. Zhang, “Comprehensive Understanding of Anchorage-Independent Survival and Its Implication in Cancer Metastasis,” Cell Death & Disease 12 (2021): 629.

[21] Y. Wang, J. Liu, X. Ying, P. C. Lin, and B. P. Zhou, “Twist-Mediated Epithelial-Mesenchymal Transition Promotes Breast Tumor Cell Invasion via Inhibition of Hippo Pathway,” Scientific Reports 6 (2016): 24606.

[22] Q. Q. Zhu, C. Ma, Q. Wang, Y. Song, and T. Lv, “The Role of TWIST1 in Epithelial-Mesenchymal Transition and Cancers,” Tumour Biology 37 (2016): 185-197.

[23] J. Yang, S. A. Mani, J. L. Donaher, et al., “Twist, a Master Regulator of Morphogenesis, Plays an Essential Role in Tumor Metastasis,” Cell 117 (2004): 927-939.

[24] H. Tang, D. Massi, B. A. Hemmings, et al., “AKT-Ions With a TWIST Between EMT and MET,” Oncotarget 7 (2016): 62767-62777.

[25] V. Infantino, A. Santarsiero, P. Convertini, S Todisco, and V. Iacobazzi, “Cancer Cell Metabolism in Hypoxia: Role of HIF-1 as Key Regulator and Therapeutic Target,” International Journal of Molecular Sciences 22 (2021): 5703.

[26] M. H. Yang, M. Z. Wu, S. H. Chiou, et al., “Direct Regulation of TWIST by HIF-1alpha Promotes Metastasis,” Nature Cell Biology 10 (2008): 295-305.

[27] G. N. Masoud and W. Li, “HIF-1α Pathway: Role, Regulation and Intervention for Cancer Therapy,” Acta Pharmaceutica Sinica B 5 (2015): 378-389.

[28] Q. Li, R. Ma, and M. Zhang, “CoCl(2) Increases the Expression of Hypoxic Markers HIF-1α, VEGF and CXCR4 in Breast Cancer MCF-7 Cells,” Oncology Letters 15 (2018): 1119-1124.

[29] X. Zheng, A. X. Zhou, P. Rouhi, et al., “Hypoxia-Induced and Calpain-Dependent Cleavage of Filamin A Regulates the Hypoxic Response,” Proceedings of the National Academy of Sciences of the United States of America 111 (2014): 2560-2565.

[30] A. Emami Nejad, S. Najafgholian, A. Rostami, et al., “The Role of Hypoxia in the Tumor Microenvironment and Development of Cancer Stem Cell: A Novel Approach to Developing Treatment,” Cancer Cell International 21 (2021): 62.

[31] D. Pradella, C. Naro, C. Sette, and C. Ghigna, “EMT and Stemness: Flexible Processes Tuned by Alternative Splicing in Development and Cancer Progression,” Molecular Cancer 16 (2017): 8.

[32] Y. Lombardo, A. de Giorgio, C. R. Coombes, J. Stebbing, and L. Castellano, “Mammosphere Formation Assay From Human Breast Cancer Tissues and Cell Lines,” Journal of Visualized Experiments 97 (2015): 52671.

[33] K. H. Baek, R. Karki, E. S. Lee, Y. Na, and Y. Kwon, “Synthesis and Investigation of Dihydroxychalcones as Calpain and Cathepsin Inhibitors,” Bioorganic Chemistry 51 (2013): 24-30.

[34] E. Lee, J. E. Eom, H. L. Kim, et al., “Effect of Conjugated Linoleic Acid, μ-Calpain Inhibitor, on Pathogenesis of Alzheimer's Disease,” Biochimica Et Biophysica Acta 1831 (2013): 709-718.

[35] H. Y. Nam, E. J. Na, E. Lee, Y. Kwon, and H. J. Kim, “Antiepileptic and Neuroprotective Effects of Oleamide in Rat Striatum on Kainate-Induced Behavioral Seizure and Excitotoxic Damage via Calpain Inhibition,” Frontiers in Pharmacology 8 (2017): 817.

[36] D. H. Kang, K. Y. Jun, J. P. Lee, C. S. Pak, Y. Na, and Y. Kwon, “Identification of 3-Acetyl-2-Aminoquinolin-4-One as a Novel, Nonpeptidic Scaffold for Specific Calpain Inhibitory Activity,” Journal of Medicinal Chemistry 52 (2009): 3093-3097.

[37] E. Lee, J. E. Eom, H. L. Kim, et al., “Neuroprotective Effect of Undecylenic Acid Extracted From Ricinus communis L. Through Inhibition of μ-Calpain,” European Journal of Pharmaceutical Sciences 46 (2012): 17-25.

[38] K. H. Jeon, E. Lee, K. Y. Jun, et al., “Neuroprotective Effect of Synthetic Chalcone Derivatives as Competitive Dual Inhibitors Against Mu-Calpain and Cathepsin B Through the Downregulation of Tau Phosphorylation and Insoluble Abeta Peptide Formation,” European Journal of Medicinal Chemistry 121 (2016): 433-444.

[39] K. H. Jeon, E. Lee, K. Y. Jun, et al., “Neuroprotective Effect of Synthetic Chalcone Derivatives as Competitive Dual Inhibitors Against μ-Calpain and Cathepsin B Through the Downregulation of Tau Phosphorylation and Insoluble Aβ Peptide Formation,” European Journal of Medicinal Chemistry 121 (2016): 433-444.

[40] I. Shapovalov, D. Harper, and P. A. Greer, “Calpain as a Therapeutic Target in Cancer,” Expert Opinion on Therapeutic Targets 26 (2022): 217-231.

[41] S. J. Storr, N. O. Carragher, M. C. Frame, T. Parr, and S. G. Martin, “The Calpain System and Cancer,” Nature Reviews Cancer 11 (2011): 364-374.

[42] D. E. Goll, V. F. Thompson, H. Li, W. Wei, and J. Cong, “The Calpain System,” Physiological Reviews 83 (2003): 731-801.

[43] P. Li, C. Miao, C. Liang, P. Shao, Z. Wang, and J. Li, “Silencing CAPN2 Expression Inhibited Castration-Resistant Prostate Cancer Cells Proliferation and Invasion via AKT/mTOR Signal Pathway,” BioMed Research International 2017 (2017): 2593674.

[44] G. Zhang, T. Fang, M. Chang, et al., “Calpain 2 Knockdown Promotes Cell Apoptosis and Restores Gefitinib Sensitivity Through Epidermal Growth Factor Receptor/Protein Kinase B/Survivin Signaling,” Oncology Reports 40 (2018): 1937-1946.

[45] F. Xu, J. Gu, C. Lu, et al., “Calpain-2 Enhances Non-Small Cell Lung Cancer Progression and Chemoresistance to Paclitaxel via EGFR-pAKT Pathway,” International Journal of Biological Sciences 15 (2019): 127-137.

[46] X. L. Ma, K. Y. Zhu, Y. D. Chen, et al., “Identification of a Novel Calpain-2-SRC Feed-Back Loop as Necessity for β-Catenin Accumulation and Signaling Activation in Hepatocellular Carcinoma,” Oncogene 41 (2022): 3554-3569.

[47] X. Peng, R. Yang, J. Song, X. Wang, and W. Dong, “Calpain2 Upregulation Regulates EMT-Mediated Pancreatic Cancer Metastasis via the Wnt/β-Catenin Signaling Pathway,” Frontiers in Medicine 9 (2022): 783592.

[48] Y. Zhang, X. Zhu, X. Qiao, et al., “LIPH Promotes Metastasis by Enriching Stem-Like Cells in Triple-Negative Breast Cancer,” Journal of Cellular and Molecular Medicine 24 (2020): 9125-9134.

[49] H. Yan, M. Guo, J. Zou, et al., “Promotive Effect of Talin-1 Protein on Gastric Cancer Progression Through PTK2-PXN-VCL-E-Cadherin-CAPN2-MAPK1 Signaling Axis,” Journal of Clinical Laboratory Analysis 34 (2020): e23555.

[50] P. Wu, X. Hou, M. Peng, et al., “Circular RNA circRILPL1 Promotes Nasopharyngeal Carcinoma Malignant Progression by Activating the Hippo-YAP Signaling Pathway,” Cell Death and Differentiation 30 (2023): 1679-1694.

[51] X. Q. Xie, M. J. Wang, Y. Li, et al., “miR-124 Intensified Oxaliplatin-Based Chemotherapy by Targeting CAPN2 in Colorectal Cancer,” Molecular Therapy Oncolytics 17 (2020): 320-331.

[52] S. Cannito, E. Novo, A. Compagnone, et al., “Redox Mechanisms Switch on Hypoxia-Dependent Epithelial-Mesenchymal Transition in Cancer Cells,” Carcinogenesis 29 (2008): 2267-2278.

[53] T. SY, V. W. C. Wu, and H. K. W. Law, “Hypoxia-Induced Epithelial-Mesenchymal Transition in Cancers: HIF-1α and Beyond,” Frontiers in Oncology 10 (2020): 486.

[54] C. Huang, R. T. Miller, and C. E. Freter, “Signaling Regulation and Role of Filamin A Cleavage in Ca²⁺-Stimulated Migration of Androgen Receptor-Deficient Prostate Cancer Cells,” Oncotarget 8 (2017): 3840-3853.

[55] R. Salimi, S. Bandaru, S. Devarakonda, et al., “Blocking the Cleavage of Filamin A by Calpain Inhibitor Decreases Tumor Cell Growth,” Anticancer Research 38 (2018): 2079-2085.

[56] V. Te Boekhorst, L. Jiang, M. Mählen, et al., “Calpain-2 Regulates Hypoxia/HIF-Induced Plasticity Toward Amoeboid Cancer Cell Migration and Metastasis,” Current Biology 32 (2022): 412-427.

[57] S. B. Kanatous, P. P. Mammen, P. B. Rosenberg, et al., “Hypoxia Reprograms Calcium Signaling and Regulates Myoglobin Expression,” American Journal of Physiology-Cell Physiology 296 (2009): C393-402.

[58] T. Arnould, C. Michiels, I. Alexandre, and J. Remacle, “Effect of Hypoxia Upon Intracellular Calcium Concentration of Human Endothelial Cells,” Journal of Cellular Physiology 152 (1992): 215-221.

[59] I. O. Donkor, “An Update on the Therapeutic Potential of Calpain Inhibitors: A Patent Review,” Expert Opinion on Therapeutic Patents 30 (2020): 659-675.

[60] K. C. Nitiss and J. L. Nitiss, “Twisting and Ironing: Doxorubicin Cardiotoxicity by Mitochondrial DNA Damage,” Clinical Cancer Research 20 (2014): 4737-4739.

[61] K. A. Won and C. Spruck, “Triple‑Negative Breast Cancer Therapy: Current and Future Perspectives (Review),” International Journal of Oncology 57 (2020): 1245-1261.

[62] G. T. Wurz and M. W. DeGregorio, “Activating Adaptive Cellular Mechanisms of Resistance Following Sublethal Cytotoxic Chemotherapy: Implications for Diagnostic Microdosing,” International Journal of Cancer 136 (2015): 1485-1493.

[63] A. J. Gooding and W. P. Schiemann, “Epithelial-Mesenchymal Transition Programs and Cancer Stem Cell Phenotypes: Mediators of Breast Cancer Therapy Resistance,” Molecular Cancer Research 18 (2020): 1257-1270.

[64] D. R. Chen, D. Y. Lu, H. Y Lin, and W. L. Yeh, “Mesenchymal Stem Cell-Induced Doxorubicin Resistance in Triple Negative Breast Cancer,” BioMed Research International 2014 (2014): 532161.

[65] A. Paramanantham, E. J. Jung, H. J. Kim, et al., “Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth With Cancer Stem Cell-Like Properties and EMT Phenotype, Which Can Be Transferred to Parental Cells Through Autocrine Signaling,” International Journal of Molecular Sciences 22 (2021): 12438.

[66] K. H. Jeon, H. V. Yu, and Y. Kwon, “Hyperactivated m-Calpain Affects Acquisition of Doxorubicin Resistance in Breast Cancer Cells,” Biochimica et Biophysica Acta General Subjects 1862 (2018): 1126-1133.

[67] S. Wu, J. Lu, H. Zhu, et al., “A Novel Axis of circKIF4A-miR-637-STAT3 Promotes Brain Metastasis in Triple-Negative Breast Cancer,” Cancer Letters 581 (2024): 216508.

[68] X. Ou, Y. Tan, J. Xie, et al., “Methylation of GPRC5A Promotes Liver Metastasis and Docetaxel Resistance Through Activating mTOR Signaling Pathway in Triple Negative Breast Cancer,” Drug Resistance Updates 73 (2024): 101063.

[69] B. Győrffy, “Transcriptome-Level Discovery of Survival-Associated Biomarkers and Therapy Targets in Non-Small-Cell Lung Cancer,” British Journal of Pharmacology 181 (2024): 362-374.

[70] P. Jézéquel, M. Campone, W. Gouraud, et al., “bc-GenExMiner: An Easy-to-Use Online Platform for Gene Prognostic Analyses in Breast Cancer,” Breast Cancer Research and Treatment 131 (2012): 765-775.

[71] P. Jézéquel, J. S. Frénel, L. Campion, et al., “bc-GenExMiner 3.0: New Mining Module Computes Breast Cancer Gene Expression Correlation Analyses,” Database 2013 (2013): bas060.

[72] T. Li, J. Fu, Z. Zeng, et al., “TIMER2.0 for Analysis of Tumor-Infiltrating Immune Cells,” Nucleic Acids Research 48 (2020): W509-W514.

[73] J. Barretina, G. Caponigro, N. Stransky, et al., “The Cancer Cell Line Encyclopedia Enables Predictive Modelling of Anticancer Drug Sensitivity,” Nature 483 (2012): 603-607.

[74] P. Jézéquel, O. Kerdraon, H. Hondermarck, et al., “Identification of Three Subtypes of Triple-Negative Breast Cancer With Potential Therapeutic Implications,” Breast Cancer Research 21 (2019): 65.

[75] P. Jézéquel, D. Loussouarn, C. Guérin-Charbonnel, et al., “Gene-Expression Molecular Subtyping of Triple-Negative Breast Cancer Tumours: Importance of Immune Response,” Breast Cancer Research 17 (2015): 43.

RIGHTS & PERMISSIONS

2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.