Targeting Calpain-1 and Calpain-2 for Prevention of Breast Cancer Metastasis: In Vivo Insights and Drug Discovery Approaches

Due to breast cancer’s aggressive behavior and lack of effective targeted therapies once it becomes metastatic, it remains a leading cause of cancer mortality among women. Our lab is in a search for novel molecular targets that can be exploited to prevent breast cancer from metastasizing, which could perhaps ameliorate the metastasis-linked decline in breast cancer patients’ survival. Calpain-1 and -2, ubiquitous calcium-dependent cysteine proteases, are a potential novel therapeutic target in cancer as they are upregulated in multiple cancer subtypes, including breast cancer, and have been implicated in tumor progression, metastasis, and therapeutic resistance. To evaluate calpains’ potential as therapeutic targets, we used CRISPR-Cas9 gene editing to knock out calpain-1 and/or calpain-2 in MDA-MB-231 triple negative breast cancer (TNBC) cells. Compared to WT cells, calpain knockouts exhibited a 79% reduction in in vitro migratory capacity and an approximately 83.4% reduction in lung metastasis in an orthotopic mouse model of breast cancer. A split-Nanoluciferase biosensor was designed and used to measure calpain-1 and calpain-2 subunit dimerization, at 185.4 nM and 508.6 nM dissociation constant (in the presence of Ca2+) and to screen for small-molecule inhibitors of calpain dimerization that could effectively block proteolytic activity in vitro. One compound inhibited calpain-2 activity by 69% at a concentration of 150 µM. I additionally designed a calpastatin-based peptide inhibitor of calpain that inhibited calpain-2 with an IC50 16.2 nM, limited calpain-dependent MDA-MB-231 cell migration to 53.5% and significantly reduced the high variance in calpain-dependent breast cancer metastasis but did not reduce average metastasis. These findings validate calpain-1 and calpain-2 as key drivers of metastasis in TNBC and support ongoing efforts to develop potent selective inhibitors of calpain that could improve outcomes in advanced breast cancer.

Department dc:contributor.department

Pathology and Molecular Medicine

Year dc:date.issued

2025

Author dc:creator

• Shapovalov, Ivan

Advisor dc:contributor.supervisor

• Greer, Peter A

• Calpain
• Breast cancer
• Cell biology
• Cancer biology
• Biosensor

Statement dc:rights

• Attribution-NonCommercial 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by-nc/4.0/

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1974/34701

OAI identifier oai:identifier

oai:queensu.scholaris.ca:1974/34701