Calpain Genetic Disruption and HSP90 Inhibition Combine To Attenuate Mammary Tumorigenesis

Abstract

Calpain is an intracellular Ca 2؉ -regulated protease system whose substrates include proteins involved in proliferation, survival, migration, invasion, and sensitivity to therapeutic drugs. Genetic disruption of calpain attenuated the tumorigenic potential of breast cancer cells and hypersensitized cells to 17AAG, an inhibitor of the molecular chaperone HSP90. Calpain-1 or -2 overexpression rendered cells resistant to 17AAG, whereas downregulation or inhibition of calpain-1/2 led to increased cell death in multiple breast cancer cell lines, including models of HER2 ؉ (SKBR3) and triple-negative basal-cell-like (MDA-MB-231) breast cancer. In an MDA-MB-231 orthotopic xenograft model, calpain knockdown or 17AAG treatment independently attenuated tumor growth and metastasis, while the combination was most effective. Calpain knockdown was associated with increased 17AAG-induced degradation of the HSP90 clients cyclin D1 and AKT and multidrug resistance protein 2, which correlated with increased expression of antimitogenic p27 KIP1 and proapoptotic BIM proteins. Like other therapeutics, 17AAG can be effluxed by specific ABC transporters. Calpain expression positively correlated with the expression of P glycoprotein in mouse embryonic fibroblasts. Importantly, we show that calpain affects ABC transporter function and efflux of clinically relevant doxorubicin. These observations provide a compelling rationale for exploring the combination of calpain inhibition with new or existing cancer therapeutics. Calpains are a family of intracellular calcium-dependent proteases. Of the 15 mammalian isoforms, calpain-1 and calpain-2 are ubiquitously expressed and have been most extensively studied (reviewed in reference 1). They are heterodimers consisting of isoform-specific catalytic subunits encoded by CAPN1 and CAPN2, respectively, and a common regulatory subunit encoded by CAPNS1. Heterodimerization is essential for both stability and enzymatic activity; hence, genetic disruption of CAPNS1 results in loss of both calpain-1 and -2 activities (2, 3).Elevated calpain expression or activity has been associated with multiple cancer types, including colon, liver, lung, prostate, ovarian, and breast cancers (reviewed in reference 4). Mechanistic understanding of calpain's involvement in cancer is confounded by its broad range of substrates, including signaling or structural proteins that regulate diverse cellular functions such as mitogenesis, migration, invasion, and death (1). Biomarker studies suggest a positive correlation between elevated calpain expression and a poor clinical outcome in breast cancer (5-7).Paradoxically, calpain is associated with prodeath (8, 9) or prosurvival (9-11), depending upon the physiologic context and specific challenges cells are exposed to. For example, calpain-mediated cleavage of PP2A supports activation of AKT and a prosurvival function in mouse embryonic fibroblasts (MEFs) (10), and in breast cancer cells and xenograft mouse models, knockdown of CAPN2 was associated with attenuated tumor growth...

Characterizations of HSP90‐Interacting Complex in Renal Cells Using Tandem Affinity Purification and Its Potential Role in Kidney Stone Formation

“…Calpain‐1 is a Ca 2+ ‐dependent proteolytic enzyme that involves in protein degradation and various signaling pathways . Interestingly, various reports have indicated that HSP90 and calpain‐1 are involved in apoptotic process and many HSP90 client proteins are also calpain substrates . These findings further support the role of calpain‐1 as a novel partner of HSP90 to regulate cellular functions.…”

Genetic disruption of calpain-1 and calpain-2 attenuates tumorigenesis in mouse models of HER2+ breast cancer and sensitizes cancer cells to doxorubicin and lapatinib

“…Calpains-1/2 have been implicated in membrane-cytoskeletal dynamics associated with cell migration and invasion [ 18 , 32 ]. In vitro migration and invasion were both compromised by capns1 KO in mouse embryonic fibroblasts [ 15 , 17 ] or capns1 knockdown in MDA-MB-231 breast cancer cells [ 25 ]; and capn2 knockdown was associated with dysregulated focal adhesion turnover [ 18 ] and lamellipodia dynamics [ 16 ] in fibroblasts, as well as attenuated migration and invasion in mammary carcinoma cells [ 9 ].…”

“…In vitro studies have linked calpain to trastuzumab resistance in HER2 + breast cancer cells through generation of a p95HER2 fragment [ 19 , 24 ], or resistance to chemotherapeutics like doxorubicin through regulating multidrug resistance protein function [ 25 ]. Thus, a growing body of research suggests that inhibition of calpain may suppress tumorigenesis and could cooperate or synergize with specific existing treatments to improve breast cancer patient outcomes.…”

Evidence for calpains in cancer metastasis

“…To allow tumor progression and dissemination in the body, apoptosis resistance is a vital determinant (Jenning, Pham, Ireland, Ruoslahti, & Biliran, 2013). CAPNs are paradoxically associated with either prosurvival or prodeath efforts (Grieve et al, 2016). Researchers revealed that in colon cancer, stress-induced CAPN activation cleaves Myc into Myc-nick, an N-terminal truncation of Myc located in the cytoplasm, and has prosurvival and metastasis capacities (Conacci-Sorrell, Ngouenet, Anderson, Brabletz, & Eisenman, 2014).…”