ABSTRACT
Introduction
Calpain-1 and calpain-2 are prototypical classical isoforms of the calpain family of calcium-activated cysteine proteases. Their substrate proteins participate in a wide range of cellular processes, including transcription, survival, proliferation, apoptosis, migration, and invasion. Dysregulated calpain activity has been implicated in tumorigenesis, suggesting that calpains may be promising therapeutic targets.
Areas covered
This review covers clinical and basic research studies implicating calpain-1 and calpain-2 expression and activity in tumorigenesis and metastasis. We highlight isoform specific functions and provide an overview of substrates and cancer-related signalling pathways affected by calpain-mediated proteolytic cleavage. We also discuss efforts to develop clinically relevant calpain specific inhibitors and spotlight the challenges facing inhibitor development.
Expert opinion
Rationale for targeting calpain-1 and calpain-2 in cancer is supported by pre-clinical and clinical studies demonstrating that calpain inhibition has the potential to attenuate carcinogenesis and block metastasis of aggressive tumors. The wide range of substrates and cleavage products, paired with inconsistencies in model systems, underscores the need for more complete understanding of physiological substrates and how calpain cleavage alters their functions in cellular processes. The development of isoform specific calpain inhibitors remains an important goal with therapeutic potential in cancer and other diseases.
1. Introduction
First identified in the 1960s, calpain-1 and calpain-2 are the founding members of a family of calcium (Ca2+)-dependent cysteine proteases that are being explored as possible therapeutic targets in diseases, including Alzheimer’s and several types of cancer [Citation1,Citation2]. A growing body of literature, spanning several cancer subtypes, supports roles for calpains in tumorigenesis and disease progression. With a diverse range of known calpain substrates involved in different cellular and physiological functions, the precise roles of calpains in different cancers appear complex and, in some cases, paradoxical. This review aims to discuss the rationale for calpain inhibition as a therapeutic strategy in cancer. We begin by summarizing the structure and regulation of calpain. We then examine translational studies focused on calpain dysregulation, biologically relevant calpain substrates, and the cellular processes they are involved in. We also discuss ongoing efforts to develop pharmacologic calpain inhibitors and the limitations that must be addressed to realize the full potential of these as therapeutic agents. We regret that we are not able to describe or acknowledge every publication that has contributed to our current understanding of the calpain system in cancer biology. For other recent reviews on the subject, we refer the reader to the following publications [Citation3-7].
1.1. Structure and activation of conventional calpains
Human calpains are a family of 15 Ca2+-activated cysteine proteases which seemingly cleave disordered or accessible peptide sequences, rather than targeting specific amino acid motifs, and thus have a myriad of peptide substrates [Citation8] . The precise biologic roles of calpains are elusive, with evidence pointing towards more than 130 substrate proteins [Citation9] involved in a wide range of cellular functions; many of which are discussed in this review.
The conventional calpain isoforms, calpain-1 and −2 (previously known as µ-calpain and m-calpain) were the first to be discovered and are the most well studied due to their abundant ubiquitous expression. Both isoforms are intracellular heterodimers consisting of a common regulatory subunit, encoded by the CAPNS1 gene (also known as CAPN4), and an isoform-specific catalytic subunit encoded by the CAPN1 or CAPN2 genes, for calpain-1 and calpain-2, respectively [Citation10,Citation11]. Calpain-1 and −2 are also considered classical calpains due to their defining domain structures. The catalytic subunit consists of an N-terminal anchor helix, a potential regulator of calpain activation [Citation12]; two protease core domains that constitute the active site (PC1 and PC2); the calpain-type beta-sandwich (CBSW) domain (previously known as a C2-like domain); and a Ca2+ binding C-terminal penta EF-hand PEF(L) domain, a mediator of dimerization and a distinguishing feature of the classical calpain isoforms. The regulatory subunit CAPNS1 consists of an unstructured glycine rich (GR) domain; and a PEF(S) domain, which is homologous to the PEF(L) domain. A crystal structure of calpain-2 and domain maps for the catalytic (CAPN1/2) and regulatory (CAPNS1) subunits are shown in .
Calpain-1 and −2 activities are tightly regulated, with only transient activation of proteolysis upon binding of Ca2+ ions [Citation14]. The required concentration of Ca2+ for in vitro activation is in the low micromolar range for calpain-1, and high micromolar to low millimolar range for calpain-2 [Citation14]. Since the intracellular Ca2+ concentration (100 nM) [Citation15] is insufficient for calpain activation, these enzymes must be activated by Ca2+ influx from the extracellular space, where Ca2+ concentration is 1.1–1.4 mM [Citation16]. Alternatively, calpains-1 and −2 may be activated by Ca2+ release from intracellular stores, such as the endoplasmic reticulum (ER) [Citation16]. Due to the cells’ propensity to rapidly ‘clean up’ free cytoplasmic Ca2+, calpains-1 and −2 are believed to be transiently activated by localized bursts of adequate concentrations of Ca2+, followed quickly by deactivation after Ca2+ levels dissipate. Excessive calpain activation is believed to occur under disease or tissue damage conditions as a result of dysregulated Ca2+ homeostasis [Citation8].
Calpain-1 and −2 are also regulated by calpastatin (encoded by the CAST gene) which binds the PEF(L) and PEF(S) domains as well as near the active site to sterically hinder substrate access () [Citation17]. The calpain-calpastatin system is regulated by phosphorylation modifications of either calpain or calpastatin. Phospho-CAST has repressed activity, and as such, dephosphorylation is required for CAST to inhibit calpain [Citation18,Citation19]. Phosphorylation of calpain may result in activation or inhibition depending on the kinase. For example, ERK- and protein kinase C-mediated phosphorylation of calpain-2 increases its activity, while phosphorylation by protein kinase A (PKA) decreases calpain-2 activity [Citation20,Citation21]. Our understanding of calpain-calpastatin cross-talk in normal health and diseases, including cancer, is incomplete. There is evidence in glioblastoma cell lines that radiation-induced CAST phosphorylation is associated with activation of calpain-1 and increased cell survival [Citation22]; and while calpain-1 is inhibited by CAST regardless of the calpain-1 phosphorylation status, PKA phosphorylated calpain-1 is more sensitive to CAST inhibition [Citation23]. In addition, the threshold for calpain-2 activation by Ca2+ can be reduced by autolysis of the N-terminus or interaction with phosphatidylinositol mono- or bis-phosphate (reviewed in [Citation2]). Binding sites on calpain-2 for Ca2+, calpastatin, and selected small molecule inhibitors (as determined in co-crystal structures) are shown in .
Calpain expression levels in cell lysates or in situ can be readily assessed by immunoblotting, immunohistochemical, or immunofluorescence methods. However, it is much more challenging to determine the activation status of calpain in cells or tissues. Biochemical zymography methods are used extensively to measure calpain activity in cell lysates [Citation27], but these are semi-quantitative and do not necessarily reflect calpain activity in situ. Fluorescent substrates have been developed as tools for measuring calpain activity in vitro. These probes typically consist of a fluorophore-quencher pair linked by a calpain-sensitive peptide [Citation28]. Such probes are not completely specific for calpain, so investigators should exercise caution when using cell permeable forms of these probes to assess cellular calpain activity. Fluorescence resonance energy transfer (FRET) probes, consisting of optimal calpain-sensitive peptides bridging CFP and YFP, have also been expressed in cells to measure in situ calpain activity [Citation29]. While these can be excellent calpain substrates in cells, there is still some concern that the apparent calpain activity could be due in part to other proteases. Many researchers assess calpain activity in cells by immunoblotting for known calpain substrates and comparing the relative level of the uncleaved substrate with the presumed calpain-generated fragments. While this approach is widely used, it is challenged by the typically low stoichiometry of substrate cleavage, as well as uncertainty about protease specificity, since calpain cleavage sites may also be targeted by other proteases. Measuring physiologically relevant calpain activity in situ remains an important challenge in calpain research. The terminal amine isotope labeling of substrates (TAILS) mass spectrometry (MS) approach [Citation30], combined with genetic model systems, has the potential to address these problems and to identify novel physiologically relevant calpain substrates.
2. Calpains
2.1. The role of calpains in human health and disease
The biological function of calpains in human health is multifaceted, which likely reflects diverse roles in cell signaling by 15 isoforms, many of which display distinct tissue-specific expression (reviewed in [Citation8]). Among the first known cellular functions was cleavage of cortical cytoskeletal proteins. Calpain-1 and −2 are involved in turnover of cell adhesion/protrusion structures via proteolytic degradation of scaffolding proteins, such as spectrin, cortactin, ezrin, and beta-catenin (see for a list of in vivo validated calpain-1 and −2 substrates). These cleavage events have been shown to promote motility of fibroblasts [Citation31] and morphological changes to platelets to facilitate clotting [Citation32]. It has also become apparent that calpain-1 and −2 modulate the activity of many substrates through limited proteolysis of regulatory domains. For example, calpain-2 mediated truncation of the androgen receptor results in ligand-independent activation in prostate cancer cells [Citation33]. In normal cells, activation of calpain-1 and −2 can potentiate pro-apoptotic cell signaling upon Ca2+ influx induced by chemical insults or ER stress [Citation2,Citation34]. However, calpains are involved in both pro- and anti-apoptotic signaling pathways. CAPNS1 gene knockout, which results in loss of both calpain-1 and −2, sensitized mouse embryonic fibroblasts to stimuli including staurosporine and tumor necrosis factor alpha, while conferring resistance to puromycin, camptothecin, etoposide, hydrogen peroxide, ultraviolet light and serum starvation [Citation35]. These findings highlight the context-dependent and opposing pro- and anti-survival roles for calpain that might be exploited for therapeutic benefits. However, this potential opportunity has been limited by questions surrounding which calpain isoforms are playing which roles, and in what specific cellular contexts.
Calpains have been implicated in the progression of several human diseases. A large body of evidence suggests that calpain-2 dysregulation contributes to the neuropathogenesis of Alzheimer’s disease, possibly through roles in amyloid beta aggregation and accumulation of tau neurofibrillary tangles (for a recent comprehensive review see [Citation51]). Recent work by Baudry and colleagues suggests that calpain-1 and −2 may have opposing roles in neuronal cell protection and degeneration, respectively [Citation52]. Consistent with this hypothesis, mutations in CAPN1 have recently been linked with cerebellar ataxia [Citation53]. Calpain-1 dysfunction has also been implicated in blood clotting disorders, with CAPN1 knockout mice displaying reduced platelet aggregation and clot retraction [Citation54].
Other calpains have also been implicated in other human diseases. Mutations in CAPN3 result in limb-girdle muscular dystrophy type 2A, and this was phenocopied in mouse CAPN3 knockout models [Citation55] (reviewed in [Citation56]). CAPN5 mutations contribute to autoimmune uveitis, retinitis pigmentosa, and retinopathies that lead to irreversible blindness [Citation57,Citation58], while CAPN14 is upregulated in response to IL-13 in esophageal epithelial cells in patients with eosinophilic esophagitis [Citation59].
An emerging area of interest in the calpain field is their potential involvement in cancer, including regulation of cancer cell survival, metastasis, and susceptibility to chemotherapeutics and targeted agents. Activating or inactivating mutations in calpain genes do not appear to play a role in cancer. However, upregulation of calpain-1 and −2 have been described in several cancers (reviewed in [Citation2]), and there is a growing appreciation for how calpain-1 and −2 are regulated by pro-oncogenic signaling pathways. These questions will be discussed in the following sections.
2.2. Evidence for calpain involvement in cancer
A growing number of translational studies suggest that aberrant calpain activity contributes to tumorigenesis and cancer progression (reviewed in [Citation3–7].). An early clinical study reported that CAPN1 transcript levels were associated with higher regional metastasis in renal cell carcinoma [Citation60], while a more recent meta-analysis showed that high CAPNS1 protein expression independently predicted shorter overall survival across eight common cancer types, including colorectal, ovarian, lung, liver, and glioblastoma [Citation61]. Similar studies suggesting pro-tumorigenic roles for calpain have emerged across various cancer subtypes. For example, high CAPNS1 transcript expression was associated with metastasis and shorter survival in gastric cancer patients [Citation62], while immunostaining has been used to show that colorectal cancer patients had poorer prognosis when tumors contained high levels of calpain-1 and low levels of its substrate filamin-A (FLNA) [Citation63]. Interestingly, another study linked calpain-1 cleavage of FLNA to enhanced migration of androgen receptor-deficient prostate cancer cells [Citation64]. Calpain-2 has been implicated in prostate cancer metastasis through regulation of gene expression by a mechanism involving cleavage of collapsin response mediator protein 4 (CRMP4), which in turn regulates DNA methyltransferase 1 expression [Citation65]. In pancreatic cancer, a subtype infamous for rapidly spawning metastases, high tumor calpain-1 expression was associated with increased metastasis and shorter overall survival [Citation66]. These authors showed that RNAi-based knockdown of calpain-1 in pancreatic cancer cell lines correlated with reduced proliferation and invasion in vitro [Citation66]. The non-specific calpain-1 and −2 inhibitor, calpeptin, suppressed pancreatic cancer cell proliferation, migration, and invasive behavior in vitro [Citation67], but the in vivo effect of calpeptin in mouse xenograft models was dependent on co-engrafted fibroblasts [Citation67], suggesting that calpain contributes to a stromal supporting role in tumorigenesis. Calpain-2 has also been identified as a potential predictive biomarker in ovarian cancer, with high expression associated with resistance to platinum-based therapies [Citation68].
Multiple research groups have published observations of increased calpain expression in breast cancer. Storr and colleagues reported that elevated levels of calpain-1 and calpain-2 were associated with poor clinical outcomes in HER2-positive and triple negative breast cancer (TNBC) subtypes, respectively [Citation69,Citation70]. High calpain-1 expression was also associated with shorter disease-free survival in breast cancer patients treated with trastuzumab [Citation71]. Conversely, low calpain-9 expression has been linked to poor outcomes in breast cancer patients who received endocrine therapy [Citation72]. Interestingly, one study reported that high calpain-1 and high calpastatin levels were associated with better survival of patients with inflammatory breast cancer, while high calpain-2 and low calpastatin was correlated with improved survival in patients with non-inflammatory breast cancer treated with neoadjuvant chemotherapy [Citation73], which invokes a more complex relationship between these calpain isoforms and their endogenous inhibitor in different types of cancer. Thus, while most of the current literature suggests a pro-tumorigenic role for calpains in cancer progression, there is conflicting evidence suggesting that, under some circumstances, calpains may have anti-tumorigenic roles. Higher calpain-1 and calpastatin levels in gastro-esophageal cancer predicted better survival in cohorts both with and without neoadjuvant chemotherapy [Citation74]. In one study of pancreatic cancer subtypes, tumors of the bile ducts and ampulla were marginally more aggressive when calpain-1 and −2 levels were low [Citation75]. In contrast, another pancreatic cancer study reported reduced survival for patients with increased calpain-1 expression [Citation66], despite both these pancreatic cancer studies analyzing protein levels and employing similar immunohistochemistry approaches [Citation66]. It has been observed that analyses of calpain protein levels and mRNA levels do not necessarily correlate. For example, in basal cell skin carcinoma, mRNA levels of CAPN1 were significantly higher than in normal tissue while protein levels of CAPN1 were reduced [Citation76]. These authors suggested that higher proteolytic and autolytic activity might be responsible for reduced calpain-1 protein, despite the increased presence of mRNA [Citation76].
In addition to looking at expression of calpains, investigators have also looked at calpain substrates as prognostic biomarkers in cancer and other diseases. For example, FLNA has been characterized as a calpain-1 target, and its degradation along with elevated calpain-1 levels has been associated with poorer outcomes in colorectal cancer [Citation63]. However, as with calpain itself, there is contrary evidence for the predictive power of FLNA. It was suggested that in glioblastoma, more FLNA cleavage predicts better patient survival and greater cancer cell apoptosis in vitro [Citation77], presumably due to co-occurrence of pro-apoptotic calpain activation and FLNA degradation. Products of calpain breakdown are used as biomarkers for cell death in other diseases. For example, troponin I degradation products are an established clinical blood biomarker for injured myocardium in a variety of cardiovascular conditions [Citation78], where it was shown that degradation is caused by hypoxia-induced calpain-mediated proteolysis [Citation79]. Spectrin degradation products have been used as biomarkers for kidney disease and traumatic brain injury (TBI) [Citation80]. Most recently, a calpain-2 mediated cleavage product of tyrosine phosphatase PTPN13, has been used as a biomarker for traumatic brain injury, where it is correlated with the severity of injury [Citation81].
2.2.1. Calpains and chemotherapy-induced toxicities
In addition to their roles in disease progression, calpains have been implicated in cancer associated diseases linked to chemotherapy. Once again, calpains have been shown to have both detrimental and protective effects on patient health in the context of chemotherapy induced toxicities. For example, Peng and colleagues reported that overexpression of calpain-2 protected the heart against doxorubicin induced cardiotoxicity [Citation82]. Calpain-2 is thought to promote AKT activation and subsequent upregulation of mitogen-activated protein kinase (MKP-1) to attenuate cardiomyocyte apoptosis in response to doxorubicin therapy [Citation82]. Perhaps unsurprisingly, calpains are also involved in chemotherapy induced peripheral neuropathy. Treatment with the microtubule stabilizing drug, Taxol, has been shown to promote calpain-1 mediated cleavage of the neuronal Ca2+ sensor-1 (NCS-1) [Citation83]. NCS-1 degradation was associated with a loss of intracellular Ca2+ signalling and irreversible damage to peripheral nerve fibers [Citation83]. Similarly, calpain activation has been identified as an important early step in cisplatin-induced neurotoxicity [Citation84]. Taken together, these observations underscore the potential of combining calpain inhibition with chemotherapy as a strategy to reduce cardiotoxicity and neurotoxicity [Citation82–85].
2.3. Calcium signaling induces calpain activation in cancer cells
One model for calpain regulation predicts activation upon influx of extracellular Ca2+ into the cytoplasm via Ca2+ channels in the plasma membrane. Substrate cleavage would be conditional upon cortical localization of calpain, its substrates, and a Ca2+ signal. A recent study in breast cancer cells showed that recruitment of calpain-1 to the plasma membrane by the ezrin adaptor protein promoted cleavage of its classical substrates talin, FAK, and cortactin [Citation86]. Other studies show that induction of Ca2+ signals by various external treatments can trigger calpain-2 activation in cancer cells. For example, exposure of cancer cells to calcium lactate increased intracellular Ca2+ levels, induced calpain-2 mediated degradation of FAK and p53; and promoted cell motility [Citation87]. Ca2+ can also be mobilized from the ER through activation of the inositol 1,4,5-trisphosphate receptor (IP3R). Such a mechanism was exploited to induce calpain-1-mediated apoptosis of an acute lymphoblastic leukemia cell line derived from a paediatric pre-B acute lymphoblastic leukemia patient [Citation88]. Induction of Ca2+ influx and the ensuing calpain activation can feed into either pro-autophagy or pro-apoptosis signaling cascades, depending on other susceptibility factors, like intracellular levels of Atg5 or Bax, respectively [Citation89]. While calpain inhibition on its own is often cytoprotective, when autophagy is induced under proteasomal stress, calpain inhibition can promote an anti-tumor effect of small-molecule cytotoxins [Citation90].
2.4. Calpain mediated pathways in cancer
Calpain activation in vivo, limited by Ca2+ signals, is transient and localized to specific subcellular domains [Citation86,Citation91]. One of the challenges associated with identifying true physiologic substrates lies in the promiscuous activity of calpain outside of the cellular context. As a result, substrates identified in vitro remain hypothetical substrates that require validation in live cell systems. In this regard, the study of calpain is analogous to the study of protein kinase A (PKA) which can phosphorylate many substrates in biochemical assays with purified proteins [Citation92]. However, its physiologic activity is closely regulated in vivo by localized cAMP signaling and the location of A kinase-anchoring proteins inside the cell [Citation92]. Therefore, researchers must be careful when discerning between possible calpain substrates and biologically validated ones. Calpain substrates are found in many cancer-related signaling pathways and include products of oncogenes and tumor suppressor genes. We next discuss several key substrates involved in signaling pathways associated with cancer progression, metastasis, and treatment response. Please refer to for a summary of several biologically relevant calpain substrates that have been validated in live cells.
2.4.1. Conventional calpain substrates associated with cell growth and death
For a comprehensive review of calpains in cancer apoptosis the reader is referred to the review by Storr and colleagues [Citation2]. Briefly, several lines of evidence suggest calpains interact with the caspase family of cysteine proteases to initiate apoptosis [Citation93]. Proteolytic cleavage mediated by conventional calpains directly activates caspases-7, −10 and −12 and inhibits caspase-9 [Citation2]. Furthermore, calpain-1 and −2 mediated cleavage of the pro-apoptotic BCL2 family member, Bax, promotes the release of cytochrome c from the mitochondria which leads to downstream activation of executioner caspase-3 [Citation94]. There is also evidence to suggest that Bax cleavage requires caspase-dependent activation of calpain [Citation95]. As summarized in , other pro-apoptotic calpain substrates include BID [Citation96,Citation97], c-FOS [Citation98,Citation99], c-JUN [Citation98,Citation99], CDK5 [Citation100], and apoptosis-inducing factor (AIF) [Citation101]. Please refer to for examples of calpain substrates involved in cell survival and apoptosis.
In some contexts, calpains contribute to pro-survival pathways and may even promote resistance to anticancer therapies. Elucidating pro-survival roles for calpain has great relevance to cancer therapeutics, as researchers work to develop calpain inhibitors that may synergize with specific cytotoxic agents. For example, calpain-mediated cleavage promotes degradation of the p53 tumor suppressor, thereby attenuating apoptosis [Citation102]. In human ovarian cancer cells, cleavage of p53-associated parkin-like cytoplasmic protein prevented nuclear localization of p53, thus inhibiting apoptosis [Citation103]. Under stressful conditions, calpain cleaved cytoplasmic Myc to produce an N-terminally truncated protein termed ‘Myc-nick’ [Citation104,Citation105]. Myc-nick attenuated cell death by promoting drug resistance and autophagy under conditions of nutrient deprivation [Citation104]. Grieve et al. demonstrated that CAPNS1 knockdown was associated with increased sensitivity to the HSP90 inhibitor 17AAG in HER2+ and TNBC cell lines [Citation106]. This observation is believed to be the result of calpain-mediated effects on ABC transporters involved in drug efflux [Citation106]. Calpain-2 has been shown to promote cancer cell survival via the PI3K-Akt-FoxO-p27Kip1 signaling pathway [Citation107]. Ho et al. demonstrated that calpain-2 knockdown was associated with reduced Akt phosphorylation, thus preventing the inhibition of Foxo3a mediated transcription of cyclin dependent kinase inhibitor p27 [Citation107]. Bertoli and colleagues had also previously demonstrated that calpain negatively regulates Foxo3a by cleaving, thereby inactivating the Akt phosphatase, PP2A [Citation108]. PP2A has also been shown to dephosphorylate calpain-1 and −2, attenuating their activation and reducing lung cancer cell migration and invasion in vitro [Citation109].
Calpain mediated proteolysis of multiple members of receptor tyrosine kinase (RTK) family has been observed, where it can release the cytoplasmic domain from the membrane. For example, calpain cleaves Met, producing a stable and pro-metastatic p45 fragment [Citation110]. Calpain-mediated cleavage in the juxta-membrane region of HER2 was also reported, which produced either the complete cytoplasmic domain or a truncated fragment [Citation111]. In that context, inhibiting calpain resulted in accumulation of more full-size RTK and greater cell survival [Citation111]. In line with such a model, MacLeod et al. have shown that knockout of calpain-1 and −2 in an activated HER2-driven transgenic mouse mammary carcinoma model changed its phospho-proteomic landscape, producing more phospho-EGFR [Citation112].
While calpains can target multiple members of RTK superfamily, alterations in RTK signaling have been implicated in the regulation of calpain as well. For example, overexpression of HER2 induced CAST transcription, which correlated with higher levels of Src, FAK, and ERK – all predicted calpain substrates [Citation113]. Another study also suggested EGF-mediated activation of calpain as a mechanism of cyclin G2 degradation [Citation114]. Cyclin E [Citation115] and cyclin D1 [Citation116] are also suspected calpain substrates. In addition, calpain activation has been observed as an effector of VEGF stimulation in endothelial cells, promoting angiogenesis [Citation117]. Miyazaki et al. reported that overexpression of calpastatin in mouse endothelial cells attenuated angiogenesis by preventing calpain-mediated cleavage of SOCS3 and downstream activation of the JAK/STAT pathway [Citation118]. Calpain involvement in tumor angiogenesis remains to be fully elucidated, but evidence including that described above supports pro-metastatic roles for calpain.
2.4.2. Conventional calpains and metastasis
Calpains contribute to tumor cell migration, invasion, and metastasis by altering focal adhesion dynamics and promoting cytoskeletal remodelling (reviewed in [Citation2,Citation3,Citation5–7]). A graphical overview of some of these processes and calpain-1 and −2 functions are illustrated in . Huttenlocher and colleagues demonstrated that calpain-2 mediated proteolysis of the cytoskeletal protein talin is required for adhesion disassembly [Citation119]. Furthermore, focal adhesion kinase (FAK) is cleaved by calpain-2 in vivo, promoting adhesion turnover, in part by altering talin dynamics [Citation120]. A more recent study found that silencing CAPNS1 in renal carcinoma cells reduced talin cleavage and significantly impaired migration and invasion in vitro [Citation121]. Calpain-2 mediated cleavage of the actin-assembly protein cortactin also promoted cell migration by modulating invadopodium formation [Citation122]. The actin-membrane linker, ezrin, has been identified as a substrate of calpain-1, but interestingly, ezrin also acts upstream of calpain to regulate focal adhesion and invadopodia turnover [Citation86]. Protein tyrosine phosphatase 1B (PTP1B) becomes hyperactivated in response to calpain-2 mediated truncation [Citation123]. Interestingly, this cleavage removes a C-terminal ER localization domain that allows PTP1B to relocalize from the ER to the cytosol, where it acts as a positive regulator of the nonreceptor tyrosine kinase c-Src to promote invadopodium formation in metastatic breast cancer cells [Citation123]. The calpain-1 isoform has been shown to negatively regulate cell adhesion by cleaving and inactivating the RhoA GTPase, a key player in the formation of stress fibers and focal adhesion complexes [Citation124]. Calpains also cleave the cell-cell adhesion molecule, E-cadherin, preventing its association with beta-catenin, gamma-catenin, and p120 catenin [Citation125]. The functional inactivation of E-cadherin has been shown to promote a metastatic phenotype through loss of epithelial cell-cell adhesion [Citation125]. Calpain has also been shown to cleave and activate beta-catenin in metastatic prostate and breast cancer cells, thereby promoting Wnt pathway activation [Citation126].
Calpain-mediated cleavage of the actin cross-linking protein FLNA also contributes to metastatic behaviour. Salimi and colleagues showed that the migratory behavior of human melanoma cells depends on a 90kDa FLNA fragment produced by calpain proteolysis [Citation131]. Calpain-mediated FLNA cleavage was also implicated in hypoxic response and tumor angiogenesis by promoting the nuclear localization of HIF-1α [Citation132]. FLNA has also been shown to regulate the transcriptional activity of androgen receptor (AR) by sequestering the FHL2 coactivator [Citation133]. Calpain cleavage of both FLNA and AR promotes association and nuclear localization of a FHL2/AR complex [Citation133]. However, there are conflicting data pertaining to whether calpain-cleaved AR is activated in a ligand-independent fashion or degraded [Citation134,Citation135]. In an animal model of prostate cancer, where the calpain-AR interaction is presumed to play a significant role, inhibition of calpain-2 resulted in less invasive cancer cells [Citation136].
Calpains also regulate membrane plasticity and protrusions through cleavage of the relevant scaffolding proteins. Dysregulation of Ca2+ homeostasis in cancer cells promotes aberrant calpain activation resulting in cleavage of cytoskeletal components [Citation137]. Spectrin, a cortical scaffolding protein, is a well-established calpain substrate, and it has frequently been used as an indicator of calpain activation in situ [Citation138]. A recent study showed a novel pathway for inducing calpain-mediated cleavage of spectrin through DCC, a netrin-1 receptor [Citation91]. Decreased cortical presence of spectrin through calpain cleavage was shown to increase the biogenesis of extracellular vesicles (EVs) [Citation139], which act as paracrine effectors of malignant cells. EV production is upregulated in cancer cells and contributes to metastatic and drug resistant phenotypes [Citation109].
Calpain-1 and −2 expression was also associated with membrane ‘blebbing’ in mouse embryonic fibroblasts, which correlated with altered protein levels of Rho GDP-dissociation inhibitor and cofilin-1 [Citation140]. Calpain-cleavage of dysferlin has also been linked with membrane repair of mechanical damage by promoting vesicle-membrane fusion [Citation141]. Together, these observations implicate calpain in disassembly of the cortical actin cytoskeleton to allow greater membrane fluidity and membrane alterations.
2.4.3. Conventional calpains and immune response
There is considerable evidence suggesting involvement of calpains in immune signaling. For example, calpain can cleave interleukin-1 alpha to produce its mature form [Citation142]; however, caspase activation may be necessary for complete activation and release of IL-1 alpha, even after calpain cleavage [Citation143]. Calpain has also been implicated in promoting inflammatory signaling of NF-kB by cleaving the IkB regulatory protein [Citation144]. Dysregulated expression of CAPN14 has also been associated with eosinophilic esophagitis [Citation59] Another study found that inhibiting calpain activation promoted autophagic degradation of PD-L1, which was associated with beneficial anti-tumor effects in an animal model. Beclin-1, a calpain substrate, is a suspected mediator of this process [Citation145]. Many other roles for calpain in regulation of inflammation have recently been reviewed [Citation146]. While many of these studies indicate calpain inhibition could protect against tissue and organ damage associated with excessive or chronic inflammation, there are also observations suggesting calpain inhibition could be detrimental [Citation146]; thus, careful study of potential side effects will need to be carried out in future clinical trials of calpain inhibitors.
2.5. Calpains as therapeutic targets
There are numerous readily available active site directed calpain inhibitors. However, there is considerable debate about the specificity of these inhibitors. Many that are described as calpain-specific inhibitors (for example, ALLN or calpeptin) exhibit inhibitory effects on other proteases, including cathepsins, the proteasome, or caspases (as disclosed in major vendor’ specifications). Peptidomimetic active-site-directed inhibitors have been developed based on the primary sequence of calpastatin. While more specific, these peptides generally lack good cell permeability and pharmacokinetic properties; however, a recent review of calpain inhibitors cites a patent on a blood-brain barrier permeable peptidomimetic inhibitor [Citation147]. The cell membrane or blood-brain barrier permeability is achieved by linking the CAST-mimetic sequence to lipid-soluble compounds, such as cholesterol, or to a cell-penetrating peptide sequence, such as from penetratin [Citation147,Citation148]. The benefit of such solubilized peptide inhibitors over conventional active site directed cysteine protease inhibitors of calpains is their high specificity, with 4–6 orders of magnitude difference in Ki between inhibiting calpains versus cathepsins, the proteasome, or caspases[Citation149].
Allosteric inhibitors of calpain exist as well. The most prominent of them, PD150606 and AMG853, are presumed to bind hydrophobic pockets in the PEF domains [Citation150]. Despite published models and co-crystal structures showing such interactions, Low et al. showed that, at least in case of PD150606, the mode of action is not through binding to a hydrophobic groove, and that PD150606 is a much weaker inhibitor compared to classic protease inhibitors [Citation151]. Currently, there are still no calpain inhibitors approved for clinical use.
2.5.1. Possible side-effects of calpain inhibition from preclinical studies
Systemic inhibition of calpain-1 or calpain-2 is likely to have distinct physiologic effects. The phenotype of calpain-1 deficient (CAPN1 mutant) mice, dogs, and humans is a predisposition to cerebellar ataxia and muscle wasting [Citation53]. Additionally, CAPN1 knockout mice have a defect in platelet aggregation and clot retraction, but surprisingly, there is no significant bleeding defect [Citation54]. There is evidence for several roles of calpain in platelet homeostasis [Citation152]. Muscle-specific deficiency in both calpain-1 and calpain-2 (through CAPNS1 knockout) caused dystrophy in aged mice, linked to the known role for calpain-1 and −2 in myoferlin cleavage during Ca2+-induced membrane repair [Citation153]. Complete germline calpain-2 deficiency in mice was associated with embryonic lethality [Citation154,Citation155]; however, ubiquitous inducible or tissue-specific CAPNS1 knockout (resulting in deficiency in both calpain-1 and −2) in adult mice is well tolerated. There are no known human pedigrees with loss-of-function mutations in CAPN2 or CAPNS1, which would likely be embryonic lethal in the homozygous state. However, there are multiple studies in human pedigrees describing loss-of-function mutations of CAPN1 that are predicted to compromise functions in PC1, PC2, CBSW and PEF(L) domains [Citation156,Citation157]. Inhibition of both calpain-1 and −2 in the brain affects long-term potentiation and susceptibility to traumatic brain injury [Citation52] and protects against cytotoxic neuronal cell death [Citation158]. Interestingly, inhibition of calpain-2 alone appears to be beneficial for neuronal survival [Citation52]. Similarly, indirect inhibition of calpains was also beneficial to survival of neuronal cells in a model of Alzheimer’s disease, where calpain activity was reduced through pharmacological inhibition of histone deacetylase and transcriptional upregulation of calpastatin [Citation159]. There is no evidence that transient calpain inhibition would have long-term detrimental effects outside of the traumatic brain injury scenario. In contrast, preclinical animal models suggest that calpain inhibition may protect against neuropathy induced by cancer chemotherapeutics [Citation84].
Other calpain isoforms with well-established roles in human disease are calpain-3, −5 and −14. Hypomorphic mutations in CAPN3 are associated with limb-girdle muscular dystrophy, observed both in human pedigrees and in mouse knockout models [Citation56], hypermorphic mutations of CAPN5 lead to retinopathy [Citation58], and CAPN14 is dynamically upregulated in patients with eosinophilic esophagitis [Citation59]. Thus, inhibition of calpain-3 could cause muscular dystrophy, while patients with mutations in CAPN5 or CAPN14 could benefit from inhibitors.
These data suggest that intermittent systemic inhibition of calpain-1 and −2 might be well tolerated, but considerations should be made for toxicities towards embryonic development, or possibly, effects of extended exposure to calpain inhibition on selected tissues. As of this writing, calpain inhibitor BLD-2660 is undergoing a clinical trial for treatment of COVID-19 to reduce tissue IL-6 and prevent lung fibrosis. Another phase-2 trial of this drug in idiopathic pulmonary fibrosis was recently withdrawn.
2.5.2. Other proteases in cancer
Calpains are not the only class of proteases studied in the context of cancer biology and therapy. Matrix metalloproteases degrade extracellular matrix (ECM) to promote cell motility (reviewed in [Citation160]). The proteasome recycles intracellular proteins, which promotes cell survival of both normal and malignant cells (reviewed in [Citation161]). Cathepsins, classically lysosomal enzymes, also participate in MHC-mediated antigen presentation, ECM degradation for cell invasion, and pro-survival autophagy [Citation162]. The plasmin protease of fibrin in blood clots can activate latent MMPs, and its activators and inhibitors have been implicated in cancer invasion [Citation163]; and caspases, general-purpose pro-apoptotic factors, can be hindered by dysregulation in calpain-mediated proteolysis [Citation2].
Perhaps the most studied among these, with promising selective anti-tumor effects in cancer models, are inhibitors of proteasomal proteases [Citation161] and matrix metalloproteases (MMP) [Citation160]; but both types are excessively vulnerable to acquired resistance and relapse in clinical settings, especially in solid tumors. However, in specific cases of myeloma and mantle-cell lymphoma, proteasome inhibitors like bortezomib, ixazomib and carfilzomib have been approved even as a first-line therapy [Citation161]. Failures of proteasome inhibitors in cancer have been traced to acquired mutations in the drug-binding pockets of the enzyme or upregulation of heat-shock and antioxidant response pathways [Citation161]. Combination of proteasome and HSP90 inhibitors might overcome such resistance, but there are no clinical data on the benefits of such combinations available yet [Citation161]. MMP inhibitors, which have similarly failed to produce reliable anti-cancer effects and also had a multitude of adverse side-effects, were speculated to be not sufficiently isoform-specific or requiring a very early intervention before the tumor has become invasive [Citation160]. Some side effects of proteasome or MMP inhibitors are diarrhea, thrombocytopenia, and painful neuropathy or dyspnea, musculoskeletal syndrome, and transaminitis, respectively [Citation160,Citation164].
In contrast, transient inhibition of calpain-1 and −2 are not expected to produce severe side effects in adults. While active-site inhibition of these calpains would also likely affect other calpain isoforms and other structurally homologous cysteine proteases, the dimeric nature of calpain-1 and −2 allows the possibility of allosteric protein-protein interaction inhibition of the PEF domains, which would likely have little off-target effect due to a limited number of other proteins containing PEF domains and due to relatively low sequence homology among them.
3. Conclusions
In summary, our growing understanding of the calpain system suggests that targeting calpain proteases represents a promising approach for the treatment of a wide range of diseases, including cancers. As we learn more about calpain biology, the complexity of calpain functions may reveal opportunities that can be exploited for therapeutic benefits. As novel calpain substrates continue to emerge, our understanding of the impact of calpain proteolysis of these substrates on pathways that they participate in may provide rationale for therapeutic strategies consisting of calpain inhibitors combined with other targeted agents. The development of isoform specific pharmacologic calpain inhibitors may be required to allow this approach to expand further into animal models, and eventually human trials.
4. Expert opinion
Calpain proteases have been studied for nearly fifty years, yet the extent to which we understand their biologic functions is still frustratingly incomplete. While they are widely conserved, studies of calpains in simpler organisms have provided limited insight into their developmental or physiologic roles. Extensive biochemical and structural studies have given us a solid understanding of their proteolytic activities and regulation by Ca2+, calpastain and other mechanisms, and crystal structures are available for some isoforms. Structure-function and genetic studies in cell systems, humans, and mice have also revealed relationships between mutations and disease phenotypes for some isoforms; and these studies continue to provide more detailed insights into their cellular and physiologic functions. However, we still do not have a good understanding of the cellular context under which the various calpain isoforms become activated, what controls their selection of specific substrates, and how substrate cleavage affects the global cell signaling network in normal biology and disease. Nevertheless, evidence of dysregulated calpain expression in diseases including cancers, fibrosis, muscular dystrophy, retinopathy, eosinophilic esophagitis, and Alzheimer’s has provided incentive to better understand potential etiologic roles for calpains in these diseases and to develop inhibitors that may be used therapeutically. In this regard, a challenging and exciting area of calpain research is the identification of physiologic substrates and elucidation of how calpain-mediated proteolytic cleavage affects their functions. Over one hundred protein substrates have been reported, and this list continues to grow. However, detailed mechanistic information regarding how calpain cleavage affects substrate functions and cellular behaviors is lacking for most of these substrates. The application of TAILS-MS or other emerging proteomic methods to quantify calpain-mediated cleavage of substrates under specific conditions has the potential to identify additional substrates and better understand calpain roles in cell signaling, as well as the implications for systemic calpain inhibition in disease contexts.
We are particularly interested in substrates that play roles in the regulation of cancer cell migration, invasion, survival, and proliferation because of the importance of these cell behaviours in metastasis and tumor progression. Emerging studies provide rationale for inhibiting calpains to interfere with these mechanisms as a strategy to suppress tumorigenesis and make cancer cells more susceptible to other therapeutics, including radiation, chemotherapies and targeted agents. Some of this insight comes from correlative translational studies that link high calpain-1 and −2 expression with poorer prognosis in different types of cancer. Preclinical studies using cultured cancer cells, engraftment, and transgenic mouse models are emerging that show genetic disruption of calpain-1 and −2 is associated with suppressed tumor growth or reduced metastatic behaviors. Beneficial effects of calpain-1 and −2 genetic disruption have also been seen in models of fibrosis. While these observations inspire efforts to develop more effective and specific calpain inhibitors, these efforts have not yet resulted in approved therapeutics. This represents an important unmet need which holds promise for the treatment of several diseases.
Recent efforts to inhibit calpains include the development of calpastatin-derived peptidomimetics and in silico-informed molecular design to exploit the hydrophobic pocket of CAPNS1. However, we still lack potent selective pharmacological inhibitors for in vivo studies and specific tools for assessing calpain activity in situ. These challenges need to be addressed using gene-specific knock-out model systems to verify that calpain is the selective target of experimental inhibitors. Another weakness is our incomplete understanding of calpain isoform-specific functions in different biological contexts, including cancers. For example, our current understanding is that calpain-1 and calpain-2 have both redundant and isoform-specific roles in terms of cleavable substrates and affected pathways; and current data suggests that disruption of either isoform can suppress tumorigenesis in some models. These observations argue that targeting either calpain-1 or calpain-2 has therapeutic potential in cancer. Not only does calpain inhibition have the potential to render cancer cells more susceptible to specific therapeutic challenges, but there is evidence that systemic calpain inhibition could also protect normal cells and tissues against the cytotoxic effects of some cancer treatments. This underscores the need to more fully elucidate the cell-specific roles that calpain isoforms are playing, especially in the context of systemic cancer therapies, and to ultimately develop isoform selective calpain inhibitors to use in rationally designed combinatory cancer therapies. The recent discovery that loss of function mutations in CAPN1 are associated with ataxia, and studies showing a protective effect of calpain-1 in brain injury suggest that calpain-2 specific inhibitors would be preferable in the treatment of neurodegenerative diseases and cancer.
Our understanding of calpain continues to grow and much of the historical and recently emerging knowledge supports the idea that isoform specific calpain inhibitors will become effective therapeutics in cancers and other diseases. The challenges going forward include improving our understanding of the effects of calpain inhibition on different cell types in various disease contexts, refining our knowledge of the structure and regulation of different calpain isoforms, developing biomarkers that reveal in vivo calpain activity, and using that knowledge to develop isoform-specific inhibitors that will be safe and effective therapeutics.
Article Highlights
Calpains are intracellular calcium-activated cysteine proteases that regulate processes, including cell survival, migration, and invasion, through a wide range of substrates.
Clinical data show that elevated expression of calpain-1 and calpain-2 isoforms in cancer is associated with shorter survival.
Genetic abrogation of calpain-1 and calpain-2 in cell and animal models correlates with anti-tumor effects.
Pharmacological inhibitors of calpain exist, but they often lack specificity. They are not approved for clinical use.
Many studies indicate that calpain inhibition could protect against tissue and organ damage associated with excessive or chronic inflammation.
Calpain inhibitor BLD-2660 is undergoing a clinical trial for treatment of COVID-19 to reduce tissue IL-6 and prevent lung fibrosis. Another phase-2 trial of this drug in idiopathic pulmonary fibrosis was recently withdrawn.
Calpain inhibition has potential side effects. For instance, inhibition of calpain activity could impact processes such as wound repair. Hence, surgery, may need to be planned carefully around calpain therapy.
This box summarizes key points contained in the article.
Declaration of interest
P Greer is the PI on a grant from the CIHR that supports this work.
The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose
Additional information
Funding
References
- Ono Y, Saido TC, Sorimachi H. Calpain research for drug discovery: challenges and potential. Nat Rev Drug Discov. 2016Dec; 15:12. 854–876. https://doi.org/10.1038/nrd.2016.212.
- Storr SJ, Carragher NO, Frame MC, et al. The calpain system and cancer. Nat Rev Cancer. 2011 May;11(5):364–374. DOI:https://doi.org/10.1038/nrc3050.
- Chen J, Wu Y, Zhang L, et al. Evidence for calpains in cancer metastasis. J Cell Physiol. 2019 Jun;234(6):8233–8240.
- LeloupL, Wells A. Calpains as potential anti-cancer targets. Expert Opin Ther Targets. 2011 Mar;15(3):309–323. DOI:https://doi.org/10.1517/14728222.2011.553611.
- Miyazaki T, Akasu R, Miyazaki A. Calpain-Associated Proteolytic Regulation of the Stromal Microenvironment in Cancer. Curr Pharm Des. 2021;27(28):3128–3138.
- Miyazaki T, Miyazaki A. Dysregulation of calpain proteolytic systems underlies degenerative vascular disorders.J Atheroscler Thromb. 2018 [Jan 1];25(1):1–15. DOI:https://doi.org/10.5551/jat.RV17008.
- Storr SJ, Thompson N, Pu X, et al. Calpain in breast cancer: role in disease progression and treatment response. Pathobiology. 2015 Sep;82(3–4):133–141.
- Ono Y, Sorimachi H. Calpains — an elaborate proteolytic system. Biochim Biophys Acta. 2012 Jan;1824(1):224–236.
- Liu Z, Cao J, Gao X, et al. GPS-CCD: a novel computational program for the prediction of calpain cleavage sites. PLoS ONE. 2011;6(4):e19001.
- Dutt P, Arthur JSC, Croall DE, et al. m -calpain subunits remain associated in the presence of calcium. FEBS Lett. 1998;436(3):367–371.
- Elce JS, Davies PL, Hegadorn C, et al. The effects of truncations of the small subunit on m-calpain activity and heterodimer formation. Biochem J. 1997;326(1):31–38.
- Campbell RL, Davies PL. Structure–function relationships in calpains.Biochem J. 2012 [Nov 1];447(3):335–351. DOI:https://doi.org/10.1042/BJ20120921.
- Strobl S, Fernandez-Catalan C, Braun M, et al. The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci U S A. 2000 [Jan 18];97(2):588–592. DOI:https://doi.org/10.1073/pnas.97.2.588.
- Moldoveanu T, Hosfield CM, Lim D, et al. A Ca2+ switch aligns the active site of calpain. Cell. 2002;108(5):649–660.
- Clapham DE. Calcium signaling. Cell. 2007;131(6):1047–1058.
- Breitwieser GE. Extracellular calcium as an integrator of tissue function. Int J Biochem Cell Biol. 2008;40(8):1467–1480.
- Hanna RA, Campbell RL, Davies PL. Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin. Nature. 2008 [Nov 20];456(7220):409–412.
- Salamino F, Averna M, Tedesco I, et al. Modulation of rat brain calpastatin efficiency by post-translational modifications.FEBS Lett. 1997 [Aug 4];412(3):433–438. DOI:https://doi.org/10.1016/S0014-5793(97)00819-3.
- Averna M, de Tullio R, Passalacqua M, et al. Changes in intracellular calpastatin localization are mediated by reversible phosphorylation.Biochem J. 2001 [Feb 15];354(1):25–30. DOI:https://doi.org/10.1042/bj3540025.
- Xu L, Deng X. Protein kinase Ciota promotes nicotine-induced migration and invasion of cancer cells via phosphorylation of micro- and m-calpains.J Biol Chem. 2006 [Feb 17];281(7):4457–4466. DOI:https://doi.org/10.1074/jbc.M510721200.
- Shiraha H, Glading A, Chou J, et al. Activation of m-calpain (calpain II) by epidermal growth factor is limited by protein kinase A phosphorylation of m-calpain. Mol Cell Biol. 2002 Apr;22(8):2716–2727.
- Bassett EA, Palanichamy K, Pearson M, et al. Calpastatin phosphorylation regulates radiation-induced calpain activity in glioblastoma. Oncotarget. 2018 [Mar 6];9(18):14597–14607. DOI:https://doi.org/10.18632/oncotarget.24523.
- Du M, Li X, Li Z, et al. Calpastatin inhibits the activity of phosphorylated μ-calpain in vitro. Food Chem. 2019;274:743–749. Feb 15. DOI:https://doi.org/10.1016/j.foodchem.2018.09.073
- Moldoveanu T, Gehring K, Green DR. Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains.Nature. 2008 [Nov 20];456(7220):404–408. DOI:https://doi.org/10.1038/nature07353.
- Moldoveanu T, Campbell RL, Cuerrier D, et al. Crystal structures of calpain–E64 and –leupeptin inhibitor complexes reveal mobile loops gating the active site.J Mol Biol. 2004 [2004 Nov 5];343(5):1313–1326. DOI:https://doi.org/10.1016/j.jmb.2004.09.016.
- Todd B, Moore D, Deivanayagam CCS, et al. A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor. J Mol Biol. 2003 [2003 Apr 18];328(1):131–146. DOI:https://doi.org/10.1016/S0022-2836(03)00274-2.
- Croall DE, Moffett K, Hatch H. Casein zymography of calpains using a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-imidazole buffer.Anal Biochem. 2002 [May 1];304(1):129–132. DOI:https://doi.org/10.1006/abio.2001.5606.
- Kelly JC, Cuerrier D, Graham LA, et al. Profiling of calpain activity with a series of FRET-based substrates. Biochim Biophys Acta. 2009 Oct;1794(10):1505–1509.
- McCartney CE, MacLeod JA, Greer PA, et al. An easy-to-use FRET protein substrate to detect calpain cleavage in vitro and in vivo. Biochim Biophys Acta Mol Cell Res. 2018 Feb;1865(2):221–230.
- Doucet A, Kleifeld O, Kizhakkedathu JN, et al. Identification of proteolytic products and natural protein N-termini by terminal amine isotopic labeling of substrates (TAILS). Methods Mol Biol. 2011;753:273–287.
- Potter DA, Tirnauer JS, Janssen R, et al. Calpain regulates actin remodeling during cell spreading. J Cell Biol. 1998 [May 4];141(3):129–132. DOI:https://doi.org/10.1083/jcb.141.3.647.
- Schoenwaelder SM, Yuan Y, Cooray P, et al. Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots.J Biol Chem. 1997 [Jan 17];272(3):1694–1702. DOI:https://doi.org/10.1074/jbc.272.3.1694.
- Chen H, Libertini SJ, Wang Y, et al. ERK regulates calpain 2-induced androgen receptor proteolysis in CWR22 relapsed prostate tumor cell lines.J Biol Chem. 2010 [Jan 22];285(4):2368–2374. DOI:https://doi.org/10.1074/jbc.M109.049379.
- Tan Y, Dourdin N, Wu C, et al. Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis.J Biol Chem. 2006 [Jun 9];281(23):16016–16024. DOI:https://doi.org/10.1074/jbc.M601299200.
- Tan Y, Wu C, De Veyra T, et al. 2006; Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli. J Biol Chem. Jun 30. 281:26. 17689–17698. https://doi.org/10.1074/jbc.M601978200.
- Pelley RP, Chinnakannu K, Murthy S, et al. Calmodulin-androgen receptor (AR) interaction: calcium-dependent, calpain-mediated breakdown of AR in LNCaP prostate cancer cells. Cancer Res. 2006 [Dec 15];66(24):1862–1869. DOI:https://doi.org/10.1158/0008-5472.CAN-06-2918.
- Yoo BH, Wu X, Li Y, et al. Oncogenic ras-induced down-regulation of autophagy mediator Beclin-1 is required for malignant transformation of intestinal epithelial cells. J Biol Chem. 2010 [Feb 19];285(8):5438–5449. DOI:https://doi.org/10.1074/jbc.M109.046789.
- Chua BT, Guo K, Li P. Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases.J Biol Chem. 2000 [Feb 18];275(7):5131–5135. DOI:https://doi.org/10.1074/jbc.275.7.5131.
- Nakagawa T, Yuan J. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis.J Cell Biol. 2000 [Aug 21];150(4):887–894. DOI:https://doi.org/10.1083/jcb.150.4.887.
- Patzke H, Tsai L-H. Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29.J Biol Chem. 2002 [Mar 8];277(10):8054–8060. DOI:https://doi.org/10.1074/jbc.M109645200.
- Huang C, Tandon NN, Greco NJ, et al. Proteolysis of platelet cortactin by calpain.J Biol Chem. 1997 [Aug 1];272(31):19248–19252. DOI:https://doi.org/10.1074/jbc.272.31.19248.
- Wang XD, Rosales JL, Magliocco A, et al. Cyclin E in breast tumors is cleaved into its low molecular weight forms by calpain.Oncogene. 2003 [Feb 6];22(5):769–774. DOI:https://doi.org/10.1038/sj.onc.1206166.
- Yao X, Thibodeau A, Forte JG. Ezrin-calpain I interactions in gastric parietal cells. Am J Physiol. 1993 Jul;265(1):C36–46.
- Kubbutat MH, Vousden KH. Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability. Mol Cell Biol. 1997 Jan;17(1):8054–8058.
- Tomita T, Huibregtse JM, Matouschek A. A masked initiation region in retinoblastoma protein regulates its proteasomal degradation.Nat Commun. 2020 [Apr 24];11(1):2019. DOI:https://doi.org/10.1038/s41467-020-16003-3.
- Tonnetti L, Netzel-Arnett S, Darnell GA, et al. SerpinB2 protection of retinoblastoma protein from calpain enhances tumor cell survival. Cancer Res. 2008 [Jul 15];68(14):460–468. DOI:https://doi.org/10.1158/0008-5472.CAN-07-5850.
- Pardo-Pastor C, Rubio-Moscardo F, Vogel-Gonzalez M, et al. Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses. Proc Natl Acad Sci U S A. 2018 [Feb 20];115(8):1925–1930. DOI:https://doi.org/10.1073/pnas.1718177115.
- Ackermann A, Brieger A. The role of nonerythroid spectrin αII in cancer. J Oncol. 2019;2019:7079604.
- Kwak H-I, Kang H, Dave JM, et al. Calpain-mediated vimentin cleavage occurs upstream of MT1-MMP membrane translocation to facilitate endothelial sprout initiation. Angiogenesis. 2012 Jun;15(2):287–303. DOI:https://doi.org/10.1007/s10456-012-9262-4.
- Hossain MI, Roulston CL, Kamaruddin MA, et al. A truncated fragment of Src protein kinase generated by calpain-mediated cleavage is a mediator of neuronal death in excitotoxicity. J Biol Chem. 2013 [Apr 5];288(14):9696–9709. DOI:https://doi.org/10.1074/jbc.M112.419713.
- Mahaman YAR, Huang F, Kessete Afewerky H, et al. Involvement of calpain in the neuropathogenesis of Alzheimer’s disease. Med Res Rev. 2019 Mar;39(2):608–630.
- Baudry M. Calpain-1 and Calpain-2 in the brain: dr. Jekill and Mr Hyde? Curr Neuropharmacol. 2019;17(9):823–829. DOI:https://doi.org/10.2174/1570159X17666190228112451.
- Wang Y, Hersheson J, Lopez D, et al. Defects in the CAPN1 gene result in alterations in cerebellar development and cerebellar ataxia in mice and humans. Cell Rep. 2016 [Jun 28];16(1):79–91. DOI:https://doi.org/10.1016/j.celrep.2016.05.044.
- Azam M, Andrabi SS, Sahr KE, et al. Disruption of the mouse mu-calpain gene reveals an essential role in platelet function. Mol Cell Biol. 2001 Mar;21(6):2213–2220.
- Richard I, Broux O, Allamand V, et al. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell. 1995 [Apr 7];81(1):27–40. DOI:https://doi.org/10.1016/0092-8674(95)90368-2.
- Ono Y, Ojima K, Shinkai-Ouchi F, et al. An eccentric calpain, CAPN3/p94/calpain-3. Biochimie. 2016 Mar;122:169–187.
- Mahajan VB, Skeie JM, Bassuk AG, et al. Calpain-5 mutations cause autoimmune uveitis, retinal neovascularization, and photoreceptor degeneration. PLoS Genet. 2012;8(10):e1003001. DOI:https://doi.org/10.1371/journal.pgen.1003001.
- Tang PH, Chemudupati T, Wert KJ, et al. Phenotypic variance in Calpain-5 retinal degeneration. Am J Ophthalmol Case Rep. 2020;18:100627.
- Kottyan LC, Davis BP, Sherrill JD, et al. Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease. Nat Genet. 2014 Aug;46(8):895–900. DOI:https://doi.org/10.1038/ng.3033.
- Braun C, Engel M, Seifert M, et al. Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type. Int J Cancer. 1999 [Feb 19];84(1):6–9. DOI:https://doi.org/10.1002/(SICI)1097-0215(19990219)84:1<6::AID-IJC2>3.0.CO;2-T.
- Tang S, Yin Q, Liu F, et al. Calpain small subunit 1 protein in the prognosis of cancer survivors and its clinicopathological correlation. Biomed Res Int. 2019;2019:8053706.
- Zhao C, Yuan G, Jiang Y, et al. Capn4 contributes to tumor invasion and metastasis in gastric cancer via activation of the Wnt/beta-catenin/MMP9 signalling pathways. Exp Cell Res. 2020 [Oct 15];395(2):112220. DOI:https://doi.org/10.1016/j.yexcr.2020.112220.
- Xu C, Yu X, Zhu Y, et al. Overexpression of calpain1 predicts poor outcome in patients with colorectal cancer and promotes tumor cell progression associated with downregulation of FLNA. Oncol Rep. 2019 Jun;41(6):3424–3434. DOI:https://doi.org/10.3892/or.2019.7121.
- Huang C, Miller RT, Freter CE. Signaling regulation and role of filamin A cleavage in Ca2+-stimulated migration of androgen receptor-deficient prostate cancer cells.Oncotarget. 2017 [Jan 17];8(3):3840–3853. DOI:https://doi.org/10.18632/oncotarget.9472.
- Gao X, Mao Y-H, Xiao C, et al. Calpain-2 triggers prostate cancer metastasis via enhancing CRMP4 promoter methylation through NF-kappaB/DNMT1 signaling pathway. Prostate. 2018 Jun;78(9):682–690. DOI:https://doi.org/10.1002/pros.23512.
- Yu LM, Zhu YS, Xu CZ, et al. High calpain-1 expression predicts a poor clinical outcome and contributes to tumor progression in pancreatic cancer patients. Clin Transl Oncol. 2019 Jul;21(7):682–690.
- Yoshida M, Miyasaka Y, Ohuchida K, et al. Calpain inhibitor calpeptin suppresses pancreatic cancer by disrupting cancer-stromal interactions in a mouse xenograft model. Cancer Sci. 2016 Oct;107(10):1443–1452. DOI:https://doi.org/10.1111/cas.13024.
- Storr SJ, Safuan S, Woolston CM, et al. Calpain-2 expression is associated with response to platinum based chemotherapy, progression-free and overall survival in ovarian cancer. J Cell Mol Med. 2012 Oct;16(10):2422–2428. DOI:https://doi.org/10.1111/j.1582-4934.2012.01559.x.
- Storr SJ, Woolston CM, Barros FF, et al. Calpain-1 expression is associated with relapse-free survival in breast cancer patients treated with trastuzumab following adjuvant chemotherapy. Int J Cancer. 2011 [Oct 1];129(7):1773–1780. DOI:https://doi.org/10.1002/ijc.25832.
- Storr SJ, Lee KW, Woolston CM, et al. Calpain system protein expression in basal-like and triple-negative invasive breast cancer. Ann Oncol. 2012 Sep;23(9):2289–2296. DOI:https://doi.org/10.1093/annonc/mds176.
- Pu X, Storr SJ, Ahmad NS, et al. Calpain-1 is associated with adverse relapse free survival in breast cancer: a confirmatory study. Histopathology. 2016 Jun;68(7):1021–1029. DOI:https://doi.org/10.1111/his.12896.
- Davis J, Martin SG, Patel PM, et al. Low calpain-9 is associated with adverse disease-specific survival following endocrine therapy in breast cancer. BMC Cancer. 2014 [Dec 23];14(1):995. DOI:https://doi.org/10.1186/1471-2407-14-995.
- Storr SJ, Zhang S, Perren T, et al. The calpain system is associated with survival of breast cancer patients with large but operable inflammatory and non-inflammatory tumours treated with neoadjuvant chemotherapy. Oncotarget. 2016 [Jul 26];7(30):47927–47937. DOI:https://doi.org/10.18632/oncotarget.10066.
- Storr SJ, Pu X, Davis J, et al. Expression of the calpain system is associated with poor clinical outcome in gastro-oesophageal adenocarcinomas. J Gastroenterol. 2013 Nov;48(11):1213–1221. DOI:https://doi.org/10.1007/s00535-012-0743-4.
- Storr SJ, Zaitoun AM, Arora A, et al. Calpain system protein expression in carcinomas of the pancreas, bile duct and ampulla. BMC Cancer. 2012 [2012 Nov 9];12(1):511. DOI:https://doi.org/10.1186/1471-2407-12-511.
- Reichrath J, Welter C, Mitschele T, et al. Different expression patterns of calpain isozymes 1 and 2 (CAPN1 and 2) in squamous cell carcinomas (SCC) and basal cell carcinomas (BCC) of human skin. J Pathol. 2003;199(4):509–516. DOI:https://doi.org/10.1002/path.1308.
- Cai L, Li Q, Li W, et al. Calpain suppresses cell growth and invasion of glioblastoma multiforme by producing the cleavage of filamin A. Int J Clin Oncol. 2020 Jun;25(6):1055–1066. DOI:https://doi.org/10.1007/s10147-020-01636-7.
- Mair J, Lindahl B, Hammarsten O, et al. How is cardiac troponin released from injured myocardium? European Heart Journal: Acute Cardiovascular Care. 2018;7(6):553–560. DOI:https://doi.org/10.1177/2048872617748553.
- Kositprapa C, Zhang B, Berger S, et al. Calpain-mediated proteolytic cleavage of troponin I induced by hypoxia or metabolic inhibition in cultured neonatal cardiomyocytes.Mol Cell Biochem. 2000 [2000 Nov 1];214(1):47–55. DOI:https://doi.org/10.1023/A:1007160702275.
- Vanderklish PW, Bahr BA. The pathogenic activation of calpain: a marker and mediator of cellular toxicity and disease states. Int J Exp Pathol. 2000 Oct;81(5):323–339.
- Wang Y, Brazdzionis J, Dong F, et al. P13BP, a calpain-2-mediated breakdown product of PTPN13, is a novel blood biomarker for Traumatic Brain Injury. J Neurotrauma. 2021 [Sep 9];38(22):3077–3085. DOI:https://doi.org/10.1089/neu.2021.0229.
- Zheng D, Su Z, Zhang Y, et al. Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity. Arch Toxicol. 2019 Apr;93(4):1051–1065. DOI:https://doi.org/10.1007/s00204-019-02405-w.
- Boeckel GR, Ehrlich BE. NCS-1 is a regulator of calcium signaling in health and disease. Biochim Biophys Acta Mol Cell Res. 2018 Nov;1865(11):1660–1667.
- Cetinkaya-Fisgin A, Luan X, Reed N, et al. Cisplatin induced neurotoxicity is mediated by Sarm1 and calpain activation.Sci Rep. 2020 [Dec 14];10(1):21889. DOI:https://doi.org/10.1038/s41598-020-78896-w.
- Wang MS, Davis AA, Culver DG, et al. Calpain inhibition protects against Taxol-induced sensory neuropathy. Brain. 2003 Mar;127(3):671–679.
- Hoskin V, Szeto A, Ghaffari A, et al. Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion.Mol Biol Cell. 2015 [Oct 1];26(19):3464–3479. DOI:https://doi.org/10.1091/mbc.E14-12-1584.
- Sundaramoorthy P, Sim JJ, Jang Y-S, et al. Modulation of intracellular calcium levels by calcium lactate affects colon cancer cell motility through calcium-dependent calpain. PLoS One. 2015;10(1):e0116984. DOI:https://doi.org/10.1371/journal.pone.0116984.
- Lee J, Rosales JL, Byun H-G, et al. L-methadone causes leukemic cell apoptosis via an OPRM1-triggered increase in IP3R-mediated ER Ca(2+) release and decrease in Ca(2+) efflux, elevating [Ca(2+)](i).Sci Rep. 2021 [Jan 13];11(1):1009. DOI:https://doi.org/10.1038/s41598-020-80520-w.
- Shi M, Zhang T, Sun L, et al. Calpain, Atg5 and Bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium. Apoptosis. 2013 Apr;18(4):435–451. DOI:https://doi.org/10.1007/s10495-012-0786-2.
- Escalante AM, McGrath RT, Karolak MR, et al. Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo. Cancer Chemother Pharmacol. 2013 Jun;71(6):1567–1576.
- Duquette PM, Lamarche-Vane N. The calcium-activated protease calpain regulates netrin-1 receptor deleted in colorectal cancer-induced axon outgrowth in cortical neurons. J Neurochem. 2020 Feb;152(3):1051–1065.
- Michel JJC, Scott JD. AKAP mediated signal transduction. Annu Rev Pharmacol Toxicol. 2002;42(1):235–257.
- Gafni J, Cong X, Chen SF, et al. Calpain-1 cleaves and activates caspase-7.J Biol Chem. 2009 [Sep 11];284(37):25441–25449. DOI:https://doi.org/10.1074/jbc.M109.038174.
- Gao G, Dou QP. N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death.J Cell Biochem. 2021 [Sep 18];80(1):53–72. DOI:https://doi.org/10.1002/1097-4644(20010101)80:1<53::AID-JCB60>3.0.CO;2-E.
- Yeo J-K, Cha S-D, Cho C-H, et al. Se-methylselenocysteine induces apoptosis through caspase activation and Bax cleavage mediated by calpain in SKOV-3 ovarian cancer cells. Cancer Lett. 2002 [Aug 8];182(1):83–92. DOI:https://doi.org/10.1016/S0304-3835(02)00075-7.
- Mandic A, Viktorsson K, Strandberg L, et al. Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis. Mol Cell Biol. 2002 May;22(9):3003–3013. DOI:https://doi.org/10.1128/MCB.22.9.3003-3013.2002.
- Andree M, Seeger JM, Schull S, et al. Bid -dependent release of mitochondrial SMAC dampens XIAP -mediated immunity against Shigella. EMBO J. 2014 [Oct 1];33(19):224–236. DOI:https://doi.org/10.15252/embj.201387244.
- Zhuang Q, Shen J, Chen Z, et al. MiR-337-3p suppresses the proliferation and metastasis of clear cell renal cell carcinoma cells via modulating Capn4. Cancer Biomark. 2018;23(4):515–525. DOI:https://doi.org/10.3233/CBM-181645.
- Hirai S, Kawasaki H, Yaniv M, et al. Degradation of transcription factors, c-Jun and c-Fos, by calpain.FEBS Lett. 1991 [Aug 5];287(1–2):57–61. DOI:https://doi.org/10.1016/0014-5793(91)80015-U.
- Pozo K, Bibb JA. The emerging role of Cdk5 in cancer. Trends Cancer. 2016 Oct;2(10):606–618.
- Norberg E, Gogvadze V, Ott M, et al. An increase in intracellular Ca2+ is required for the activation of mitochondrial calpain to release AIF during cell death. Cell Death Differ. 2008 Dec;15(12):1857–1864. DOI:https://doi.org/10.1038/cdd.2008.123.
- Gonen H, Shkedy D, Barnoy S, et al. On the involvement of calpains in the degradation of the tumor suppressor protein p53.FEBS Lett. 1997 [Apr 7];406(1–2):17–22. DOI:https://doi.org/10.1016/S0014-5793(97)00225-1.
- Woo MG, Xue K, Liu J, et al. Calpain-mediated processing of p53-associated parkin-like cytoplasmic protein (PARC) affects chemosensitivity of human ovarian cancer cells by promoting p53 subcellular trafficking.J Biol Chem. 2012 [Feb 3];287(6):3963–3975. DOI:https://doi.org/10.1074/jbc.M111.314765.
- Conacci-Sorrell M, Ngouenet C, Anderson S, et al. Stress-induced cleavage of Myc promotes cancer cell survival.Genes Dev. 2014 [Apr 1];28(7):689–707. DOI:https://doi.org/10.1101/gad.231894.113.
- Conacci-Sorrell M, Ngouenet C, Eisenman RN. Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.Cell. 2010 [Aug 6];142(3):480–493. DOI:https://doi.org/10.1016/j.cell.2010.06.037.
- Grieve S, Gao Y, Hall C, et al. 2016; Calpain genetic disruption and HSP90 inhibition combine to attenuate mammary tumorigenesis. Mol Cell Biol. Aug 1. 36:15. 2078–2088. https://doi.org/10.1128/MCB.01062-15.
- Ho W-C, Pikor L, Gao Y, et al. Calpain 2 regulates Akt-FoxO-p27(Kip1) protein signaling pathway in mammary carcinoma.J Biol Chem. 2012 [May 4];287(19):15458–15465. DOI:https://doi.org/10.1074/jbc.M112.349308.
- Bertoli C, Copetti T, Lam EWF, et al. Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A.Oncogene. 2009 [2009 Feb 1];28(5):721–733. DOI:https://doi.org/10.1038/onc.2008.425.
- Xu L, Deng X. Suppression of cancer cell migration and invasion by protein phosphatase 2A through dephosphorylation of mu- and m-calpains.J Biol Chem. 2006 [Nov 17];281(46):15458–15465. DOI:https://doi.org/10.1074/jbc.M607702200.
- Fernandes M, Duplaquet L, Tulasne D. Proteolytic cleavages of MET: the divide-and-conquer strategy of a receptor tyrosine kinase. BMB Rep. 2019 Apr;52(4):239–249.
- Kulkarni S, Reddy KB, Esteva FJ, et al. Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer.Oncogene. 2010 [Mar 4];29(9):1339–1350. DOI:https://doi.org/10.1038/onc.2009.422.
- MacLeod JA, Gao Y, Hall C, et al. 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.Oncotarget. 2018 [Sep 7];9(70):33382–33395. DOI:https://doi.org/10.18632/oncotarget.26078.
- Ai M, Qiu S, Lu Y, et al. HER2 regulates Brk/PTK6 stability via upregulating calpastatin, an inhibitor of calpain. Cell Signal. 2013 Sep;25(9):1754–1761.
- Bernaudo S, Khazai S, Honarparvar E, et al. Epidermal growth factor promotes cyclin G2 degradation via calpain-mediated proteolysis in gynaecological cancer cells. PLoS One. 2017;12(6):e0179906.
- Libertini SJ, Robinson BS, Dhillon NK, et al. Cyclin E both regulates and is regulated by calpain 2, a protease associated with metastatic breast cancer phenotype. Cancer Res. 2005 [Dec 1];65(23):10700–10708. DOI:https://doi.org/10.1158/0008-5472.CAN-05-1666.
- Choi YH, Lee SJ, Nguyen P, et al. Regulation of cyclin D1 by calpain protease. J Biol Chem. 1997 [Nov 7];272(45):28479–28484. DOI:https://doi.org/10.1074/jbc.272.45.28479.
- Zhang Y, Liu NM, Wang Y, et al. Endothelial cell calpain as a critical modulator of angiogenesis. Biochim Biophys Acta Mol Basis Dis. 2017 Jun;1863(6):1326–1335.
- Miyazaki T, Taketomi Y, Saito Y, et al. Calpastatin counteracts pathological angiogenesis by inhibiting suppressor of cytokine signaling 3 degradation in vascular endothelial cells. Circ Res. 2015 [Mar 27];116(7):1170–1181. DOI:https://doi.org/10.1161/CIRCRESAHA.116.305363.
- Franco SJ, Rodgers MA, Perrin BJ, et al. Calpain-mediated proteolysis of talin regulates adhesion dynamics. Nat Cell Biol. 2004 [2004 Oct 1];6(10):977–983. DOI:https://doi.org/10.1038/ncb1175.
- Chan KT, Bennin DA, Huttenlocher A. Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK).J Biol Chem. 2010 [Apr 9];285(15):11418–11426. DOI:https://doi.org/10.1074/jbc.M109.090746.
- Zhuang Q, Luo W, Zhang M, et al. Capn4 contributes to tumor invasion and metastasis in clear cell renal cell carcinoma cells via modulating talin-focal adhesion kinase signaling pathway. Acta Biochim Biophys Sin (Shanghai). 2018 [May 1];50(5):465–472. DOI:https://doi.org/10.1093/abbs/gmy031.
- Perrin BJ, Amann KJ, Huttenlocher A. Proteolysis of cortactin by calpain regulates membrane protrusion during cell migration. Mol Biol Cell. 2006 Jan;17(1):239–250.
- Cortesio CL, Chan KT, Perrin BJ, et al. Calpain 2 and PTP1B function in a novel pathway with Src to regulate invadopodia dynamics and breast cancer cell invasion. J Cell Biol. 2008 [Mar 10];180(5):957–971. DOI:https://doi.org/10.1083/jcb.200708048.
- Kulkarni S, Goll DE, Fox JE. Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading.J Biol Chem. 2002 [Jul 5];277(27):24435–24441. DOI:https://doi.org/10.1074/jbc.M203457200.
- Rios-Doria J, Day KC, Kuefer R, et al. The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells. J Biol Chem. 2003 [Jan 10];278(2):1372–1379. DOI:https://doi.org/10.1074/jbc.M208772200.
- Rios-Doria J, Kuefer R, Ethier SP, et al. Cleavage of beta-catenin by calpain in prostate and mammary tumor cells.Cancer Res. 2004 [Oct 15];64(20):7237–7240. DOI:https://doi.org/10.1158/0008-5472.CAN-04-1048.
- Franco S, Perrin B, Huttenlocher A. Isoform specific function of calpain 2 in regulating membrane protrusion.Exp Cell Res. 2004 [Sep 10];299(1):179–187. DOI:https://doi.org/10.1016/j.yexcr.2004.05.021.
- Serrano K, Devine DV. Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton. Cell Motil Cytoskeleton. 2004 Aug;58(4):242–252.
- Chang S-J, Chen Y-C, Yang C-H, et al. Revealing the three dimensional architecture of focal adhesion components to explain Ca(2+)-mediated turnover of focal adhesions. Biochim Biophys Acta Gen Subj. 2017 Mar;1861(3):624–635. DOI:https://doi.org/10.1016/j.bbagen.2017.01.002.
- J-c W, Chen Y-C, Kuo C-T, et al. Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration. Sci Rep. 2015 [2015 Dec 18];5(1):18476. DOI:https://doi.org/10.1038/srep18476.
- Salimi R, Bandaru S, Devarakonda S, et al. Blocking the cleavage of filamin a by calpain inhibitor decreases tumor cell growth. Anticancer Res. 2018 Apr;38(4):957–971. DOI:https://doi.org/10.21873/anticanres.12447.
- Zheng X, Zhou AX, Rouhi P, et al. Hypoxia-induced and calpain-dependent cleavage of filamin A regulates the hypoxic response. Proc Natl Acad Sci U S A. 2014 [Feb 18];111(7):2560–2565. DOI:https://doi.org/10.1073/pnas.1320815111.
- McGrath MJ, Binge LC, Sriratana A, et al. Regulation of the transcriptional coactivator FHL2 licenses activation of the androgen receptor in castrate-resistant prostate cancer. Cancer Res. 2013 [Aug 15];73(16):5066–5079. DOI:https://doi.org/10.1158/0008-5472.CAN-12-4520.
- Libertini SJ, Tepper CG, Rodriguez V, et al. Evidence for calpain-mediated androgen receptor cleavage as a mechanism for androgen Independence.Cancer Res. 2007 [Oct 1];67(19):9001–9005. DOI:https://doi.org/10.1158/0008-5472.CAN-07-1072.
- Sivanandam A, Murthy S, Chinnakannu K, et al. Calmodulin protects androgen receptor from calpain-mediated breakdown in prostate cancer cells. J Cell Physiol. 2011 Jul;226(7):1889–1896. DOI:https://doi.org/10.1002/jcp.22516.
- Mamoune A, Luo J-H, Lauffenburger DA, et al. Calpain-2 as a target for limiting prostate cancer invasion. Cancer Res. 2003 [Aug 1];63(15):4632–4640.
- Taylor J, Azimi I, Monteith G, et al. Ca(2+) mediates extracellular vesicle biogenesis through alternate pathways in malignancy. J Extracell Vesicles. 2020;9(1):1734326.
- Goll DE, Thompson VF, Li H, et al. The calpain system. Physiol Rev. 2003 Jul;83(3):731–801.
- Taylor J, Patio K, De Rubis G, et al. Membrane to cytosol redistribution of αII-spectrin drives extracellular vesicle biogenesis in malignant breast cells. Proteomics. 2021 Apr 19;21(13–14):e2000091.
- Larsen AK, Lametsch R, Elce J, et al. Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin. Biochem J. 2008 [May 1];411(3):657–666. DOI:https://doi.org/10.1042/BJ20070522.
- Redpath GM, Woolger N, Piper AK, et al. Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair. Mol Biol Cell. 2014 [Oct 1];25(19):3037–3048. DOI:https://doi.org/10.1091/mbc.e14-04-0947.
- Tsuchiya K, Hosojima S, Hara H, et al. Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes. Cell Rep. 2021 [Mar 23];34(12):108887. DOI:https://doi.org/10.1016/j.celrep.2021.108887.
- Wiggins KA, Parry AJ, Cassidy LD, et al. IL-1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence-associated secretory phenotype. Aging Cell. 2019 Jun;18(3):e12946. DOI:https://doi.org/10.1111/acel.12946.
- Shumway SD, Maki M, Miyamoto S. The PEST domain of IkappaBalpha is necessary and sufficient for in vitro degradation by mu-calpain.J Biol Chem. 1999 [Oct 22];274(43):30874–30881. DOI:https://doi.org/10.1074/jbc.274.43.30874.
- Li C, Yao H, Wang H, et al. Repurposing screen identifies amlodipine as an inducer of PD-L1 degradation and antitumor immunity. Oncogene. 2021 Feb;40(6):1128–1146.
- Ji J, Su L, Liu Z. Critical role of calpain in inflammation. Biomed Rep. 2016 Dec;5(6):647–652.
- Donkor IO. An update on the therapeutic potential of calpain inhibitors: a patent review. Expert Opin Ther Pat. 2020 Sep;30(9):659–675.
- Fiorino F, Gil-Parrado S, Assfalg-Machleidt I, et al. A new cell-permeable calpain inhibitor. J Pept Sci. 2007 Jan;13(1):70–73.
- Gil-Parrado S, Assfalg-Machleidt I, Fiorino F, et al. Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: enhancing cell permeability by conjugation with penetratin. Biol Chem. 2003 Mar;384(3):395–402. DOI:https://doi.org/10.1515/BC.2003.045.
- Kalash L, Cresser-Brown J, Habchi J, et al. Structure-based design of allosteric calpain-1 inhibitors populating a novel bioactivity space. Eur J Med Chem. 2018 [Sep 5];157:1264–1275.
- Low KE, Karunan Partha S, Davies PL, et al. Allosteric inhibitors of calpains: reevaluating inhibition by PD150606 and LSEAL. Biochim Biophys Acta. 2014 Dec;1840(12):3367–3373.
- Kuchay SM, Chishti AH. Calpain-mediated regulation of platelet signaling pathways. Curr Opin Hematol. 2007 May;14(3):249–254.
- Piper A-K, Sophocleous RA, Ross SE, et al. Loss of calpains-1 and −2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy. Am J Physiol Cell Physiol. 2020 [Jun 1];318(6):C1226–c37. DOI:https://doi.org/10.1152/ajpcell.00408.2019.
- Takano J, Mihira N, Fujioka R, et al. Vital role of the calpain-calpastatin system for placental-integrity-dependent embryonic survival. Mol Cell Biol. 2011 Oct;31(19):4097–4106.
- Dutt P, Croall DE, Arthur JS, et al. m-calpain is required for preimplantation embryonic development in mice. BMC Dev Biol. 2006 [Jan 24];6(1):3. DOI:https://doi.org/10.1186/1471-213X-6-3.
- Lai -L-L, Chen Y-J, Li Y-L, et al. Novel CAPN1 mutations extend the phenotypic heterogeneity in combined spastic paraplegia and ataxia. Ann Clin Transl Neurol. 2020 Oct;7(10):1862–1869. DOI:https://doi.org/10.1002/acn3.51169.
- Kim A, Kumar KR, Davis RL, et al. Increased diagnostic yield of spastic paraplegia with or without cerebellar ataxia through whole-genome sequencing. Cerebellum. 2019 Aug;18(4):781–790. DOI:https://doi.org/10.1007/s12311-019-01038-0.
- Sedarous M, Keramaris E, O’Hare M, et al. Calpains mediate p53 activation and neuronal death evoked by DNA damage. J Biol Chem. 2003 [Jul 11];278(28):26031–26038. DOI:https://doi.org/10.1074/jbc.M302833200.
- Seo J, Jo SA, Hwang S, et al. Trichostatin A epigenetically increases calpastatin expression and inhibits calpain activity and calcium-induced SH-SY5Y neuronal cell toxicity. Febs J. 2013 Dec;280(24):6691–6701. DOI:https://doi.org/10.1111/febs.12572.
- Winer A, Adams S, Mignatti P. Matrix metalloproteinase inhibitors in cancer therapy: turning past failures into future successes. Mol Cancer Ther. 2018 Jun;17(6):1147–1155.
- Manasanch EE, Orlowski RZ. Proteasome inhibitors in cancer therapy.Nat Rev Clin Oncol. 2017 [2017 July 1];14(7):417–433. DOI:https://doi.org/10.1038/nrclinonc.2016.206.
- Tan G-J, Peng Z-K, Lu J-P, et al. Cathepsins mediate tumor metastasis.World J Biol Chem. 2013 [Nov 26];4(4):91–101. DOI:https://doi.org/10.4331/wjbc.v4.i4.91.
- Kwaan HC, McMahon B. The role of plasminogen-plasmin system in cancer. Cancer Treat Res. 2009;148:43–66.
- Tan CRC, Abdul-Majeed S, Cael B, et al. Clinical pharmacokinetics and pharmacodynamics of bortezomib. Clin Pharmacokinet. 2019 Feb;58(2):157–168.