Calpains are intracellular cysteine-proteases described as being calcium-dependent in vitro
, though the mechanisms of activation in cells is varied, not always involving calcium. The calpain family is currently constituted by 15 members, classified according to their localization (ubiquitous or tissue-specific) or to the presence or the absence of EF-hands, structures allowing calcium binding (calpains are typical if they encode EF-hands, atypical if they do not) (, ) [1
]. The calpain system also includes the endogenous inhibitor of calpains 1 and 2, calpastatin, and two regulatory small subunits carrying EF-hands, calpains S1 and S2 [2
]. Calpain S1 is known to interact with calpain 1 and calpain 2 to form heterodimers called µ-calpain and m-calpain, respectively (). This regulatory subunit is required for the proper folding of the heterodimers as well as in the regulation of their activity. The role(s) played by the S2 subunit and its ability to heterodimerize currently remain unknown. Calpains have a broad spectrum of substrates, from proteins of the cytoskeleton (such as talin, vinculin, …) to transcription factors (p53, c-fos, …), and enzymes (PKC, caspases, RhoA/Rac …) (). The proteolysis actuated by calpains is not degradative but rather signaling in that the fragments generated act as dominant-negative or –positive elements (cleavage of RhoA [3
], talin [4
], EGFR [5
], …). Thus, the inhibition of calpains should be considered analogous to abrogation of diverse signaling pathways.
Schematic diagram of the structure of µ- and m-calpain, calpain-3, -6, and -9.
Schematic representation of ubiquitous calpain regulation and key substrates.
By cleaving these numerous substrates, calpains are involved in a large number of physiological and pathological phenomena, from embryogenesis to cell adhesion, diabetes, and Alzheimer’s disease [1
] (). Key to this discussion, several studies have also linked the calpain system to cancer development and progression. Indeed, the expression and/or activity of several members of the calpain system have been shown to be strongly altered in different types of cancer cells or transformed cells (). Recent publications have shed light on possible molecular mechanisms; with these focusing mainly on the ubiquitous calpains 1 and 2 ().
Alterations of calpain system in different types of cancer
Implications of calpain family members in cancer
Among the 15 members of the calpain family, the ubiquitous calpains 1 and 2 are the most intensely studied. The heterodimers µ- and m-calpain that they form are known to regulate and even be required for numerous physiological processes. It is important to note that in cells, the contribution of µ-calpain versus m-calpain is often not evident or experimentally distinguished; it is not determined whether this is due to co-regulation or compensation. Several of these processes, such as regulation of cell cycle and apoptosis, adhesion, migration, invasion, and also angiogenesis, are critical for the formation, progression, and growth of cancers (see & ).
Schematic representation of the implication of the ubiquitous calpains in the different mechanisms leading to cancer progression or suppression.
The ubiquitous calpains were notably shown to regulate the cell cycle, particularly the transition between the phases G1
and S [9
]. Indeed several crucial regulators of this phase of the cell cycle are substrates of µ- and m-calpain, including two key cyclins, the cyclin D1 and the cyclin E [12
]. In normal cells, the degradation of these two cyclins by the ubiquitous calpains induces the cell cycle arrest. But in cancer cells the opposite occurs; calpains can promote the progression of the cell cycle in transformed cells or tumor cells. Indeed, in these cells calpains were shown to proteolyze two inhibitors of cyclin-dependent kinases (Cdk), p21Cip1
, thus leading to the activation of the complexes cyclinD-Cdk4 and cyclinE-Cdk2 [14
]. Cdk5, a cyclin-dependent kinase implicated in the regulation of neuronal cell cycle was also shown to be a substrate of calpains [16
]. By regulating these proteins, ubiquitous calpains are thus strongly involved in the progression of the cell cycle and in cell proliferation.
Ubiquitous calpains are also known to be involved in the regulation of cell death. In fact, calpain was the first protease identified in initiating apoptosis [17
]. Several studies have notably highlighted how closely these proteases are linked to caspases. Mu- and m-calpain cleave several members of caspase family, thus activating the caspase-3, -7 and -12 and inactivating the caspase-8 and -9 [18
]. By regulating caspases, calpains can thus control indirectly apoptosis. Also, in situations of mass calcium influx, membrane transection or ischemia/reperfusion injury, the ubiquitous calpains are activated and in turn trigger caspase-3 [20
]. Ubiquitous calpains were also shown to regulate cell proliferation and apoptosis through the tumor suppressor protein p53 [21
]. Indeed µ- and m-calpain can cleave this transcription factor inducing its degradation; the implications of loss of p53 regulation of genomic repair for tumor genetic instability are obvious.
Ubiquitous calpains were shown to control cell adhesion and migration in different types of normal cells (such as fibroblasts, endothelial cells, myoblasts, …). Indeed, in physiological conditions, µ-calpain controls the formation of focal adhesion complexes through the regulation of Rho GTPases, thus allowing cell adhesion [3
], while m-calpain controls cell de-adhesion by cleaving several components of these complexes (such as paxillin, talin, vinculin, FAK) [22
]. Ubiquitous calpains regulate cell adhesion to the substratum, and thereby cell migration as this process was previously described as a succession of adhesion and de-adhesion steps [24
]. Recent publications have shown that the regulation of migration by calpains depends on a compartmentalization of these enzymes: in adherenet cells, µ-calpain is localized at the front of the cells (in the lamellipodia) where it induces focal adhesion complexes, while m-calpain is concentrated at the rear of the cells where it induces cell retraction [25
]. However, the distribution of the calpain isoforms in cells of the immune system may be different [27
], as motility in these cells qualitatively and quantitatively distinct. By regulating cell migration, calpains can also control tumor dissemination. Several studies have shown that these proteases can regulate the invasiveness of isolated tumor cells by modulating cell migration [28
], invadopodia dynamics [29
] and MMP (matrix metalloproteinase) expression and activity [30
These critical implications show how dramatic could be a deregulation of calpain activity. Alterations of the calpain activity balance has been observed in numerous cancer types () can reduce apoptosis, increase cell proliferation and stimulate cell migration and invasiveness (). For these reasons, calpains are considered potential therapeutic targets to treat cancer and to limit its progression. In this review we present how and why these proteases can be targeted as well as the pros and the cons of such an approach.