Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer

Abstract

Resistance to anti-HER2 (human epithelial growth factor receptor 2) trastuzumab therapy occurs commonly in HER2-positive breast cancer and involves overactivation of HER2 and/or AKT1. Using the model of trastuzumab-sensitive or trastuzumab-resistant HER2-positive cells with wild-type PTEN, negative regulator of AKT1, we explore the involvement of cysteine protease calpain in mechanisms of trastuzumab resistance. Overexpression of calpain1 or activation of endogenous calpain during adhesion or trastuzumab treatment of trastuzumab-sensitive cells induces cleavage of cytoplasmic domains of HER2/phospho-HER2; cleavage occurs in HER2-positive tumors. Expression of the catalytically inactive mutant of calpain1 reduces the cleavage to enhance the activity of HER2, inactivates PTEN to enhance the activation of AKT1, induces desensitization to trastuzumab and promotes survival of trastuzumab-sensitive cells. In the model of trastuzumab resistance, constitutive overactivation of HER2 and AKT1 correlates with reduced activation of calpain. Moreover, inhibitors of the catalytic site of calpain reduce the increase in constitutive activity of AKT1 and survival of trastuzumab-resistant cells selectively. Together, by regulating the activation of HER2 and PTEN/AKT1, calpain regulates trastuzumab sensitivity and survival, and the deregulation of the activation of calpain promotes trastuzumab resistance. Trastuzumab-resistant cells activate AKT1 in a mechanism dependent on the residual calpain activity, inhibition of which restores trastuzumab sensitivity and rescues resistance. These data identify calpain as a new therapeutic target in HER2-positive breast cancer.

Introduction

The family of human epithelial growth factor receptor (HER1–4) regulates transmission of signals of adhesion, growth, survival and metastasis in breast cancer. Members are structurally similar containing an extracellular region with ligand binding and dimerization domains, a single transmembrane domain and a cytoplasmic region with tyrosine kinase and regulatory domains. Binding of ligand induces homo- or heterodimerization of members and activation of cytoplasmic domains, which recruit GRB2/RAS/ERK, phosphoinositide-3 (PI3) kinase/AKT1 and SRC to ensure transmission of signals or ubiquitin ligase to suppress the transmission (Yarden and Sliwkowski, 2001).

Overexpression of HER2 due to gene amplification is observed in 20–30% HER2-positive breast cancer patients. HER2-positive cancer is aggressive with high risk of recurrence, propensity for brain metastasis and poor outcome. Treatment with the recombinant, humanized, anti-HER2 antibody trastuzumab (Herceptin) for the extracellular region administered alone or combined with chemotherapy arrests growth of cancer (Slamon et al., 2001; Vogel et al., 2002; Piccart-Gebhart et al., 2005). Trastuzumab disrupts transmission of signals to arrest growth initially but within a year patients acquire resistance, and evidence points to the overactivation of HER2 and/or AKT1 (Clark et al., 2002; Vogel et al., 2002; Yakes et al., 2002; Nagata et al., 2004; Chan et al., 2005; Berns et al., 2007; Ishizawar et al., 2007; Ritter et al., 2007). As HER2 is a ligand-less receptor, the regulation of activity typically occurs through cross-talk with epithelial growth factor receptor (EGFR)/HER1 or HER3 and deregulation of cross-talk is believed to promote activity and resistance. Activity of AKT1 is regulated by both, PI3 kinase and the tumor suppressor, PTEN; PIP3 generated by PI3 kinase recruits AKT1 to the cell membrane for activation, whereas dephosphorylation of PIP3 by PTEN suppresses the activation (Hennessy et al., 2005). The loss of phosphatase activity of PTEN promotes activation of AKT1 and trastuzumab resistance (Nagata et al., 2004). The mechanisms that alter the activation of HER2 or PTEN/AKT1 during acquisition of trastuzumab resistance are little understood.

Calpains are a family of ubiquitous or tissue-specific intracellular, cysteine proteases implicated in the regulation of transmission of signals in cancer. Ubiquitous forms, calpain1 (μ) and calpain2 (m) are activated during integrin-induced adhesion (Fox et al., 1993), growth factor (EGF) stimulation (Glading et al., 2004), calcium influx or cellular stress, and regulate growth, cytoskeletal organization or survival (Goll et al., 2003). Both forms contain the cysteine protease domain, which catalyzes cleavage that often alters activity of substrates during transmission of signals (Kulkarni et al., 1999, 2002; Raynaud et al., 2003; Perrin et al., 2006). Intramolecular interactions within domains outside the protease domain and the regulatory form, calpain4, influence the catalytic activity (Goll et al., 2003). Deletion of calpain4 in mouse eliminates catalytic activity of both forms and is lethal (Arthur et al., 2000). Fibroblasts lacking calpain4 show defective regulation of AKT1, sensitivity to staurosporine and tumor necrosis factor-α, but resistance to DNA-damaging agents, implicating a role in positive and negative regulation of cell survival (Tan et al., 2006).

New evidence supports involvement of calpain1 and calpain2 in regulation of the mechanisms of survival in cancer cells that have acquired resistance to therapeutic drugs. Pharmacological inhibitors of the catalytic activity of calpains overcome resistance to TRAIL in colon cancer (Zhu et al., 2004), cisplatin in melanoma (Mlynarczuk-Bialy et al., 2006) or androgen independence of prostate cancer (Libertini et al., 2007). In this study, we provide evidence that calpain regulates trastuzumab sensitivity and survival of HER2-positive breast cancer cells, deregulation of calpain promotes deregulation of the activation of HER2 and PTEN/AKT1, and inhibitors of calpain activity rescue trastuzumab resistance.

Results

Calpain cleaves HER2 in vitro and in vivo

Calpain induces cleavage of the cytoplasmic region of EGFR/HER1, the prototype of HER family in vitro (Gregoriou et al., 1994). To investigate cleavage of HER2, glutathione-S-transferase (GST)-fusion protein of the C-terminal, cytoplasmic region (Figure 1a, lane 1) was incubated with calpain1 alone (lane 2) or along with calpeptin, inhibitor of the catalytic site (lane 3). Aliquots were detected with antibody for the C-terminus of HER2. Calpain1 cleaved the cytoplasmic region to generate 75 or 42 kDa fragments, and the cleavage was inhibited in the presence of calpeptin. Similar fragments were detected in HER2-positive skbr3 cells (lane 4).

Figure 1
figure1

Calpain induces cleavage of human epithelial growth factor receptor 2 (HER2) in vitro and in skbr3 cells. (a) The glutathione-S-transferase (GST)-cytoplasmic region (residues 679–1255) of HER2 (lane 1, 200 ng) was incubated with purified human calpain1 alone (lane 2, 10 ng) or along with inhibitor calpeptin (lane 3, 0.25 μg/μl) for 5 min at room temperature. Cleavage was initiated with the addition of 5 mM CaCl2, terminated with SDS sample buffer followed by denaturation. Aliquots were separated in 8% acrylamide gel and detected in western blot with a monoclonal antibody for the C-terminus of HER2 (1242TAENPEYLGLDVPV1255). Lysates of skbr3 cells were separated alongside (lane 4). (b) skbr3 cells were spread on fibronectin-coated dishes for 1 (lane 1), 4 (lane 2) or 6 h (lane 3) alone or along with inhibitors, calpeptin (lane 4) or MDL28170 (lane 5), 100 μM each for 6 h and lysates were prepared. Equal amount of protein was separated in 8% acrylamide gel and detected in western blot with a monoclonal antibody for the C-terminus of HER2. The intensity of bands was quantitated by Scion Image analysis. (c) Nontransfected skbr3 cells (lane 1) or skbr3 cells transfected with plasmid expressing wild-type calpain1 (lane 2) were spread on fibronectin-coated dishes for 2 h and lysates were prepared. Equal amount of protein was separated in 8% gel and detected in western blots for HER2 (top) or calpain1 (bottom). Overexpression appeared to increase the accessibility of calpain allowing cleavage of HER2 at several closely situated sites. (d) Lysates of skbr3 cells, untreated (lane 1) or treated with trastuzumab (10 μg/ml) for 24 (lane 2) or 48 h (lane 3), were prepared and equal amount of protein was analyzed in western blot with a monoclonal antibody for the C-terminus of HER2.

Calpain1 is activated during adhesion to matrix proteins (Fox et al., 1993). To explore cleavage of HER2, skbr3 cells were spread on fibronectin alone or along with membrane-permeable inhibitors of the catalytic site of calpain, calpeptin or MDL28170 for 1–6 h (Figure 1b). Fragments of HER2, 75 and 42 kDa, were detected (lanes 1–3) and inclusion of calpeptin (lane 4) or MDL28170 (lane 5) resulted in 3.4- and 2.1-fold reduction in the generation of 42 kDa fragment respectively. The generation of 75 kDa fragment was not significantly altered. In another approach, cells transfected with plasmid expressing wild-type calpain1 (Kulkarni et al., 1999) were analyzed (Figure 1c). Compared with nontransfected control (lane 1), overexpression of calpain1 (lane 2, bottom) overproduced 75 and 50–42 kDa fragments (top). Moreover, compared with untreated cells (Figure 1d, lane 1), the treatment of cells with trastuzumab for 24 h (lane 2) or 48 h (lane 3) also overproduced 50–42 kDa fragments.

Analysis of HER2-positive BT474 cells spread on fibronectin showed cleavage of HER2 (Figure 2a, lane 1) and inclusion of calpeptin (lane 2) or MDL28170 (lane 3) reduced the cleavage. Compared with untreated (Figure 2b, lane 1), the treatment of BT474 cells with trastuzumab (lane 2) also increased cleavage. Analysis of eight human HER2-positive invasive ductal carcinomas showed the presence of several C-terminal fragments (Figure 2c, lanes 1–8). Fragments (150–90 kDa) spanning transmembrane and the cytoplasmic region proposed to be generated by protein translation or cleavage of extracellular region were detected (Molina et al., 2001; Anido et al., 2006). Smaller fragments, 75 or 50–42 kDa, similar to those induced by calpain were also detected. On the basis of biochemical analysis, two regions of cleavage were predicted (Figure 2d). Cleavage within the region I close to the membrane would produce 75 kDa fragments with intact cytoplasmic domains, whereas cleavage within the region II would disrupt the kinase domain to release 50–42 kDa fragments.

Figure 2
figure2

Cleavage of human epithelial growth factor receptor 2 (HER2) occurs in HER2-positive cells and tumors. (a) HER2-positive BT474 cells were spread on fibronectin-coated dishes alone (lane 1) or along with inhibitors, calpeptin (lane 2) or MDL28170 (lane 3), each 100 μM, overnight and lysates were prepared. Equal amount of protein was separated in 8% acrylamide gel and detected in western blot with a monoclonal antibody for the C-terminus of HER2. (b) BT474 cells were left untreated (lane 1) or treated with trastuzumab (1 μg/ml) for 48 h (lane 2). Lysates were prepared and equal amount of protein was detected in western blot with a monoclonal antibody for the C-terminus of HER2. (c) Lysates of eight different human HER2-positive invasive breast carcinoma tissues (lanes 1–8) were separated in 8% acrylamide gel and detected in western blot with a monoclonal antibody for the C-terminus of HER2. (d) Scheme of proposed sites of calpain1-induced cleavage in HER2. Cleavage is proposed to occur within two critical regions within cytoplasmic domains. The cleavage within region I close to the membrane is expected to generate 75 kDa fragments, and the cleavage of closely situated cluster of sites within region II in the kinase domain is expected to release 50–40 kDa fragments. Tyrosine residue within the kinase (Y877) or the regulatory (Y1221/22) domain is indicated with an asterisk.

To investigate the cleavage of phospho-HER2, HER2 was immunoprecipitated with a monoclonal antibody (Figure 3a, top, lanes 1, 3 and 4) or mouse IgG (lane 2). Aliquots were incubated with calpain1 alone (lane 3) or along with MDL28170 (lane 4) and detected with a polyclonal phospho-Y1221/22 HER2 antibody (bottom). Immunoprecipitates contained 150–42 kDa phospho-fragments of HER2; calpain1 increased the generation of 50–42 kDa phospho-fragments, which was reduced by the inclusion of MDL28170. In another experiment, cells were spread on fibronectin alone or along with calpeptin or MDL28170 and used to immunoprecipitate HER2 (Figure 3b, top, lanes 1, 3 and 4); mouse IgG was used as a control (lane 2). Detection with phospho-Y1221/22 HER2 antibody showed 50–42 kDa phospho-fragments co-immunoprecipitated with HER2 (bottom, lane 1); inclusion of calpeptin (lane 3) or MDL28170 (lane 4) during adhesion reduced the co-immunoprecipitation. Taken together these data show calpain induces cleavage of cytoplasmic domains of HER2 and phospho-HER2 and suggest the cleavage perhaps regulates transmission of downstream signals.

Figure 3
figure3

Calpain induces phosphorylated fragments of human epithelial growth factor receptor 2 (HER2) and the fragments co-immunoprecipitate with HER2. (a) HER2 was immunoprecipitated with the monoclonal antibody from precleared lysates of exponentially growing skbr3 cells. Immunoprecipitate was resuspended in calpain activity buffer. Equal volume of immunoprecipitate was left as control (lane 1) or incubated with purified human calpain1 (lane 3, 5 ng) alone or along with the inhibitor MDL28170 (lane 4, 0.25 μg/μl) at room temperature. Calpain1 was activated with the addition of 5 mM CaCl2 for 2 min and reactions were terminated with the addition of SDS sample buffer followed by denaturation. Immunoprecipitate with mouse IgG is shown in lane 2. Reactions were separated in 8% acrylamide gel and detected for HER2 with the monoclonal antibody (top) or phospho-HER2 with the polyclonal phospho-Y1221/22 antibody (bottom) in western blots. (b) skbr3 cells were spread on fibronectin-coated dishes alone (lane 1) or along with inhibitor calpeptin (lane 3) or MDL28170 (lane 4), each 150 μM, for 2 h. Cell lysates were prepared, precleared and immunoprecipitated with the monoclonal HER2 antibody. Mouse IgG was used as a control (lane 2). Immunoprecipitates were detected for HER2 with the monoclonal antibody (top) or phospho-HER2 with the polyclonal phospho-Y1221/22 antibody (bottom) in western blots.

Activation of calpain exerts influence on cell survival

To understand functional significance of calpain, skbr3 cells were spread on fibronectin alone or along with calpeptin or MDL28170, and activity was measured (Figure 4a) as described (Edelstein, 2000). Activity was also assessed in cells treated with trastuzumab alone or along with calpeptin or MDL28170 (Figure 4b). Calpain was activated during adhesion and treatment with trastuzumab, and activity was inhibited by the inclusion of each inhibitor. To study the consequences, cells were spread on fibronectin in the presence of trastuzumab alone as a control or along with each inhibitor and viability was measured (Figure 4c). Compared with control, inclusion of each inhibitor increased the viability by 15% (P<0.01), suggesting an involvement in regulation of survival.

Figure 4
figure4

Activation of calpain exerts influence on cell survival. (a) skbr3 cells were spread on fibronectin-coated dishes alone or along with inhibitors, calpeptin or MDL28170 (100 μM each) for 1 h in the serum-free medium. Lysates were prepared and protein was estimated. Equal amount of protein was plated in 96-well plates in triplicate and incubated with the fluorescently labeled calpain substrate II (100 μM) at 37 °C. Fluorescence of the cleaved substrate II was measured at excitation 380 nm/emission 460 nm. Average fluorescence units indicative of the activity of calpain with error bars are shown. (b) skbr3 cells were left untreated as control or treated with trastuzumab (10 μg/ml) alone or together with inhibitors, calpeptin or MDL28170 (100 μM each) for 1 h in the serum-free medium. Lysates were prepared and the activity of calpain was measured with substrate II as described above. (c) skbr3 cells were spread in fibronectin-coated wells in quadruplicate. Cells were left untreated as controls or treated with trastuzumab (10 μg/ml) alone or together with inhibitors, calpeptin or MDL28170 (each 25 μM) for 48 h at 37 °C. Cell viability was measured in a one-step MTS assay and normalized to the control (P<0.01).

Expression of the catalytically inactive calpain1 alters trastuzumab sensitivity and cell survival

Although calpeptin or MDL28170 is a potent inhibitor of the active site of calpain1 and calpain2 (Goll et al., 2003), to rule out nonspecific effects and gain insights into mechanisms, skbr3 cells were transfected with plasmid expressing HA-tagged catalytically inactive calpain1 with mutation in the active site (His 272 to Ala) (Kulkarni et al., 1999) to obtain two clones by selection with G418. Nontransfected cells (lane 1), clone #1 (lane 2) or clone #2 (lane 3) were spread on fibronectin and analyzed for the expression of endogenous calpain1 (Figure 5a, top) or catalytically inactive calpain1 (bottom). Comparison of the viability (Figure 5b) of spread cells treated with trastuzumab (38% in nontransfected cells against 85% in clone #1 or 87% in clone #2) indicated loss of trastuzumab sensitivity and increase in survival of transfected cells.

Figure 5
figure5

Expression of the catalytically inactive calpain1 alters trastuzumab sensitivity and cell survival. (a, c) Nontransfected skbr3 cells (lane 1) or clone #1 (lane 2) and clone #2 (lane 3) of skbr3 cells transfected with the plasmid expressing HA-tagged catalytically inactive calpain1 were spread on fibronectin-coated dishes overnight and lysates were prepared. (a) Protein was detected for expression of endogenous calpain1 (top) or catalytically inactive calpain1 with the HA antibody (bottom) in western blots. (c) Protein was detected for human epithelial growth factor receptor 2 (HER2) (top) or phospho-Y1221/22 HER2 (bottom). (b) Nontransfected skbr3 cells, clone #1 or clone #2 were spread in fibronectin-coated wells in triplicate. Cells were left untreated as controls or treated with trastuzumab (10 μg/ml) for 72 h at 37 °C. Cell viability was measured in a one-step MTS assay and normalized to the control.

To understand the mechanisms involved in the above, calpain-induced regulation of cleavage or activity of HER2 was analyzed (Figure 5c). In nontransfected cells, cleavage of the cytoplasmic region occurred (top, lane 1) and activity was reduced (bottom, lane 1), whereas in clones #1 and #2 cleavage was reduced (top, lanes 2 and 3) and activity was increased (bottom, lanes 2 and 3). Together, catalytically inactive calpain1 altered endogenous regulation of cleavage and the activity of HER2, thus providing an advantage for survival.

Transmission of signals from HER family converges on the regulation of ERK1/2 and AKT1. ERK1/2 promotes growth while AKT1 promotes survival (Yarden and Sliwkowski, 2001). To understand consequences of the expression of catalytically inactive calpain1 on transmission of signals, expression or activity of ERK1/2 and AKT1 was investigated. Expression of ERK1/2 (Figure 6a, top) or AKT (Figure 6b, top) remained comparable in nontransfected cells (lane 1), clone #1 (lane 2) or clone #2 (lane 3), but the activity of each was increased in clone #1 and clone #2 (respective bottom). The increase in the activity of AKT1 was accompanied by an increase in expression of PTEN (Figure 6c, top) but not of PTP1B (bottom). Despite the increase in expression the phosphatase activity of PTEN immunoprecipitated from spread cells (Figure 6d, top, lanes 1–4) was reduced 2.5-fold in clone #1 and clone #2 compared with nontransfected cells (Figure 6d, bottom). Thus, catalytically inactive calpain1 increases the activation of AKT1 by reducing the phosphatase activity of PTEN.

Figure 6
figure6

Expression of the catalytically inactive calpain1 influences transmission of signals. Equal amount of protein from lysates of nontransfected skbr3 cells (lane 1) or from cells transfected with the plasmid expressing HA-tagged catalytically inactive calpain1, clone #1 (lane 2) or clone #2 (lane 3) from the experiment described in Figure 5 was detected for ERK1/2 (top) or phospho-ERK1/2 (bottom) (a), AKT (top) or phospho-S473 AKT1 (bottom) (b) and PTEN (top) or PTP1B (bottom) (c). (d) Nontransfected skbr3 cells, clone #1 or clone #2 were spread on fibronectin for 1 h and lysates were prepared in the buffer containing 20 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); pH 7.5), 50 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.1% NP40 and inhibitors. Lysates were precleared with the addition of rabbit IgG and protein G-agarose beads, and immunoprecipitated with the polyclonal PTEN antibody or rabbit IgG for control. Immunoprecipitates of nontransfected skbr3 cells (lane 1), clone #1 (lane 2) or clone #2 (lane 3) were detected for PTEN in western blot (top). Immunoprecipitate with rabbit IgG is shown in lane 4. PTEN immunoprecipitates were washed three times in the buffer containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 0.1 mM EDTA and 2.5 mM MgCl2 and once in the activity buffer containing 100 mM Tris-HCl (pH 8.0) and 10 mM dithiothreitol (DTT), divided in triplicate in 96-well plates and incubated with water-soluble PIP3 (1 μg per well) for 1 h at 37 °C (bottom). Release of phosphate indicative of PTEN activity was estimated with the addition of BIOMOL GREEN Reagent and calibrated against the phosphate standard (0.3–3.0 nM). Similar results were obtained in two independent experiments.

Taken together, the activation of endogenous calpain1 exerts influence on at least two sites of transmission of signals. At site 1, calpain1 regulates cleavage and activity of HER2 to influence the transmission of GRB2/RAS/ERK signals, whereas at the site 2, by exerting influence on the mechanisms of activation and/or stabilization of PTEN, calpain1 regulates the activity of AKT1. Consistently, catalytically inactive mutant of calpain1 alters trastuzumab sensitivity and survival by influencing calpain1-induced regulation at both sites.

Overactivation of HER2 in the model of acquired trastuzumab resistance is associated with the deregulation of activation of calpain

Previously described model of acquired resistance of trastuzumab in skbr3 cells (Nahta and Esteva, 2004) was used to assess the regulation by calpain. Trastuzumab-sensitive (Figure 7a, lanes 1 and 2) or trastuzumab-resistant (lanes 3 and 4) skbr3 cells were spread on fibronectin for 1–2 h and compared for regulation of HER2 by cleavage or phosphorylation. In the trastuzumab-resistant cells, cleavage was reduced (top) while activity at Y877 (middle) or Y1221/22 (bottom) was increased. Comparison of trastuzumab-sensitive (lanes 1 and 2) and trastuzumab-resistant (lanes 3 and 4) cells treated with trastuzumab (Figure 7b) for 1–2 h also showed reduction in cleavage (top) and an increase in activity at Y877 (middle) or Y1221/22 (bottom) in the trastuzumab-resistant cells. Thus, the intracellular mechanism that regulated cleavage and activity of HER2 was impaired in trastuzumab-resistant cells. To investigate this further, expression or activity of calpain was compared in trastuzumab-sensitive and trastuzumab-resistant cells. Expression of calpain1 (Figure 7c, top, lanes 1 and 2), calpain 2 (middle) or integrin β1 (bottom) was not altered significantly. Surface expression of integrin β1 also remained unaltered (data not shown). Analysis of activity during adhesion (Figure 7d) or trastuzumab treatment (Figure 7e) of trastuzumab-sensitive cells showed an increase, which was inhibited by inclusion of calpeptin. In contrast, in trastuzumab-resistant cells, the activation of calpain was reduced during adhesion and trastuzumab treatment did not significantly increase the activity. Thus, trastuzumab sensitivity correlated with the activation of calpain, cleavage and low activation of HER2, whereas trastuzumab resistance correlated with the impaired activation of calpain, reduced cleavage and overactivation of HER2. Moreover, these data imply adhesion- or trastuzumab-induced deregulation of the activation of calpain, which allows the deregulation of HER2 associated with trastuzumab resistance.

Figure 7
figure7

Deregulation of human epithelial growth factor receptor 2 (HER2) in the model of acquired trastuzumab resistance is associated with the deregulation of activation of calpain. (a, b) Trastuzumab-sensitive (lanes 1 and 2) or trastuzumab-resistant (lanes 3 and 4) skbr3 cells were spread on fibronectin-coated dishes for 1 h (lanes 1 and 3) or 2 h (lanes 2 and 4) in panel a. (b) Trastuzumab-sensitive (lanes 1 and 2) or trastuzumab-resistant (lanes 3 and 4) skbr3 cells were treated with trastzumab (10 μg/ml) for 1 h (lanes 1 and 3) or 2 h (lanes 2 and 4). Lysates of all cells were prepared and equal amount of protein was detected for HER2 (top), phospho-Y877 HER2 (middle) or phospho-Y1221/22 HER2 (bottom) in western blots. (c) Equal amount of protein from lysates of trastuzumab-sensitive (lane 1) or trastuzumab-resistant (lane 2) skbr3 cells was detected for calpain1 (top), calpain2 (middle) or integrin β1 (bottom) in western blots. (d) Trastuzumab-sensitive or trastuzumab-resistant skbr3 cells were spread on fibronectin-coated dishes alone or along with the inhibitor, calpeptin (150 μM), for 1 h. (e) Trastuzumab-sensitive or trastuzumab-resistant skbr3 cells were left untreated, treated with trastuzumab (10 μg/ml) alone or trastuzumab and the inhibitor, calpeptin (100 μM) together for 1 h. Lysates were prepared and protein was estimated. Equal amount of protein was plated in 96-well plates in triplicate and incubated with the fluorescently labeled calpain substrate II (100 μM) at 37 °C. Fluorescence of the cleaved substrate II was measured at excitation 380 nm/emission 460 nm. Average fluorescence units with error bars indicative of the activity of calpain are shown.

Trastuzumab-resistant cells require calpain activity for survival and activation of AKT1

Elimination of catalytic activity of calpain1 and calpain2 is lethal, indicating at least some activity is essential (Arthur et al., 2000). Our data indicated the activation of calpain occurred during adhesion of trastuzumab-sensitive and trastuzumab-resistant skbr3 cells. To investigate significance, trastuzumab-sensitive or trastuzumab-resistant cells spread on fibronectin were treated with calpeptin or MDL28170 and assessed for morphology or viability. Trastuzumab-sensitive cells (Figure 8a, top) remained adherent and refractile in the presence of inhibitor (60% viable), but trastuzumab-resistant cells were rounded and shrunk (bottom). Consistently, the survival of trastuzumab-sensitive cells (Figure 8b) treated with trastuzumab along with calpeptin or MDL28170 was increased by 17 or 33% respectively. In contrast, the survival of trastuzumab-resistant cells (95%) treated with trastuzumab along with calpeptin or MDL28170 was reduced to 33 or 40% respectively.

Figure 8
figure8

Trastuzumab-resistant cells require calpain activity for survival and activation of AKT1. (a) Trastuzumab-sensitive or trastuzumab-resistant skbr3 cells were spread on fibronectin-coated dishes alone or along with inhibitors, calpeptin or MDL28170 (50 μM each), for 48 h and photographed. (b) Trastuzumab-sensitive or trastuzumab-resistant skbr3 cells were spread in fibronectin-coated wells in triplicate and treated with trastuzumab (10 μg/ml) alone or trastuzumab and the inhibitor, calpeptin or MDL28170 (50 μM each) together for 72 h at 37 °C. Cell viability was measured in one step MTS assay. (c) Trastuzumab-sensitive (lanes 1–3) or trastuzumab-resistant (lanes 4–6) skbr3 cells were spread on fibronectin-coated dishes alone as control (lanes 1 and 4) or treated with the inhibitor, calpeptin (lanes 2 and 5) or MDL28170 (lanes 3 and 6), each 25 μM, overnight. Lysates of cells were prepared, equal amount of protein was separated in 8% acrylamide gel and detected for AKT (top) or phospho-S473 AKT1 (bottom) in western blots.

Constitutive overactivation of AKT1 promotes cell survival and resistance to trastuzumab (Clark et al., 2002; Yakes et al., 2002). To explore involvement of calpain, expression (Figure 8c, top) or activity (bottom) of AKT1 in trastuzumab-sensitive (lanes 1–3) or trastuzumab-resistant (lanes 4–6) cells spread on fibronectin alone or along with calpeptin or MDL28170 was compared. The expression of AKT or activity of AKT1 in trastuzumab-sensitive cells (lane 1) remained comparable by the inclusion of calpeptin (lane 2) or MDL28170 (lane 3). In trastuzumab-resistant cells, expression of AKT was comparable, but the constitutive activity of AKT1 was increased (bottom, lane 4) and inclusion of calpeptin (lane 5) or MDL28170 (lane 6) reduced the increase. Thus, the increase in constitutive activity of AKT1 associated with trastuzumab resistance was dependent on the activity of endogenous calpain. Taken together, survival of trastuzumab-sensitive cells appears to be regulated by several other mechanisms, but survival and activation of AKT1 during adhesion of trastuzumab-resistant cells is dependent on the activity of calpain, inhibition of which reduces constitutive overactivation of AKT1, restores trastuzumab sensitivity and rescues resistance.

Discussion

The ability of cancer cells to acquire alternate traits of growth and survival to escape therapy or become resistant poses a significant challenge in treatment. Trastuzumab is the anti-HER2 therapy offered to patients with HER2-positive breast cancer. About 12–34% of patients respond to the therapy and the remainder are resistant. Moreover, an initial response almost always develops into resistance. Combining trastuzumab with cytotoxic agents improves the rate of response; however, resistance still develops (Slamon et al., 2001; Vogel et al., 2002; Piccart-Gebhart et al., 2005). In this study, we explore the involvement of the cysteine protease calpain in the mechanisms of resistance to trastuzumab. Our observations provide the first evidence that by regulating the activation of HER2 and PTEN/AKT1, calpain regulates trastuzumab sensitivity and survival pathway. These findings implicate calpain in molecularly targeted therapy of HER2-positive breast cancer and support several possibilities including (1) the activation of calpain during transmission of signals or treatment with trastuzumab inactivates HER2 in a feedback mechanism to exert influence on trastuzumab sensitivity, (2) the deregulation of activation of calpain alters trastuzumab sensitivity and survival, and (3) trastuzumab-resistant cells become increasingly dependent on the residual activity of calpain for activation of AKT1 and survival.

Regulation of HER2

In the absence of mutations in kinase domain (Zito et al., 2008) or changes in expression, the activity of HER2 in acquired resistance of trastuzumab is subject to regulation by cross-talk with the members of HER family and/or downstream molecules (Anastasi et al., 2005; Ishizawar et al., 2007; Ritter et al., 2007). Cross-talk with ligand-activated EGFR/HER1 or HER3 activates HER2 (Yarden and Sliwkowski, 2001) while activation of SRC promotes activity by promoting heterodimer formation (Ishizawar et al., 2007). Cross-talk with other receptors including endocrine receptors, IGFR or Met also appears to have a role in resistance (Nahta et al., 2005; Tao et al., 2007; Shattuck et al., 2008). Integrins or mechanisms downstream of integrins are believed to modulate sensitivity to cancer therapy (Zutter, 2007). New evidence supports the involvement of integrins in clustering of HER2 (Tan et al., 2005; Wang et al., 2006, 2009). We show that integrin-induced adhesion in HER2-positive breast cancer activates calpain, which contributes to the transmission of signals of trastuzumab sensitivity and survival.

Several observations support the possibility that one mechanism by which calpain influences trastuzumab sensitivity and survival is by regulating cleavage and activity of HER2. First, calpain1-induced cleavage fragments the cytoplasmic region, which couples HER2/phospho-HER2 to downstream molecules. Second, activation of calpain occurs following interaction of HER2 with the HER2-specific antibody trastuzumab, indicating that the activation and subsequent cleavage functions as a feedback mechanism to inactivate HER2. The presence of small fragments of HER2/phospho-HER2 in positive cell lines and human tumors suggests that the feedback mechanism is functional in vivo. Third, expression of catalytically inactive calpain1 relieves the feedback inhibition to induce overactivation of HER2 and ERK1/2. Finally, catalytically inactive calpain1 increases survival of cells exposed to trastuzumab. These findings together with observations that calpain is activated following the activation of EGFR/HER1 (Glading et al., 2004) and that calpain induces cleavage of the kinase domain of EGFR/HER1 (Gregoriou et al., 1994) support the role of calpain in regulation of activity and function of HER family and transmission of downstream signals.

In other examples of cancer therapy including TRAIL, cisplatin or androgens, calpain-induced proteolytic events alter regulation of caspases, NF-κβ or androgen receptor, and thereby contribute to resistance (Zhu et al., 2004; Mlynarczuk-Bialy et al., 2006; Libertini et al., 2007). Here, analysis of trastuzumab sensitivity in the model of acquired trastuzumab resistance indicates that the deregulation of the activation of calpain occurs during acquisition of resistance that correlates with the deregulation of cleavage and activity of HER2. Optimal activation of calpain ensures optimal cleavage of HER2/phospho-HER2 and perhaps disengagement from the downstream mechanisms of transmission of signals; as a result, trastuzumab sensitivity is retained high and cell survival low. Consistently, the suboptimal activation of calpain appears responsible for suboptimal cleavage of HER2, an increase in the activity of HER2 and for desensitization of cells.

Events involved in activation of calpain, sequence of occurrence or mechanisms of regulation, are not clear but involve calcium binding, dimerization and translocation to membrane. In the inactive conformation of calpain, catalytic residues cysteine, histidine and asparagine in protease domain II are held apart. Binding of calcium to domains I–III alters the conformation to assemble the active site. Interaction of domain III with phospholipids or dimerization through EF hand containing domain IV in calpain1/2 and domain VI in calpain4 influences the activity (Goll et al., 2003). Our data implicate that, trastuzumab alters or disables the mechanisms of regulation of activation of calpain to allow an early advantage for survival and selection of resistant cells. Moreover, as trastuzumab alters activation of one or more forms of calpain, regulation of critical downstream substrates other than HER2 may also be altered. Activation of calpain in other cells regulates components of cytoskeleton, talin, cortactin or α-actinin, or Rac1 and RhoA to influence transmission of signals (Kulkarni et al., 1999, 2002; Raynaud et al., 2003; Perrin et al., 2006). Recent studies show involvement of known substrates of calpain including SRC or α-actinin in transmission of signals, cytoskeletal organization and clustering of HER2 in breast cancer (Tan et al., 2005; Wang et al., 2006, 2009). Much work is required to understand the regulation of activity of calpain as well as calpain-induced regulation of other molecules in HER2-positive breast cancer.

Regulation of AKT1

Overactivation of AKT1 is often observed in cancers of prostate, ovary, thyroid and breast, and correlates with the ability of cells to escape cancer therapy. The regulation of activity of AKT1 by PI3 kinase and PTEN is therefore a critical site for therapeutic intervention in cancer (Clark et al., 2002; Hennessy et al., 2005).

Mutations in PI3 kinase or PTEN are known to occur in some breast cancers (Hennessy et al., 2005; Stemke-Hale et al., 2008). About 1.4% of breast cancers also contain a mutation in AKT1 (E17 K) that confers constitutive activity (Carpten et al., 2007; Stemke-Hale et al., 2008). Other regulators, 3-phosphoinositide-dependent protein kinase 1 (PDK1) or integrin-linked kinase (ILK) activate AKT1 by inducing phosphorylation of S308 or S473 (Lawlor and Alessi, 2001; Troussard et al., 2006). The role of PDK1 or ILK in the mechanisms of resistance to trastuzumab is not known at present. Recent evidence suggests that altered regulation of PTEN expression, stability or function, rather than mutations of PI3 kinase, contributes to the overactivation of AKT1 (Stemke-Hale et al., 2008) and trastuzumab resistance (Nagata et al., 2004; Berns et al., 2007).

Using the model of HER2-positive skbr3 cells that are trastuzumab-sensitive or trastuzumab-resistant and contain wild-type forms of PI3 kinase, PTEN and AKT1 (Lacroix and Leclercq, 2004), we provide evidence for involvement of calpains in the activation as well as the inactivation of AKT1. Involvement in the activation is supported by the observation that inhibitors of the catalytic site of calpain1/2 lower the activity of AKT1 in trastuzumab-resistant cells. Consistently, fibroblasts lacking calpain4 that lack catalytic activity of both calpain1 and calpain2 show compromised activation of AKT1 (Arthur et al., 2000; Tan et al., 2006). Involvement in the inactivation is supported by the observation that expression of catalytically inactive calpain1 reduces the phosphatase activity of PTEN to promote activation of AKT1. PTEN is regulated by transient inter- and/or intramolecular mechanisms of localization, phosphorylation or protein–protein interactions that influence stability, expression and activity (Gericke et al., 2006). Our data are consistent with the possibility that by exerting influence on one or more mechanisms of regulation, calpain1 promotes the activation of PTEN to inactivate AKT1, whereas catalytically inactive calpain1 influences the regulation, increases expression of PTEN by stabilizing the inactive state and promotes the activity of AKT1.

In summary, this study supports a model in which the intracellular activities of ubiquitous calpains together regulate the mechanisms of transmission of signals that influence trastuzumab sensitivity and pathway of survival in HER2-positive breast cancer. Activation of calpain regulates at least two parallel mechanisms. In the first mechanism, calpain regulates cleavage and activity of the oncogene HER2, and in the second mechanism, calpain exerts influence on the activation of the tumor suppressor PTEN to regulate the activity of AKT1. Calpain-induced inactivation of HER2 and activation of PTEN ensure that the transmission of signals of survival is low and trastuzumab sensitivity is high. Deregulation of the activation of calpain causes an imbalance in the regulation of one or both mechanisms and produces sustained activation of HER2 and AKT1 to desensitize cells. In this model, by deregulation of calpain, cells gain an advantage for survival and also acquire a mechanism of activation of AKT1 that is critically dependent on minimally available residual activity of calpain, the inhibition of which rescues trastuzumab resistance.

Materials and methods

Antibodies and reagents

Antibody for C-terminus of HER2 (1242TAENPEY-LGLDVPV1255), purified calpain1, substrate II, calpeptin and MDL28170 were purchased from Calbiochem (San Diego, CA, USA). Antibodies for phospho-HER2 (Y877 or Y1221/22), AKT, phospho-S473 AKT1, PTEN (polyclonal) and GST-HER2 protein (residues 676–1255) were purchased from Cell Signaling (Danvers, MA, USA); integrin β1 antibody was purchased from Chemicon (Danvers, MA, USA); FITC-labeled integrin β1 antibody from BD (San Jose, CA, USA); antibody for calpain1 from Alexis (Plymouth Meeting, PA, USA); antibodies for calpain2, HA epitope and ERK1/2 from Sigma (St Louis, MO, USA); antibody for phospho-ERK1/2 from New England Biolabs (Beverly, MA, USA); antibody for PTEN (monoclonal) from Millipore (Danvers, MA, USA) or for PTP1B from Upstate (Danvers, MA, USA). Fibronectin was purchased from Roche (Indianapolis, IN, USA); EasyTransgator-si reagent from America Pharma Source (Gaithersburg, MD, USA); and MTS reagent from Promega (Madison, WI, USA). Trastuzumab was purchased from Genentech (South San Francisco, CA, USA); protein G-agarose beads from Santa Cruz Biotechnology (Santa Cruz, CA, USA); PIP3 from Echelon Biosciences (Salt Lake City, UT, USA); and BIOMOL GREEN Reagent from Biomol Research Laboratories (Plymouth Meeting, PA, USA). Plasmids expressing HA-tagged wild-type or catalytically inactive calpain1 were described (Kulkarni et al., 1999).

Cell culture, flow cytometry and transfection

skbr3 or BT474 cells were from ATCC. Generation of trastuzumab-resistant skbr3 cells was described (Nahta and Esteva, 2004). Cells were cultured in the medium (Dulbecco's modified Eagle's medium/F12) containing 10% serum, penicillin and streptomycin, and for trastuzumab-resistant cells trastuzumab (4 μg/ml) was included. For activation of calpain, cells were plated on fibronectin-coated dishes alone or along with calpeptin or MDL28170, or cells were treated with trastuzumab (10 μg/ml) alone or along with calpeptin or MDL28170. Cells were pretreated with inhibitors for 30–45 min before adhesion or treatment with trastuzumab. For morphology, cells were photographed under light microscope. For flow cytometry, cells were incubated with FITC-labeled integrin β1 antibody, washed and analyzed. For transient transfection, cells were transfected with 9 μg of plasmid expressing wild-type calpain1 and 24 μl of EasyTransgator-si reagent for 4 h, recovered for 48 h and spread on fibronectin for 2 h. For expression of catalytically inactive calpain1, cells were transfected and selected in the presence of G418 (400 μg/ml) for 6 weeks. Transfected cells from two different 100 mm plates were cloned by serial dilution to obtain clones #1 and #2.

Cell viability

Cells (1–10 × 103) were plated in triplicate in fibronectin-coated wells and allowed to spread. Cells were treated with trastuzumab (10 μg/ml), calpeptin or MDL28170 individually or together as indicated at 37 °C for 48–72 h, incubated with MTS reagent (Promega) for 1 h at 37 °C and read at 490 nm to assess viability. In other experiments, viability was assessed by staining with trypan blue.

Western blot

Lysates were prepared in RIPA buffer containing Tris (20 mM, pH 7.6), NaCl (150 mM), SDS (0.1%), DOC (0.1%), Triton X-100 (1.0%) and protease inhibitors (Roche). Frozen sections (20 μM) of human HER2-positive breast cancer tissues containing 80% invasive carcinoma as assessed by hematoxylin–eosin staining of consecutive sections were homogenized in the above buffer. Lysates or homogenates were centrifuged, protein was estimated (Bradford reagent, Bio-Rad, Hercules, CA, USA), denatured with 2 × SDS sample buffer, separated through 8% SDS gels (Invitrogen, Eugene, OR, USA), transferred to polyvinylidene fluoride membrane and detected.

Immunoprecipitation

Lysates were precleared with mouse IgG and protein G-agarose, and incubated with monoclonal HER2 antibody at 4 °C followed by incubation with protein G-agarose beads and detection for phospho-Y1221/22 HER2 in western blots.

Cleavage

GST-cytoplasmic region or HER2-immunoprecipitates were incubated with calpain1 alone or along with calpeptin in activity buffer (20 mM Tris-HCl (pH 7.2), 50 mM NaCl, 2 mM MgCl2 and 5 mM dithiothreitol) for 5 min at room temperature. Cleavage was initiated with 5 mM CaCl2 and terminated with 2 × SDS sample buffer.

Calpain activity

Activity was measured as described (Edelstein, 2000). Lysates were prepared in buffer (20 mM Tris–HCl (pH 7.2), 50 mM NaCl, 2 mM MgCl2 and 0.1% NP40) and protein was estimated. Equal amount of protein was incubated with fluorescently labeled substrate II at 37 °C in triplicate in 96-well plates. Fluorescence was measured at excitation 380 nm/emission 460 nm.

PTEN phosphatase activity

Activity was measured as described (Nagata et al., 2004). PTEN was immunoprecipitated with a polyclonal antibody from lysates precleared with rabbit IgG and protein G-agarose beads, and incubated with PIP3 in 96-well plates. Release of phosphate was measured using BIOMOL GREEN Reagent.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

First author thanks Dr James H Finke, Department of Immunology Cleveland Clinic, for critical review of data and paper.

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Correspondence to S Kulkarni.

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Kulkarni, S., Reddy, K., Esteva, F. et al. Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer. Oncogene 29, 1339–1350 (2010). https://doi.org/10.1038/onc.2009.422

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Keywords

  • HER2
  • AKT1
  • survival
  • resistance
  • calpain

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