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

Abstract

Calpains are a family of calcium activated cysteine proteases which participate in a wide range of cellular functions including migration, invasion, autophagy, programmed cell death, and gene expression. Calpain-1 and calpain-2 isoforms are ubiquitously expressed heterodimers composed of isoform specific catalytic subunits coupled with an obligate common regulatory subunit encoded by capns1. Here, we report that conditional deletion of capns1 disrupted calpain-1 and calpain-2 expression and activity, and this was associated with delayed tumorigenesis and altered signaling in a transgenic mouse model of spontaneous HER2⁺ breast cancer and effectively blocked tumorigenesis in an orthotopic engraftment model. Furthermore, capns1 knockout in a tumor derived cell line correlated with enhanced sensitivity to the chemotherapeutic doxorubicin and the HER2/EGFR tyrosine kinase inhibitor lapatinib. Collectively, these results indicate pro-tumorigenic roles for calpains-1/2 in HER2⁺ breast cancer and provide evidence that calpain-1/2 inhibitors could have anti-tumor effects if used either alone or in combination with chemotherapeutics and targeted agents.

Keywords: HER2; breast cancer; calpain; capns1.

Figure 1. Deletion of capns1 in the mammary epithelium delays Her2/Neu-driven tumorigenesis

NIC capns1^flox/flox (KO) or NIC capns1^+/+ (WT) female mice were assessed for tumor onset by weekly palpitation. Median tumor onset was 318 vs 300 days, (n = 43 vs 48, respectively, p = 0.0277^* Gehan-Breslow-Wilcoxon Test).

Figure 2. Deletion of capns1 in Her2/Neu-driven mouse mammary tumors correlates with a differential phosphoproteome

Spontaneous tumors arising in NIC capns1^flox/flox (KO, n = 9) or NIC capns1^+/+ (WT, n = 10) female mice were subject to RPPA analysis with 128 antibodies. The indicated phosphoproteins displayed significantly different signal intensities (A.U.).

Figure 3. CAPNS1 protein and calpain-1/2 activities are ablated by capns1 deletion

(Upper) Lysates from capns1^flox/flox MTECs transduced with Cre-expressing (KO) or control (WT) retroviruses; or capns1^+/+ (WT) or capns1^−/− (KO) mouse embryonic fibroblasts (MEFs) were subjected to immunoblotting analysis with antibodies recognizing CAPNS1 or tubulin. (Lower) Casein zymography assessment of calpain-1 and calpain-2 activities in lysates from the same panels of MTEC and MEF cells.

Figure 4. capns1 KO is associated with attenuated MTEC in vitro invasion but no effect on migration

(A) Temporal profiles of capns1 KO and WT MTEC migration behavior in scratch-wound healing assays. Fifteen thousand MTECs were seeded on ImageLock 96-well plates and confluent monolayers were scratch-wounded after an overnight incubation. Migration was monitored in an IncuCyte system for 24 h. Data are the means ± SD of three independent experiments run in sextuplicate. (B) Boyden chamber assays measured capns1 KO and WT MTEC invasion after 24 h through 8 μm pores of transwells coated with 15 μg of matrigel. Data are means ± SD of two independent experiments run in triplicate (p = 0.006, student's t-test).

Figure 5. capns1 KO correlates with enhanced sensitivity to lapatinib and doxorubicin

Two × 10⁴ capns1 KO or WT MTECs were seeded on 96 well plates, then cultured overnight. The following day, cells were treated for 72 h with (A) lapatinib or (B) doxorubicin at the indicated concentrations. Cell viability was assessed at endpoint using the PrestoBlue viability reagent. Data are means ± SD of three independent experiments with experimental triplicates (p, student's t-test).

Figure 6. capns1 KO is associated with reduced MTEC tumorigenic potential

One × 10⁶ capns1 KO or WT MTECs were engrafted into the number four mammary glands of female Rag2^−/− IL-2Rgc^−/− mice (n = 6 for each cohort). (A) Caliper measurements of tumor volumes. Median time to onset of palpable tumors at the engraftment site were 79 or 29 days for KO and WT, respectively (p = 0.0009^***, Log-rank (Mantel-Cox) test). (B) Late arising tumors in mice engrafted with capns1 KO MTECs display CAPNS1 expression. Immunoblot analysis of tumor lysates reveals CAPNS1 expression in tumors from both capns1 WT and KO engrafted MTECs. Lysates from WT and KO MEF cells served as positive and negative controls for CAPNS1. Immunoblotting for tubulin confirmed comparable sample loading.

Figure 7. Knockout of calpain correlates with enhanced EGFR expression and sustained MAPK signaling

7.5 × 10⁴ WT or capns1 KO MTECs were seeded overnight in 60 mm dishes. The following day cells were washed with PBS and changed to serum-free media. MTECs were stimulated with 50 ng/mL of EGF for the indicated times, lysed in RIPA buffer and assessed by immunoblotting. (A) capns1 KO correlates with activation of the MAPK pathway in response to EGF. (B) Full length EGFR levels are elevated in capns1 KO MTECs after overnight serum starvation. (C) capns1 KO is associated with greater EGF-induced MEK1/2 phosphorylation. (D) capns1 KO is associated with greater EGF-induced ERK1/2 phosphorylation.