Abstract 890: The role of calpastatin isoforms in breast cancer progression
Authors: Sukkarn Bhudsaban , Storr Sarah , Ellis Ian O. , Jewell Kirsty , Parr Tim , Martin Stewart G. 
Published: July 2017

Calpastatin is the endogenous inhibitor of the ubiquitously expressed m-calpain and μ-calpain. Both of them are important regulators of various cellular pathways including proliferation, apoptosis, adhesion, and migration. Dysregulation of the calpain system is associated with a wide range of pathologies including tumour invasion and metastasis. In breast cancer, down-regulation of calpastatin mRNA, in particular an exon 3 containing variant, is associated with lymphatic vessel invasion which is the predominant form of lymphovascular invasion (LVI) (96%), a critical initial step of metastases. The current project seeks to determine the role that individual calpastatin isoforms play in regulating breast cancer cell migration and endothelial interactions to understand processes involved in regulating LVI.

Seven breast cancer cell lines were assessed for calpain system protein expression (calpain-1, -2, -4 and calpastatin) and calpastatin (CAST) mRNA expression (total CAST, CASTexon3, CAST I, II and III) by Western blotting and qRT-PCR, respectively. T47D and MDA-MB-231 cell lines showed low calpastatin but high calpain expression. From haptotaxis cell migration data, T47D migrated more slowly than MDA-MB-231 (35.49±6.14 and 80.34±5.52 percent wound closure at 24 hours post-scratch respectively) (P value=0.0233). These cell lines, that represent different subtypes of breast cancer (luminal and basal respectively), were therefore selected to study the role of differential calpastatin type/isoform expression.

GFP-tagged XL and Leader domains of calpastatin type I, II and III, with or without exon 3 were overexpressed in each cell line. The different calpastatin types show differential subcellular localization. In MDA-MB-231, type I, III, IΔ3, IIIΔ3 were expressed generally in cytoplasm whereas type II and IIΔ3 were located as punctate nuclear invaginations. To assess if overexpression of the different calpastatin types cause phenotypic changes, full length HA-tagged calpastatin type I, II and III were transfected into MDA-MB-231 and T47D and stable clones produced. Thus far stable type II overexpression in MDA-MB-231 and type III in T47D have been obtained with others under single cell selection. The localization of full length type II in MDA-MB-231 was similar to the localization of the truncated form that showing a strong single perinuclear signal. In T47D, the full length and truncated type III also show similarly localization that expressed throughout the cytoplasm. For the effect of calpastatin types on cell proliferation, calpastatin type II in MDA-MB-231, and type III in T47D, had no significant effect on cell doubling time when compared to respective controls (P value=0.6400 and 0.8874, respectively). According to preliminary studies, calpastatin type II seems to have no effect on clonogenicity of MDA-MB-231. Such effect on T47D and other phenotypic changes involved in regulating LVI of transfected cell are being examined.

Citation Format: Bhudsaban Sukkarn, Sarah Storr, Ian O. Ellis, Kirsty Jewell, Tim Parr, Stewart G. Martin. The role of calpastatin isoforms in breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 890. doi:10.1158/1538-7445.AM2017-890