Sukkarn, Bhudsaban
(2020)
Role of calpastatin isoforms in breast cancer lymphovascular invasion.
PhD thesis, University of Nottingham.
Abstract
Expression of calpastatin protein or exon3-containing variant (CASTexon3 mRNA) is negatively associated with various clinicopathological features of the high-risk group for breast cancer recurrence, including the presence of lymphatic invasion, a predominant form of lymphovascular invasion (LVI). It is mostly unknown how individual calpastatin isoforms originated from a single gene regulate such processes. This study, therefore, aimed to assess the role that individual calpastatin isoforms, encoded by CASTexon3 mRNA, play in regulating calpain activity as well as breast cancer progression and LVI in vitro.
Stably transfected monoclonal cells overexpressing calpastatin Type 1 (231CAST1 and T47DCAST1), Type 2 (231CAST2), Type 3 (T47DCAST3) and the mock-transfected controls (231Ctrl and T47DCtrl) were generated from MDA-MB-231 and T47D lines using DNA transfection and single-cell selection techniques. By immunofluorescence confocal microscopy, Type 1 and 3 were expressed mainly in the cytoplasm and partially in the nuclei. Type 2 uniquely showed a predominant perinuclear expression. This suggested to select Type 1 and 2 to fully examine and compare their roles in regulating breast cancer progression and LVI due to the differences between their intracellular localisation and mRNA sequences by only one exon. The cell models overexpressing Type 1 and 2 were examined for calpain activity using the t-BOC assay. Growth curve and clonogenic survival assays were used to assess the effect of calpastatin isoform overexpression on cancer progression-related phenotypic endpoints, i.e., cell proliferation and clonogenicity, respectively. For LVI-related phenotypic features, the cell models were assessed for cell migration and interactions to blood vascular hMEC-1 vs lymphatic hTERT-LEC endothelial cells (BECs vs LECs) using scratch-wound migration and static adhesion assays, respectively.
While calpain inhibitor calpeptin significantly suppressed calpain activity, cell proliferation, migration, and interactions to hMEC-1 model in MDA-MB-231 cells, upregulation of both Type 1 and 2 unexpectedly contributed to a significant increase of calpain activity (39.82 and 70.78 %increase respectively) and promotion of cancer progression- and LVI-related phenotypic endpoints of the cells. Type 1 overexpression seemed to shorten the doubling time of MDA-MB-231 and T47D cells relative to controls (29.13 vs 38.41 hours and 33.11 vs 41.97 hours, respectively), although a significant difference was not observed. MDA-MB-231 doubling time was not affected by Type 2 overexpression (35.63 hours), while MDA-MB-231 clonogenic survival was not altered by both Type 1 and 2 overexpression (survival fraction = 1.04 and 0.96 respectively). Type 1 overexpression significantly enhanced MDA-MB-231 and T47D cell migration by 42.61% and 93.30%, respectively, at 24 hours post-scratch, with a similar increase in migration observed at other time-points. Type 2-overexpressing MDA-MB-231 migration was also enhanced with statistical significance observed at 2 hours after wounding (37.44% increase). Type 1 and 2 overexpression showed differential effects on tumour cell adhesion to both EC models - inducing and suppressing effects, respectively. However, a significantly increased adhesiveness of 231CAST1 cells was only observed with hTERT-LEC cells (41.47% increase), while a significantly reduced adhesion of 231CAST2 cells was only detected with the hMEC-1 model (49.80% decrease). T47D cells did not adhere firmly to either EC model limiting the ability to study the role of Type 1 in regulating cell adhesion. With limited upregulation of calpain activity by a 24-hour treatment with 0.5 µM A23187 in both 231CAST1 (35.39% increase) and 231CAST2 cells (10.12% increase), A23187 significantly inhibited the interactions previously observed between hMEC-1 and tumour cells, including 231CAST1 (42.83% inhibition), 231CAST2 (12.86% inhibition), and the mock-transfected cells (35.42% inhibition), but did not affect other phenotypic endpoints.
This study presented the opposing role of calpastatin to what has been published before in regulating calpain activity and cancer progression. Type 1 may induce tumour cell proliferation, migration, and endothelial interactions, in particular to LECs. Type 2 may enhance breast cancer cell migration but suppress tumour cell adhesion, specifically to BECs. These differential effects of Type 1 and 2 probably related to their distinct subcellular localisation.
Actions (Archive Staff Only)
 |
Edit View |
Tools
Tools
![[img]](/style/images/fileicons/application_pdf.png)