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Stewart Martin - Understanding calpastatin’s role in regulating breast cancer metastasis 2020 Pilot Grant

Lay summary

Background: Metastasis is when tumour cells gain the ability to move and start growing at a different site within the body, developing into secondary cancer. An early step is development of a process called lymphovascular invasion; when the cancer cells enter into blood or lymphatic vessels to be carried elsewhere. We previously examined breast cancer patients’ tumours that did, or did not have lymphovascular invasion (LVI) and identified that patients’ tumours with lymphovascular invasion had low levels of a protein called calpastatin. Calpastatin stops a family of proteins, called calpains, from working. Calpains normally act like scissors within the cell, cutting up important proteins; low levels of calpastatin therefore means that the calpains are free to cut (calpain cleavage), when they should be controlled and inhibited. Calpain cleavage allows cells to move, migrate and invade into the blood and lymphatic vessels. We have also shown that the cancer cells, with functional calpains, also become resistant to various treatments. There are 3 different types of calpastatin but how each regulates LVI is unknown – understanding calpastatin regulation of LVI related pathways will ultimately allow development of novel agents to target secondary, metastatic breast cancer, and improve treatment response.
Aims: We will examine the changes that happen in cells that we force to overproduce different types of calpastain and assess how often such changes happen in patient tumours.
Techniques and Methodology: We will use breast cancer cell lines that we have altered so that they express different calpastatin types and assess how they change cell behavior and growth; we will then look in a large number of breast cancer patient tumours to see how often this happens.
Impact on breast cancer research: This work will allow us to understand ‘how’ calpastatin regulates lymphovascular invasion, an important first step in metastasis.

Scientific summary

Background: Breast cancer metastasis is a major cause of mortality. Despite advances in modern therapies, a significant number of patients respond only transiently. The calpain system has been implicated in breast cancer progression and response to current forms of therapy. Calpains are a family of cysteine proteases and are endogenously inhibited by calpastatin. Our group has previously shown that both calpastatin mRNA and protein levels are significantly associated with the presence of lymphovascular invasion; in particular an exon three containing variant. Calpastatin has three ubiquitously expressed isoforms; resultant from a single gene subject to differential splicing and promoter usage. Isoform differences occur in the XL and L domains; it is unclear what functional consequence this may have; however, they are postulated to effect cellular localisation.
Aims: Breast cancer cell lines, MDA-MB-231 and T47D, overexpressing calpastatin type 1, type 2 and type 3 have been generated; experiments on GFP tagged Page 3 of 7 versions have shown differential cellular localization. We hypothesise that such differential localisation alterssubcellular calpain activity and function. This proposal will define what changes associate with differential expression of calpastatin isoforms in vitro and validate idetified targets in breast cancer tumour specimens.
Techniques and Methodology: Breast cancer cells overexpressing calpastatin type 1, 2 or 3 will be subject to transcriptome sequencing using RNA-seq. Identified gene targets will be verified in breast cancer tissue microarray using Immunohistochemistry; with a particular focus on determining links with lymphovascular invasion.
Impact on breast cancer research: This study will provide knowledge on how calpastatin isoforms can influence the process of lymphovascular invasion. Whilst the calpains are well studied in breast cancer, and their effect on migration and invasion is well documented, there no information on the role that calpastatin isoforms have on this process

Nottingham Breast Cancer Research Centre (NBCRC)

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