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Title: Defining regulation of Herstatin and novel HER2 splice variants in breast cancer
Author: Silipo, Marco
ISNI:       0000 0004 6425 6515
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2017
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Alternative splicing is a co-transcriptional process which contributes to protein diversity in eukaryotes because it can generate different mRNAs arising from the same gene. Resulting proteins can play either redundant or counteracting roles, or they can lack functionality. Changes in the alternative splicing process have been associated to several diseases including breast cancer. The Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase (RTK) which is activated through hetero-dimerisation with other receptors including members of its own family (HERs). No ligand binds directly to the HER2 receptor; however, it is the favoured hetero-dimerisation partner amongst other HER family members. Following phosphorylation, HER2 can activate several signalling pathways, including RAS/MAPK (ERK1/2) and PI3/AKT, which promote cell proliferation and inhibit apoptosis. Therefore, HER2 is a breast cancer biomarker and its overexpression is commonly assessed in patients. HER2 is a target for therapies including the monoclonal antibody Trastuzumab (Herceptin®) and the tyrosine kinase inhibitor Lapatinib (Tykerb). Nevertheless, these therapies are not always efficacious and their efficiency decreases with the aggressiveness of the disease leading to drug resistance. Besides the HER2 (wild type) receptor, the HER2 gene can produce three functional alternative splicing variants. HER2Δ16, arising from skipping of exon 16, shows high oncogenicity and it is associated with drug resistance. Conversely, Herstatin and p100, arising from inclusion of intron 8 and intron 15 of HER2 respectively, have shown anti-oncogenic properties. In this study a speculative working model was developed; this partially explains regulation of the alternative splicing of Herstatin in the HER2 positive breast cancer cell line SKBR3. In this model the Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 promotes the inclusion of intron 8, whereas the SR protein SRSF1 prevents Herstatin formation. Moreover, using a HER2 minigene, RNA secondary structure and somatic mutations were shown not to affect the splicing of Herstatin. Also, two novel alternative splicing variants of HER2, arising from intron inclusion, were identified. Their protein expression was subsequently confirmed via an in vitro translation system using plasmid expression vectors. Furthermore, these novel variants were shown to enhance phosphorylation of ERK1/2 in the HER2 negative breast cancer cell line MCF-7. iii Finally, a CRISPR/Cas9 system was developed with the aim of promoting Herstatin expression in breast cancer cell lines by modifying the splicing of HER2. Expression of Cas9 protein, as well as its ability to cleave the DNA, was confirmed in the HER2 negative breast cancer cell lines MCF-7 and T47D. Here, modification of HER2 splicing and increased Herstatin expression was not confirmed, but this can be the subject of future research.
Supervisor: Not available Sponsor: RVI Breast Cancer Appeal
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available