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Title: Deregulation of the folate pathway in respiratory tract cancer : molecular and functional consequences
Author: Al-Humairi, I. A.
ISNI:       0000 0004 7657 9901
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Respiratory tract (lung and head and neck) cancers constitute a significant public health burden, contributing greatly to the cancer-related mortality worldwide. Folate is an essential nutrient for genome stability through its role in regulating DNA biosynthesis, repair and methylation processes. The deregulation of specific genes encoding for enzymes in this pathway has been implicated in several types of cancer including respiratory tract cancer and might be critical for cancer cell growth. Certain folate-related genes are targets of very common anticancer drugs and their expression has been associated associated with the relevant chemotherapeutic response. The current study sought to expand the current knowledge on folate pathway deregulation in respiratory tract cancer and shed light on as yet unexplored areas. In particular it aims to: 1) explore the expression profile of the core genes of the folate pathway in lung cancer. 2) investigate the potential of sensitising respiratory tract cancer cells to antifolates using well-known epigenetic drugs. 3) assess the impact of manipulating the activity of previously unexplored folate genes on cancer cell response to antifolates. We evaluated the mRNA expression levels of MTR, AHCY, MAT2A, CBS, DHFR, MTHFR, TYMS, DNMT1 and SHMT1 genes in 104 primary non-small cell lung carcinomas (NSCLC) and adjacent normal tissues, utilising quantitative real time PCR (qPCR). Expression analysis revealed significant expression differences between normal and tumour lung tissues in only six of the examined genes (DHFR, DNMT1, SHMT1, CBS, TYMS and MTHFR) (p=0.003, p < 0.001, p=0.015, p=0.002, p < 0.001 and p=0.014) respectively. The relative expression of CBS and SHMT1 splice variants was assessed in 53 NSCLC, paired with adjacent normal tissues using relative fluorescent quantification and qPCR, respectively. CBS variant 3 (CBSv3) and SHMT1 variant 2 (SHMT1v2) were found to be differentially expressed in NSCLC tumour samples compared to adjacent normal tissues. A significant reduction was observed in the viability of A549 and SK-MES NSCLC cell lines when exposed to the HDAC inhibitor sodium valproate 48 hours prior to treatment with 5-Fluorouracil (5-FU), while no epigenetic sensitisation was established for methotrexate and pemetrexed. Finally, SHMT1 expression was knocked down in the PE/CA-PJ15 head and neck squamous cell carcinoma (HNSCC) cell line, using short hairpin RNA (shRNA). SHMT1-low clones consistently showed increased sensitivity to the 5-FU anticancer drug in a n expression dose dependent manner. Inhibition of SHMT1 enzymatic activity by the selective inhibitor aminomethylphosphonic (AMPA) acid also potentiated 5-FU activity in the same cell line. In conclusion, the data generated from this study provides compelling new evidence into the functional role of CBS and SHMT1 splice variants that might influence NSCLC pathogenesis. Moreover, this study uncovers a new role of SHMT1 as a potential modulator of 5-FU based chemotherapy and points to a new strategy for exploiting SHMT1 as a valuable metabolic target for precision 5-FU therapy.
Supervisor: Liloglou, Triantafillos ; Risk, Janet ; Shaw, Richard ; Field, John K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral