Novel role of PKC isoforms in regulation of disintegrin and metalloproteinase (ADAM) activity - Impact on tyrosine kinase signalling in head and neck cancer

Antwi, Frank 2023. Novel role of PKC isoforms in regulation of disintegrin and metalloproteinase (ADAM) activity - Impact on tyrosine kinase signalling in head and neck cancer. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Objective: Head and neck cancer (HNC) stands as the 7th most common cancer in the world. EBC-46 is a phorbol ester that activates classical PKC isoforms currently in development for cancer treatment. The PKC pathway activates ADAM metalloproteinases, which cleave several proteins. The cancer markers, Trop2 and receptor tyrosine kinase (RTK), MET are over-expressed in HNC, implicative of increased tumorigenesis, metastases and recurrence. This study investigated the molecular mechanism of EBC-46 in regulating ADAM-dependent proteolysis of Trop2, total and phosphorylated MET in HNC. global cellular changes induced by EBC-46 were investigated using phosphoproteome, proteome and secretome analysis. Methods: Using a Trop2 over-expressed cell line (HEK-Trop2) and a HNC cell line (H357), flow cytometry was performed for EBC-46-dependent cell surface loss or shedding of Trop2 and MET. Cell lysates were investigated with Western blots for Trop2, total and phosphorylated MET. Media investigations were also performed for soluble MET and Trop2. Mass spectrometry was adopted to determine differentially altered proteins and phosphoproteins in H357 cell lysate and media. Results: Flow cytometry showed EBC-46-dependent loss of Trop-2 which was rescued by the PKC inhibitors BIM-1 and Enzastaurin. ADAM inhibitors showed partial rescue of Trop-2 in HEK-Trop2 cell lines. Additionally, western blot analysis showed the generation of an 11 kDa C-terminal fragment in response to EBC-46 due to the activation of ADAM proteinases, which was blocked by both PKC and ADAM inhibitors. EBC-46-dependent MET cell surface loss from H357 cells by flow cytometry was inhibited by PKC inhibitor treatment. EBC-46 induced phosphorylation on Ser 985 but loss of Tyr 1234/5 and Tyr 1003 phosphorylated MET species. ADAM inhibitors blocked formation of a 55kDa fragment formation upon EBC-46 treatment and soluble MET release. Mass spectrometry for EBC-46 treated H357 cells showed several alterations in the phosphoproteome, proteome and secretome. Ingenuity Pathway Analysis (IPA) showed differential changes in several proteins and phosphoproteins associated with the RTK pathway. Phosphoproteomic analysis done for an earlier time point (10 mins) showed an increase in RTK signalling, while later proteome and secretome analysis (1h) showed a decrease in signalling. Secretome analysis showed several ADAM substrates increased in EBC-46 treated media. Conclusion: Altogether, the data indicate that EBC-46 regulates Trop2 and MET-dependent signalling as well as other proteins and phosphoproteins in Head and neck cancers.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Dentistry
Subjects:	R Medicine > R Medicine (General) R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer) R Medicine > RK Dentistry
Date of First Compliant Deposit:	10 May 2023
Last Modified:	01 Jun 2023 08:08
URI:	https://orca.cardiff.ac.uk/id/eprint/159388

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