Integrating kinome dynamics and protein shedding after tigilanol tiglate treatment in head and neck Cancer

Awad, Tufaha 2025. Integrating kinome dynamics and protein shedding after tigilanol tiglate treatment in head and neck Cancer. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Head and neck cancer (HNC) is the seventh most common cancer worldwide. Tigilanol tiglate (TT) is a powerful molecule involved in cell signalling with anticancer effects. TT's anti-cancer mechanism has been linked to protein kinase C (PKC) activation, but detailed kinase activity remains unknown. Members of the PKC family are believed to activate ADAMs, which cleave cell surface proteins like c-MET, Nectin-1, syndecan-1, NGFR, and PTPRU into the surrounding medium. These proteins are vital in cancer development. This study characterized the kinome of H357 cells treated with TT using PamGene technology and confirmed the shedding of transmembrane proteins through Western blot and ELISA. The impact of NGFR shedding on apoptosis was examined using LDH assays and Western blots for active caspase-3, -7, and PARP. Given the established roles of ADAM10 and ADAM17 in shedding, the potential contribution of ADAM15 was investigated by creating H357 cells with ADAM10, 15, and 17 knocked out using CRISPR-Cas9. QRT-PCR was used to analyse ADAM15 splice variants, and Western blot confirmed protein expression before the knockouts. The results showed TT activates various S/T kinases, with TKL and AGC-CAMK groups prominent after 10 min, but after 1 h, AGC group members are more active. PKC isoforms also show stronger activation at 1 h. This was confirmed by increased phosphorylation of MARCK protein at serine 167 and 170. TT also promoted the shedding of multiple transmembrane proteins, with the shedding of total c-MET and p-Y1003 c-MET significantly affected by TT application. p-Y1234/5 and p-Y1349 c-MET levels remained unchanged. Nectin-1, NGFR, and SDC-1 were clearly cleaved after TT. These findings highlight a novel mechanism by which TT alters HNC cell signalling, through modulation of ADAM-mediated ectodomain shedding. ADAM15 expression in H357 cells was confirmed, and ADAM10 and ADAM15 levels decreased after 24 h TT treatment, but ADAM17 levels remained the same. The first evidence of differential ADAM15 splice variant expression in HNC cells was provided, with ADAM15-A being the most expressed and ADAM15-C the least expressed. Flow cytometry revealed that ADAM17 was absent in ADAM17 CRISPR-Cas9 knockout cells. In contrast, ADAM10 and ADAM15 remained present in ADAM10 and ADAM15-null cells, respectively. Although 1 μg/ mL TT is 95% cytotoxic to H357 cells, caspase-3/-7 and PARP activation were only observed at high doses (168 μg/mL). This suggests that apoptosis is not the primary mode of cell death at low doses, pointing instead to alternative cell death pathways. Collectively, these results extend the current understanding of TT’s molecular mechanism of action and establish links between PKC activation, kinase signalling, and transmembrane protein shedding. The results also suggest new opportunities to target ADAM15 and related proteases in HNC.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Dentistry
Subjects:	Q Science > QR Microbiology R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Date of First Compliant Deposit:	4 March 2026
Last Modified:	04 Mar 2026 17:11
URI:	https://orca.cardiff.ac.uk/id/eprint/185464

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