GENERATE MOLECULAR BIOLOGY TOOLS TO STUDY THE CROSSTALK BETWEEN CDK2 AND TFGb SIGNALLING

Abstract

Cancer remains one of the leading causes of death globally, because of its complex progression and metastasis. Among the key signalling pathways implicated in cancer is the Transforming Growth Factor Beta (TGFβ) pathway, known for its paradoxical role. It acts as a tumour suppressor in early stages of cancer however promotes tumour progression and metastasis in the later stages. Interestingly, recent research found crosstalk between TGFβ signalling and Cyclin-Dependent Kinase 2 (CDK2), a critical cell cycle regulator. This thesis investigates the hypothesis that CDK2 activity is essential for TGFβ signalling and aims to establish molecular biology tools to study this interaction. Preliminary findings from our lab showed that pharmacological inhibition of CDK2 by CVT-313 suppresses TGFβ-induced transcriptional activity. In addition, various molecular tools were developed, including CRISPR/Cas9-mediated CDK2 knockout in HEK293T and A549 cells, CDK2 activity biosensors (DHB-mCherry), and luciferase reporter assays to assess TGFβ transcriptional induction. The results confirmed that both pharmacological and genetic inhibition of CDK2 significantly reduce TGFβ-mediated luciferase activity, and that CDK2 inhibition particularly by the degrader CPS2 lowers levels of phosphorylated SMAD2, a downstream marker of TGFβ activity. Interestingly, while CDK2 knockout did not alter the morphological or molecular EMT phenotype in A549 cells post TGFβ treatment, this may be due to incomplete pathway inhibition or compensatory mechanisms. Nonetheless, the development of live-cell imaging biosensors to monitor CDK2 activity in real time presents a powerful platform for further exploration. Collectively, this research establishes and validates essential tools to dissect the molecular interplay between CDK2 and TGFβ signalling. The findings offer insights for a better understanding of the mechanistic basis of the crosstalk between TGFβ pathway and CDK2.

Date of Award	2025
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences