Identification of novel protein kinase regulators of TGFβ signalling

Abstract

The signalling pathways initiated by members of the transforming growth factor-β (TGFβ) family of cytokines perform critical functions during embryonic development and adult tissue homeostasis. Accordingly, TGFβ family members are involved in the regulation of a diverse array of cellular processes including epithelial-mesenchymal transition (EMT), differentiation, cellular proliferation, extracellular matrix (ECM) production and apoptotic cell death. Hence, genetic malfunctions in the TGFβ-induced signalling pathways are implicated in the pathogenesis of multiple somatic and inherited human pathologies, most notably fibrosis and tumourigenesis and metastasis. Accumulating evidence has illuminated the context-dependent nature of TGFβ signalling and provided a molecular framework by which to explain how a conceptually simple signal transduction pathway is able to control diverse cellular responses. Thus, further elucidation of the molecular basis underlying the contextual determinants of TGFβ signalling will evidently provide a greater understanding of how TGFβ signalling operates in different cellular contexts and inform research focused on pharmacologically targeting the pathway effectively.

This thesis project began by conducting a pharmacological screen using an endogenous TGFβ-dependent transcriptional reporter cell line in order to identify potential novel regulatory components of the TGFβ signalling pathway. From this screen and subsequent validation experiments, I observed that three structurally unrelated small-molecule inhibitors that target the salt-inducible kinases (SIKs) are able to attenuate the TGFβ-mediated transcriptional regulation of specific target genes. Moreover, this transcriptional attenuation occurred without affecting the receptor-mediated phosphorylation of the R-SMAD transcription factors SMAD2 or SMAD3 nor the ability of activated SMAD transcriptional complexes to translocate into the nucleus.

The SIKs, of which there are three isoforms, are serine-threonine specific protein kinases that belong to the AMPK-related family of kinases. To date, the reported cellular functions of SIK isoforms are primarily in the control of innate immune signalling and metabolic regulation. The SIKs primarily function by phosphorylating and hence regulating the activity of the transcriptional coregulators CREB-regulated transcriptional coactivators (CRTCs) and the class IIa histone deacetylases (HDACs) HDAC4 and HDAC5. From the research conducted during this thesis project, it is now evident that SIKs also function to modulate TGFβ-dependent transcriptional responses. However, the precise molecular mechanisms underlying this observation remain to be elucidated.

In addition to small-molecule kinase inhibition of SIK isoforms, I have demonstrated that genetic inactivation of SIK2 and SIK3 results in the attenuation of TGFβ-dependent transcriptional induction of the target gene PAI-1. Moreover, both PAI-1 transcript and protein expression are attenuated in cells which are deficient in the endogenous expression of LKB1, the upstream activating kinase of SIK isoforms. Crucially, restoration of wild type, but not kinase inactive LKB1, potentiates both basal and TGFβ-induced PAI-1 expression. The effect of small-molecule SIK inhibitors was also investigated in the context of the TGFβ-dependent cellular proliferation and apoptotic responses. Inhibition of SIKs was sufficient to enhance the anti-proliferative response in certain cell lines. Furthermore, treatment of cells with small-molecule SIK inhibitors potentiated TGFβ-mediated apoptotic cell death. Collectively, these findings not only identify SIK isoforms as novel regulators of TGFβ signalling but also imply that inhibiting SIKs might sensitise certain cancers for TGFβ-mediated apoptosis.

Date of Award	2019
Original language	English
Awarding Institution	University of Dundee
Supervisor	Gopal Sapkota (Supervisor)