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Receptor tyrosine kinases (RTKs) are crucial regulators of cellular processes, including growth, differentiation, and survival. They function by transmitting extracellular signals through membrane receptors to intracellular signaling pathways. Among these RTKs, the platelet-derived growth factor receptors (PDGFRs) and the epidermal growth factor receptor (EGFR) are vital for maintaining cellular homeostasis and are implicated in various pathological conditions, including cancer. In addition to these pathways, nuclear receptors such as the vitamin D receptor (VDR) also play a significant role in modulating cellular functions. The VDR regulates gene expression in response to the active form of vitamin D, and its cross-talk with RTK pathways offers a complex layer of regulatory control that affects cellular proliferation and differentiation.

This thesis investigates the complex signaling mechanisms of PDGFRs and EGFR, emphasizing the influence of lipid rafts, Rho GTPases, and cross-talk with vitamin D receptor (VDR) signaling.

Paper I focuses on the consequences of lipid raft disruption on PDGFR-β signaling. The research highlights that disrupting lipid rafts alters the association of PDGFR-β with some of its downstream signaling components, reducing the activity of ERK1/2 and AKT in BJ-hTERT fibroblasts and AKT and Src in U2OS osteosarcoma cells. 

Paper II explores the differential impact of cholesterol depletion on EGFR and PDGFR-β signaling. We found that EGFR and PDGFR-β internalize into distinct compartments after activation, converging only after prolonged stimulation. Cholesterol depletion enhanced EGFR dimerization and activation while reducing downstream AKT and ERK1/2 phosphorylation, suggesting distinct membrane microdomain dependencies for these receptors.

Paper III explores the impact of Rho GTPase depletion on the phosphorylation and internalization of PDGFR-α and -β. The findißngs suggest that the depletion of Cdc42, Rac1, or RhoA significantly diminishes PDGFR phosphorylation, and downstream stability of STAT1 and activation of STAT1 and STAT3 indicating that these Rho GTPases are integral for the optimal signaling of PDGFRs.

Paper IV examines the cross-talk between VDR and RTK signaling. We discovered that EGF, but not PDGF-BB, enhances VDR-mediated CYP24A1 expression, indicating a selective interaction between these pathways. Additionally, 1,25(OH)2-vitamin D₃ inhibited PDGF-BB-induced proliferation and PDGFR-β phosphorylation, revealing a complex interplay.

Collectively, these studies elucidate the nuanced regulatory mechanisms of PDGFRs and EGFR, emphasizing the roles of lipid rafts, Rho GTPases, and cross-talk with nuclear receptors. 

Cell communication mediated by growth factors and steroid hormones plays a critical role in regulating proliferation, migration and survival. Dysregulation of these signaling pathways is commonly implicated in the progression of cancer. Among steroid hormones, vitamin D metabolites, in particular 1α,25-dihydroxyvitamin D₃ have demonstrated anti-tumor properties. In paper I, we investigated the effects of 1α,25-dihydroxyvitamin D₃ and its analog tacalcitol on cell signaling and anchorage-independent growth in glioblastoma cell lines T98G and U251. Both compounds modulated key signaling proteins, including STAT3 and suppressed anchorage-independent growth, supporting their potential to reduce tumorigenicity in glioblastoma. The effects of tacalcitol were stronger than for 1α,25-dihydroxyvitamin D₃, indicating that even minor modifications of vitamin D can impact its effect on signaling. In paper II, we explored the role of protein disulphide-isomerase A3 (PDIA3), an alternative receptor for 1α,25-dihydroxyvitamin D₃-mediated signaling. Silencing PDIA3 in prostate cancer (PC3, DU145) cells disrupted proliferation, increased migration, and altered the expression of vitamin D-regulating enzymes and transcription factors. Cellular thermal shift assay indicated that 1α,25-dihydroxyvitamin D₃ directly or indirectly interacts with PDIA3, suggesting PDIA3 involvement in mediating 1α,25-dihydroxyvitamin D₃ responses. In paper III, we studied the crosstalk between 1α,25-dihydroxyvitamin D₃ and the growth factors epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) -BB in U2OS cells. EGF enhanced vitamin D-induced expression of the catabolic enzyme CYP24A1 via AKT and ERK1/2 pathways, while 1α,25-dihydroxyvitamin D₃ suppressed PDGF-induced proliferation and receptor phosphorylation, highlighting reciprocal regulation between 1α,25-dihydroxyvitamin D₃ and growth factor signaling. In paper IV, we examined the effects of the oxidized cholesterol derivative 24-hydroxycholesterol in T98G glioblastoma cells. 24-hydroxycholesterol altered the cell morphology, suppressed proliferation and enhanced migration. 24-hydroxycholesterol increased the expression of epithelial-to-mesenchymal transition markers in a liver X receptor (LXR)-dependent manner. This thesis provides insight into the effects of 1α,25-dihydroxyvitamin D₃-mediated signaling in the regulation of cancer cell behavior and crosstalk with growth factor signaling involving EGF and PDGF. The thesis supports the role of PDIA3 in 1α,25-dihydroxyvitamin D₃-signaling responses. By modulating key pathways that control proliferation, migration, and differentiation, 1α,25-dihydroxyvitamin D₃ and vitamin D analogs demonstrate mechanisms that may contribute to anti-cancer activity and offer potential targets for therapeutic intervention.