Regulation of sphingosine kinase 1 oncogenic signalling by calcium and integrin binding proteins

Abstract

Sphingosine kinase 1 (SK1) is one of the key enzymes that regulate the cellular balance of pro-apoptotic and pro-survival sphingolipids. By catalysing formation of the versatile signalling molecule sphingosine 1-phosphate (S1P), SK1 has clear roles in a wide range of cellular processes. The dysregulation of SK1 signalling has been broadly implicated in the pathogenesis of various cancer types, although better understanding of the regulation of SK1 and how defects in this exerts oncogenic effects is still required. Previously, studies by the Pitson Laboratory found that oncogenic signalling of SK1 is reliant on its localisation at the plasma membrane, which is mediated by calcium and integrin binding protein 1 (CIB1) through its Ca2+-myristoyl switch function. This raises the possibility that CIB1, and potentially other members of the CIB family, may contribute to oncogenesis through regulating the subcellular localisation of SK1. In light of this, the studies detailed in this thesis aimed to examine if and how CIB family proteins are involved in cancer via the regulation of SK1. In this study, by mining gene expression databases and examining tissue samples from cancer patients, I demonstrated that CIB1 is widely upregulated in a wide range of cancer types. In some cases, this upregulation of CIB1 was significantly associated with oncogenic mutation of KRas. Ectopic expression of oncogenic Ras also led to upregulation of CIB1 expression. In line with a direct role for CIB1 in initiating oncogenesis, I found that overexpression of CIB1 was sufficient to drive the plasma membrane localisation of SK1 and to induce full neoplastic transformation of mouse fibroblast cells in an SK1-dependent manner. SK1 was previously found to be required for the oncogenic function of Ras. In agreement with this finding, I demonstrated that targeting CIB1 also significantly inhibited the oncogenic signalling by Ras. Together, these findings indicated that the upregulation of CIB1 was not only a consequence of the oncogenic Ras pathway, but also a critical downstream mediator of the Ras-induced oncogenesis via the regulation of SK1. Examination of CIB2, a member of the CIB protein family closely related to CIB1 but lacking the Ca2+-myristoyl switch function, demonstrated that it played an opposite role to CIB1 in the regulation of SK1. CIB2 blocked agonist- and oncogene-induced relocalisation of SK1 to the plasma membrane and the associated pro-survival and pro-oncogenic signalling. In further examining the potential role of CIB2 as a tumour suppressor, I found that CIB2 was significantly downregulated in ovarian cancer tissues and lower levels of CIB2 expression was associated with poor patient prognosis. In line with this observation, re-expression of CIB2 in ovarian cancer cells blocked the plasma membrane localisation of endogenous SK1, and suppressed the neoplastic growth as well as cell motility and invasiveness both in vitro and in vivo. In agreement with the synergistic effects observed between SK1 inhibitors and standard chemotherapeutics, re-expressing CIB2 also sensitised ovarian cancer cells to carboplatin. Together, this evidence suggested CIB2 acts as a novel tumour suppressor in ovarian cancer via inhibiting the oncogenic signalling of SK1. Since targeting the interaction between SK1 and CIB1 appeared to be a promising approach to inhibit the oncogenic function of SK1, I also examined the SK1-binding interface on CIB1. Using mutagenesis followed by an in vitro pull-down approach, I identified the key SK1-binding residues in the hydrophobic groove of CIB1. Furthermore, in light of a recent study that demonstrated the phosphorylation of a longer isoform of CIB1, CIB1a, by protein kinase D2 (PKD2), I further investigated the role of this phosphorylation on SK1 signalling. Observations from these studies suggested that PKD2-mediated phosphorylation of CIB1 enhanced its interaction with SK1 and promoted relocalisation of SK1 to the plasma membrane, suggesting PKD2 as a novel regulator of SK1 signalling. Together, by providing further understanding of the SK1-CIB1 interaction, these findings shed light on future development of novel SK1-targeting agents as potential anti-cancer therapies. Finally, I also generated CIB1 transgenic mice, which exhibited ubiquitous expression of the CIB1 transgene in various tissues. Although histological analysis of male and female CIB1 transgenic mice, at both 12 and 52 weeks of age, found no overt phenotypes nor signs of sporadic neoplasms, these animals remain a useful tool for future evaluation of the oncogenic roles of CIB1 in vivo. In summary, the studies detailed in this thesis demonstrated that CIB1 and CIB2 played opposite roles in cancer biology via differential regulation of SK1 signalling. These findings not only further highlight the importance of plasma membrane-associated SK1 in promoting oncogenesis, but also indicate the therapeutic potential of targeting SK1/CIB1 signalling in cancer.