CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1.

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this upregulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca2+-myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane-associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.

Enhanced expression of transferrin receptor 1 contributes to oncogenic signalling by sphingosine kinase 1.

Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cell-surface G-protein-coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a gene expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of transferrin receptor 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing antibody inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis.

Guanine nucleotides regulate sphingosine kinase 1 activation by eukaryotic elongation factor 1A and provide a mechanism for eEF1A-associated oncogenesis.

Sphingosine kinase 1 (SK1) catalyses the formation of bioactive phospholipid sphingosine 1-phosphate (S1P). Elevated cellular SK1 activity and S1P levels enhance cell proliferation and survival, and are strongly implicated in tumourigenesis. Regulation of SK1 activity can occur through various mechanisms, including phosphorylation and protein-protein interactions. We have previously shown that eukaryotic elongation factor 1A (eEF1A) interacts with and directly activates SK1, but the mechanisms regulating this were undefined. Notably, eEF1A has GTPase activity and can exist in GTP- or GDP-bound forms, which are associated with distinct structural conformations of the protein. Here, we show that the guanine nucleotide-bound state of eEF1A regulates its ability to activate SK1, with eEF1A.GDP, but not eEF1A.GTP, enhancing SK1 activity in vitro. Furthermore, we show that enhancing cellular eEF1A.GDP levels through expression of a guanine nucleotide dissociation inhibitor of eEF1A, translationally controlled tumour protein (TCTP), increased SK1 activity in cells. We also examined a truncated isoform of eEF1A1, termed prostate tumour inducer-1 (PTI-1), which can induce neoplastic cell transformation through undefined mechanisms. PTI-1 lacks the G protein domain of eEF1A1 and is therefore unable to undergo the GTP-binding-induced conformational change. Notably, we found that PTI-1 can directly activate SK1 and that this seems to be essential for neoplastic transformation induced by PTI-1, as chemical SK1 inhibitors or overexpression of a dominant-negative SK1 blocked this process. Thus, this study defines the mechanism regulating eEF1A-mediated SK1 activation, and also establishes SK1 as being integral for PTI-1-induced oncogenesis.