Regulation of cell proliferation and apoptosis in neuroblastoma cells by ccp1, a FGF2 downstream gene

A previously uncharacterized gene named Coiled-coil domain containing 115 (Ccdc115) or coiled coil protein 1 (ccp1) (GeneID: 69668), has been recently identified downstream of Fibroblast Growth Factor 2 (FGF2) by microarray analysis and its expression pattern was characterized [1]. The ccp1 transcript was up-regulated upon FGF2 stimulation in primary cortical neuron culture (CNC) derived from mouse embryonic telencephalon at embryonic day 14.5 (E14.5) and in neuroblastoma cell line, SK-N-SH. In situ hybridizations revealed that ccp1 was expressed in the ventricular zone (VZ), a region of the developing cerebral cortex known to be composed of progenitor cells undergoing proliferation [2].

The mechanism by which cell proliferation is controlled in the VZ is still not fully understood. A number of factors, including FGFs, have been shown to regulate the proliferation of progenitor cells in embryonic Central Nervous System (CNS) in vitro [3‐7]. FGFs are a family of 22 polypeptides known to play various roles in neural development [8, 9]. FGF signals are mainly mediated by high-affinity receptor-type tyrosine kinases, FGF receptors (FGFRs). FGF signaling plays variety of roles in neural development and in pathogenesis of developmental diseases. FGFs are a class of molecules that regulate proliferation by controlling the length of the G1 phase. Addition of FGF2 in primary culture prepared from developing cortex at E14-E16 showed shortening of the G1 length and increase in proliferative divisions, indicating that FGF2 controls cell proliferation via its control of G1 length [10]. Regulation of cell proliferation is mediated by a complex system of signaling pathways. One of the core pathways downstream of FGF is the mitogen-activated protein kinase (MAPK) pathway [11], which has a central role in transmitting cell proliferation and survival signals [12]. In this pathway, RAS promotes activation of the serine/threonine protein kinases Raf1 and MEK1. In addition to controlling RAF kinases, MAPK may also directly regulate several other signaling pathway, such as the phosphatidylinositol 3 (PI3) kinase [13].

In this study, ccp1 function was investigated using a retroviral over-expression system and RNA interference (RNAi) in vitro. We analysed the effects of altered ccp1 expression in cell proliferation and apoptosis in mouse embryonic fibroblast (MEF), a neuroblastoma cell line SK-N-SH, and in NIH3T3. Since ccp1 expression is found regulated by FGF2, we also investigated the effects of FGF2 and compared them to those of the serum in ccp1-expressing cells. Furthermore, by specifically inhibiting the MAPK pathway with the pharmacological inhibitor U0126, we further investigated the involvement of this pathway in ccp1-induced cell proliferation. Our results showed that ccp1 regulates cell number by promoting proliferation and suppressing cell death.

In this study, we have investigated the potential role of ccp1, with a hypothesis that ccp1 may be a downstream effecter of FGF2, promoting cell proliferation and protecting from apoptosis. We show here that ccp1, a gene expressed in embryonic and adult brain [1], may regulate cell morphology, proliferation and programmed death in normal fibroblast and in neuroblastoma cells.

We have demonstrated that morphological transformation occurred in MEF and SK-N-SH cells under stable over-expression of ccp1 (Figure 1). This effect was observed both in the presence or absence of FGF2 or serum (Figure 3). A long-term culture, for example, of neurite outgrowth or growth of cells in soft agar, may further clarify the effect of ccp1 in morphological transformation in the presence or absence of FGF2 in the future. Growth curve and BrdU incorporation in the presence of serum showed that ccp1 expression is able to promote proliferation up to 5 days in culture (Figure 2). Similar results were observed in the absence of serum, suggesting that ccp1 is able to induce proliferation without mitogen stimulation (Figure 2 and 3). Reduction of ccp1 level by RNAi dramatically reduced SK-N-SH cell proliferation, providing further evidence that ccp1 can induce cell proliferation (Figure 4). In addition, we have showed that ccp1 plays a role in suppressing cell death (Figure 5). Whether extrinsic from intrinsic pathways are involved in the suppression of apoptosis by ccp1 is unknown and will need further investigation.

Although both FGF2 and serum treatment enhanced the increase in cell proliferation upon ccp1 over-expression, the effect of FGF2 did not reach that of the serum (Figure 3 and 6). Therefore it is likely that other mitogenic factors present in the serum can also enhance this ccp1 activity. In addition, it was shown that in the absence of serum, ccp1-induced proliferation was partially inhibited by U0126 (Figure 6B). In contrast, in cells treated with FGF2, the inhibition was less prominent in MEF expressing ccp1 than control MEF (Figure 6C, D). This indicates that MAPK may play only a partial role in cell proliferation promoted by ccp1 over-expression, and that there are signaling pathways other than MAPK that are likely to be also involved in this process. For example, ccp1 activity could be regulated by several signaling pathways, such as the PI3K/AKT pathway. The use of inhibitors of the AKT pathway, such as an mTor inhibitor, rapamycin, may be useful in the future to clarify this point.

It is still unclear how ccp1-induced proliferation is enhanced by FGF2 or serum, in particular, either as a consequence of an increased expression of endogenous ccp1 induced by mitogens such as FGF2 [1], or functional enhancement of ccp1 activity by these factors.

Ccp1 expression was able to increase ERK phosphorylation immediately after the treatment with FGF2 (Figure 7B). This could be the bases of ccp1-induced cell proliferation observed in Figure 6. However, ccp1-expressing MEF showed a decrease in ERK phosphorylation in the steady state culture under serum (Figure 7A). As the decrease in MAPK signaling could be due to the presence of the feedback loop such as Sprouty (SPRY) proteins [17], we analysed the SPRY levels (Figure 7D). In MEF expressing ccp1, the level of SPRY was already higher than in the control MEF at the time point 0, however at 6 hours, it reached the maximum level observed in the presence of FGF2. This may indicate that ccp1 expression accelerated the induction of SPRY level, possibly due to activation of MAPK signaling. However it is remains unclear, why pERK was suppressed upon ccp1 over-expression in the steady state of culture. On the other hand, maintained increase in SPRY levels in ccp1-overexpressing cells (Figure 7D) may explain the decrease in cell growth observed in the absence of serum at 4 days of treatment (Figure 2C).

Aberrant activation of FGFs and their receptors lead to several pathologies, including cancer [19]. Study of ccp1 function in promoting proliferation and suppressing cell death would be interesting in aiming a better understanding of tumor formation. Further experiments using knockdown system of ccp1 are necessary to address the requirement of ccp1 in mediating FGF signaling in cell proliferation and apoptosis, possibly using multiple cell lines. Although unlikely, a potential cannot be excluded that the siRNA regulates the protein level of ccp1 differently from that of the mRNA. This has to be addressed in the future experiments.

This study has shown that ccp1 regulates cell proliferation and cell death. Although FGF2 enhanced the effects of ccp1, other mitogenic factors such as MAPK, are likely taking part in enhancing the effects of ccp1.