Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

GSK-3 is a pleiotropic serine-threonine kinase discovered for its involvement in insulin signaling. Two major isozymes (GSK-3α and GSK-3β) are known and conserved throughout the species [1]. This kinase is involved in cell proliferation and survival by controlling the Wnt/β-catenin and growth factors (GFs)-dependent pathways [2]. Constitutive GSK-3-mediated phosphorylation directs β-catenin to proteasome-mediated degradation [3]. Upon activation of Wnt signalling, GSK-3 activity is hampered and unphosphorylated β-catenin accumulates in the cytosol, translocates to the nucleus and promotes gene transcription and cell growth by acting as a co-activator of the transcription factors TCF/LEF [3, 4]. GSK-3 is also inhibited by the action of the Phosphatidylinositol 3-OH kinase (PI3K)/AKT cell-survival pathway through phosphorylation on serine 21 (GSK-3α) and serine 9 (GSK-3β) [2]. Since Wnt/β-catenin and PI3K/AKT-dependent signaling pathways promote cell growth, GSK-3 has been considered a growth-suppressor. By contrast, GSK-3β has been found essential for cell survival by critically regulating NF-κB transcription factor activity and by protecting cells from TNFα [5] and TRAIL-induced apoptosis [6‐9]. Moreover, while a number of studies demonstrated that GSK-3 may favor intrinsic apoptosis [10‐12], other work showed that its inhibition could result in cancer cell apoptosis and growth arrest, in some cases due to an impaired NF-κB activity [8, 13‐15]. Taken together, several lines of evidence indicate that GSK-3 could play a twofold role in cell survival, depending on the different contexts (for instance, malignant versus non-malignant cells) or on whether apoptosis is started by intrinsic or extrinsic mechanisms [16]. However, a caveat for many of these studies is that GSK-3α and β isoforms were not evaluated separately.

Multiple myeloma (MM) is an incurable malignancy of plasma cells (PC) that clonally expand in the bone marrow (BM) [17]. Signaling pathways that might lead to GSK-3 inactivation and to NF-κB activation have been implicated in MM pathogenesis [18, 19]. For instance, interleukin-6 (IL-6) production by BM stromal cells (BMSC), which stimulates malignant PC growth and expression of adhesion molecules, is NF-κB-dependent [20]. Insulin-like Growth Factor-I (IGF-I), an important growth and chemotactic factor for MM cells [21, 22], can activate both PI3K/AKT and NF-κB. Also, Tumor Necrosis Factor-α (TNFα) produced in the tumor microenvironment can lend MM cells the ability to escape apoptosis by up regulating NF-κB dependent anti-apoptotic molecules [23]. Whether GSK-3 plays a role in NF-κB activation in MM and other blood tumors upon these and other stimuli is largely unknown. Moreover, the Wnt/β-catenin pathway causes MM cells proliferation [24], suggesting that secreted Wnt proteins in the BM microenvironment may act as GFs for malignant plasma cells. Furthermore, active Wnt signaling is also crucially involved in osteoblast differentiation [25, 26]. Interestingly, the Wnt antagonist DKK1 is over expressed in MM patients displaying impaired bone formation and bone lytic lesions [27]. In this scenario, GSK-3, by inhibiting Wnt signaling, should be a growth brake for MM cells but also a negative regulator of osteoblastogenesis. Thus, the use of GSK-3 inhibitors to by-pass the DKK1-mediated Wnt signaling block on osteoblast precursors - that has been proposed with the aim to slow down the progression of myeloma bone disease [28, 29] - could be hazardous. It is hence important to study both the role of GSK-3 in MM cell growth as well as the effects of its inhibition on the MM cell-tumor microenvironment interactions. Likewise, the investigation of the potential regulation of drug-induced cytotoxicity by GSK-3 - expected to occur in light of its role in the NF-κB signaling - would provide hints to determine the feasibility of this potentially useful therapeutic approach in MM therapy.

With this as a background, the aim of our study was to analyze GSK-3 role in MM cell growth and survival. By using selective ATP-competitive small chemical GSK-3 inhibitors as well as gene knock down by RNA interference, we investigated the consequences of GSK-3 (α and β) down regulation in MM cells. We found that treatment of MM cells with GSK-3 inhibitors and GSK-3β knock down caused growth arrest and apoptosis by perturbing pivotal signaling pathways. We also found that the GSK-3 inhibitors and GSK-3α knock down enhanced the anti-MM cytotoxic effect of bortezomib, a clinically used proteasome inhibitor.

The involvement of GSK-3 in cancer cell biology has recently been demonstrated. GSK-3 sustains the growth of malignant blood cells, like chronic lymphocytic leukemia (CLL) [34] and acute myeloid leukemia cells (AML) [49]. Our data demonstrated that GSK-3 regulates MM cell growth and survival. Importantly, we also found that GSK-3 modulates MM cell sensitivity to BZ-induced apoptosis.

The expression analysis revealed that, in most MM samples from patients and even more, in all the MMCLs, GSK-3β protein levels were lower than GSK-3α. This was not evident in normal controls (PBMC, nPC). GSK-3β also resulted more phosphorylated in Ser 9 than GSK-3α in Ser 21 both in normal and malignant samples; moreover, in the MM samples analyzed for GSK-3 Tyr phosphorylation, we found that GSK-3β was much less Tyr phosphorylated than GSK-3α (Fig. 1A). Since GSK-3β Ser 9-phosphorylation is associated with a reduction in the enzymatic activity whereas GSK-3β Tyr 216-phosphorylation indicates activation [30], these findings, even if to be validated on a larger number of primary MM samples, indicate that the GSK-3β isoform may be less active than GSK-3α in normal PBMC and nPC as well as in MM cells. In these latter cells, however, we observed also a reduction of GSK-3β expression that could contribute to a further down-regulation of its activity. Interestingly, GSK-3α and GSK-3β do not share the same physiological functions [50]. In particular, GSK-3α does not supply for the loss of GSK-3β-dependent NF-κB regulation in GSK-3β knock out cell, whereas it does in the regulation of the Wnt/β-catenin pathway [5]. Our expression studies also showed that in MM plasma cells from patients and some MMCLs (OPM-2, INA-6), GSK-3 was present in the cytosol and partly close to the cell surface, but rarely in the nucleus (Fig. 1B and 1C). Although in other types of cancer cells GSK-3 localization in the nucleus has been associated to an aggressive behavior, our data indicate that in MM cells at resting conditions GSK-3 nuclear functions might not be selected.

GSK-3 was demonstrated to play an important role in modulating cell growth in several solid tumors and two recent studies have shown that B-CLL and Mixed Lineage Leukemia-associated AML cell survival is greatly impaired by the inhibition of GSK-3 [34, 49]. Our data indicate that GSK-3 promotes MM cell survival both at basal and under stressed (proteasome inhibition) growing conditions (Fig. 2, 3 and 4). Firstly, the results with the chemical inhibitors SB216763 and SB415286 indicate that GSK-3 inhibition in MM cells leads to a reduction of MM cell growth. This effect is achieved despite the fact that GSK-3 inhibition causes an increase of β-catenin and phospho-ERK levels, which is expected to result in a growth advantage. Moreover, we have observed that exposing MM cells to GSK-3 inhibitors could augment the response to the cytotoxic effects of BZ, a clinically used chief therapeutic agent in MM therapy. BZ regulates several signaling pathways important for cell survival, among which are the AKT, MCL-1, NF-κB and the Wnt/β-catenin-regulated cascades [42]. Our results suggest that GSK-3 might positively regulate the AKT and MCL-1-dependent survival pathways which are normally targeted by BZ, since GSK-3 inhibitors cause a reduction of the BZ-triggered AKT phosphorylation and MCL-1 degradation. However, even if selective and extensively tested, these inhibitors act indistinctly on GSK-3α and GSK-3β, rendering difficult to study isoform-specific functions; additionally, "off-target" GSK-3-independent effects could also occur. Nevertheless, GSK-3α and GSK-3β down modulation by RNA interference in MM cells substantiated the results found with the inhibitors. In fact, our data indicated that GSK-3α and GSK-3β might control MM cell growth, although in a distinct manner. GSK-3β but not GSK-3α knock down caused basal MM cell apoptosis whereas GSK-3α but not GSK-3β knock down was associated with a trend towards increased BZ-induced apoptosis: this would suggest unexpected different roles of the two GSK-3 isoforms in MM cell survival. As mentioned above, the two GSK-3 isoforms can have overlapping functions in regulating the Wnt/β-catenin signaling pathway but not other cascades, such as the NF-κB one, which is mostly dependent on GSK-3β and is critical for MM cell survival [5, 16]. Even if the analysis of the pre-transcriptional steps of the NF-κB pathway did not reveal significative differences upon GSK-3α or β inactivation (data not shown), it is still possible, as already reported by other studies [34], that, even in MM cells, GSK-3β regulates NF-κB transcriptional activity directly on DNA. Moreover, our data suggest that GSK-3α might control AKT protein turnover, while GSK-3β could modulate MCL-1 protein stability. Clearly, this latter effect of GSK-3β - already described in previous studies [12] - is in contradiction with the evidence that GSK-3β knock down leads to basal MM cell reduced viability. Other signaling pathways should therefore be involved here. In agreement with previous studies, we have shown that BZ induces a reduction of total AKT (with the paradoxical effect of increasing its Ser 473 phosphorylation) and MCL-1 protein levels in MM cells. Interestingly, these BZ-induced biochemical changes were amplified by the treatment with the GSK-3 inhibitors, which were also able to cause a reduction of AKT Ser 473 phosphorylation (Fig. 4). Remarkably, we demonstrated that the siRNA-mediated knock down of GSK3α and of both the GSK-3 isoforms but not of GSK-3β alone, was associated with the same changes of AKT and MCL-1 levels as seen with the inhibitors. Therefore, our experiments suggest that GSK-3β could influence MM cell survival through MCL-1 and AKT-independent mechanisms, while GSK-3α could modulate BZ-induced MM cell apoptosis interfering with pathways controlling AKT and MCL-1 protein levels. To note, AKT levels have been demonstrated to be controlled by GSK-3 in previous studies [51].

Furthermore, we provided evidence for a functional link between BZ and GSK-3. Indeed, despite the fact that BZ induces Ser 473 AKT phosphorylation, it caused GSK-3 dephosphorylation in Ser 9/21 and its nuclear migration. However, only a little increase in the GSK-3 Tyr phosphorylation (especially in the nucleus) was seen. It is possible that the BZ-induced modifications are not sufficient to trigger a marked Tyr autophosphorylation; alternatively, a full Tyr autophosphorylation could take place at different time points which were not explored in the current study. To note, these alterations of GSK-3 function have also been demonstrated to occur after cell exposure to other apoptotic noxae and have been related to the pro-apoptotic effects of this kinase [48]. Interestingly, BZ-induced GSK-3 nuclear accumulation seems to depend on GSK-3 activity, since treatment with SB216763 impaired this intracellular shift. However, the exact significance of GSK-3 nuclear migration upon BZ treatment remains to be elucidated. Since GSK-3 inhibition is accompanied by an increased sensitivity to BZ-triggered cell death, it is unlikely that BZ-induced GSK-3 nuclear accumulation triggers a pro-apoptotic pathway. Oppositely, it is possible that nuclear GSK-3 antagonizes a BZ-dependent nuclear apoptotic pathway. Nonetheless, BZ could trigger GSK-3 activation as a feedback loop to protect MM cells from apoptosis. AKT might be Ser 473 phosphorylated through an unknown, GSK-3-dependent pathway. GSK-3 inhibition would therefore lead to a reduction of phospho-Ser 473 AKT and increased susceptibility to BZ-induced cell death. This unexpected scenario would imply that GSK-3, at basal conditions (to a lesser extent) and in the presence of BZ (to a larger extent), might positively regulate the PI3K/AKT pathway, which in turn is a negative regulator of GSK-3 - as also suggested by the significant amount of GSK-3 (especially GSK-3β) Ser-phosphorylation found in MM cells (Fig. 1A and 6D). In resting conditions this could represent a regulatory loop whereby GSK-3 might buffer down its own activity. However, upon BZ treatment, being Ser 473 AKT phosphorylation increased, other PI3K/AKT-independent mechanism should take place that justify the reduction of GSK-3 Ser-phosphorylation.

In conclusion, we have herein demonstrated distinct functions of GSK-3α and GSK-3β in MM cell survival. We have employed two strategies to inhibit GSK-3α and β function: the use of selective chemical compounds, which were already extensively characterized, as well as the more specific RNA interference technology. We have shown that GSK3α and β might promote MM cell survival under different conditions and we have described a link between the action of BZ and GSK-3 providing a mechanistic clue to sustain a viewpoint whereby this cross-talk may occur. Our data could be helpful to improve our knowledge of MM pathogenesis and of the mechanisms of response to novel treatments, as well as to develop novel anti-MM targeted therapies.