br g Quantification of total red dots from
(g) Quantification of total red dots from 30 NCT-501 from randomly selected images for target and blank samples of 3 independent sets. All bar chart displaying mean and SD. One way-ANOVA and Tukey's multiple comparisons tests were used. ** indicates P-value < 0.02. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
for Bcl-2/Rac1 binding. On the other hand, as Rac1 status is implicated
in the interaction between Rac1 and Bcl-2, interests arise if other gain-of-function Rac1 mutations, such as Rac1P29S and Rac1b [6,27–30], could also contribute to the interaction between Rac1 and Bcl-2 via a potential change in Rac1 conformation, its activation status or affinity towards Bcl-2. With that said, narrowing down the actual interacting domains of Bcl-2 and Rac1 would undoubtedly provide important in-formation in the specific targeting of Rac1/Bcl-2 interaction and the resultant S70pBcl-2.
Notably, our data also demonstrated that Rac1V12 could stabilize Bcl-2 protein. Indeed, Rac1V12 could induce an increase in T69pBcl-2, which has been shown to stabilize Bcl-2 protein via Pyruvate Kinase M2 (PKM2) . Nevertheless, our data showed that T69pBcl-2 is not regulated by O2.-, suggesting that while T69pBcl-2 could stabilize Bcl-2, it does not take part in the redox regulation of Bcl-2/Rac1 interaction and S70pBcl-2 induction. Nonetheless, as JNK and PKM2 could phos-phorylate Bcl-2 [25,31], it is possible that T69pBcl-2 is a function of active Rac1-induced JNK or PKM2 activation .
4.2. Active Rac1-induced S70pBcl-2 involves the interaction of Rac1 with Bcl-2 as well as its redox activity to inhibit PP2A assembly
While S70pBcl-2 is phosphorylated by JNK , we demonstrated that Rac1-induced S70pBcl-2 via O2.- is not JNK-dependent but more relevantly the redox-dependent inhibition of PP2A holoenzyme as-sembly. To that end, PP2A functions as a putative tumor suppressor by regulating the phosphorylation-dependent activation/stability of on-cogenic proteins such as c-Myc, Akt, IKK and Bcl-2 [5,33–35]. More-over, PP2A has been shown to bind to and regulate Rac1 activity, thus influencing Rac1-mediated migration/invasion . Intrigued by the involvement of active Rac1 in not only creating a ‘pro-oxidant’ milieu
but also in promoting sustained S70pBcl-2, we questioned whether the latter was a function of disrupting PP2A-mediated dephosphorylation of S70pBcl-2. Our present work indeed re-capitulated the redox-depen-dent inhibition of PP2A-B56δ assembly , resulting in an increase in S70pBcl-2 in a Rac1-dependent manner. In addition, as S70pBcl-2 binds to active Rac1 and B56δ, it is plausible that B56δ-bound S70pBcl-2 places B56δ in close proximity of active Rac1 and amenable to redox-dependent modification. This hypothesis is plausible as our previous in vivo work demonstrated that B56δ-bound S70pBcl-2 has disrupted in-teraction with PP2A C-subunit , which is in accordance to our pre-sent in vivo evidence that S70pBcl-2 is highly bound to Rac1.
4.3. Increased Rac1 and S70pBcl-2 as predictive marker(s) of advanced disease as well as potential nodes for therapeutic strategy against refractory tumors
Our findings demonstrate that active Rac1, S70pBcl-2 and their physical interaction function in tandem to promote survival and drug resistance. Of note, this functional synergy is verified in clinical samples obtained from patients with lymphoma; levels of Rac1, S70pBcl-2 (and Bcl-2) and their interaction are positively correlated with higher stage lymphomas, thereby underscoring the presence of a novel crosstalk that maintains a conducive cellular redox environment for the growth and progression of cancer cells. Based on the strong clinical correlation, one is tempted to speculate that the co-expression of Rac1 and S70pBcl-2 as well as their physical interaction could serve as markers for disease stratification and potential nodes for therapeutic intervention. The latter would highlight the potential of novel combinational approaches to target S70pBcl-2, particularly with drugs that are FDA-approved [37–41]. As a proof-of-concept, we provide evidence that combina-tional approach using tiron or ABT199 with staurosporine, significantly