Recently, in the context of lung cancer, palbociclib has been reported to induce an apoptotic response in lung adenocarcinoma cell line [18]. Another study reported that dual CDK4/6 inhibition by palbociclib induced apoptosis and senescence in esophageal squamous cell carcinoma cells [19]. In our study, we found that palbociclib dose-dependently induced apoptosis of LUSC cells. These works suggest that palbociclib has anti-cancer effects by inducing cell apoptosis and not restricting to growth arrest. Given the current inefficiency in therapy of LUSC, palbociclib might be an effective alternative therapeutic strategy for LUSC patients, at least in part, by inducing cell apoptosis.
STAT are highly conserved family of transcription factors that are activated by phosphorylation to regulate gene expression [20]. Among the seven STATs, STAT3 signaling activation contributes to progression of cancers [20], while inhibition of STAT3 signaling leads to apoptosis of various cancer cells including lung cancer [21]. To our knowledge, we for the first time found that palbociclib effectively inhibited the STAT3 activity in LUSC cells, which was further confirmed by decreased expression of its downstream, survivin. Further experiments showed that STAT3 inhibitor did not induce additional increase in apoptosis compared with palbociclib alone. These works suggested that STAT3 inactivation play important role in palbociclib-induced apoptosis in LUSC cells. Previous studies have indicated that Rb is a direct downstream protein of CDK4/6 complex and its action determined anti-cancer activity of palbociclib [22]. Our results indicate that when Rb expression was silenced, palbociclib treatment also exert inhibition of STAT3 phosphorylation in LUSC cells. These data strongly support that palbociclib mediated LUSC cells apoptosis via inhibition of STAT3 phosphorylation but not classic Rb signaling. This finding is in keeping with previous work, which reported that palbociclib consistently suppresses tumor growth under Rb-deficient conditions. These results together suggested that Rb dependence was not general for CDK4/6 inhibitor treatment, and palbociclib exerted its cytotoxic effects at least partly by targeting STAT3 signaling beyond canonical CDK4/6-Rb signaling. Our findings suggested that palbociclib treatments might benefit STAT3-positive patients with LUSC, and provide useful information for ongoing and future clinical studies.
A previous study showed that CDK4 inhibition directly inactivated Smad1/5/9 and subsequently dephosphorylated STAT3 in human mesenchymal stem cells [13]. In this study, palbociclib had no influence on Smad1/5/9 phosphorylation in LUSC cells. This may be cell context dependent, perhaps reliant on the necessity of CDK4 versus CDK6 function for different cell types. STAT3 is mainly activated by phosphorylation of tyrosine, which can be mediated by many tyrosine kinases including the Src and JAK families [23, 24]. Our study found palbociclib could reduce Src phosphorylation while have no effects on JAK2 phosphorylation in H520 and H226 cells. Further study showed that palbociclib could not induce additional decrease in STAT3 phosphorylation in cells treated with Src inhibitor. To sum up, these findings reveal that palbociclib suppresses STAT3 activity via Src/STAT3 axis in Rb-independent manner.
Human lung cancer cell-related inflammatory signaling can promote cell proliferation and survival [25, 26]. Some inflammatory cytokine signature predicted the effectiveness of the anti-cancer medication, for example, IL-6 level is a prognostic marker for survival in advanced NSCLC patients or those treated with chemotherapy [27]. In the current study, palbociclib treatment dose-dependently reduced expression and secretion of IL-1β and IL-6 while has no effects on IL-8 and IL-10, indicating that the decrease of IL-6 or IL-1β, but not IL-8 and IL-10, may be serve as a potential biomarker to predict clinical response to palbociclib for LUSC patients. A previous study indicated that palbociclib improved cardiac dysfunction in diabetic cardiomyopathy by preventing inflammatory cytokine release [28]. These studies suggested that palbociclib resulted in not only cell cycle arrest but also decreased inflammatory response, which hinted that palbociclib might be beneficial not only cancers but also inflammatory-related diseases. Future studies are warranted to test this hypothesis. Most importantly, recombinant human IL-1β or IL-6 could rescue the inhibitory effects on Src and STAT3 activity in palbociclib-treated LUSC cells. These observations strongly demonstrated the palbociclib inhibited Src/STAT3 signaling in LUSC cells probably suppression of IL-1β and IL-6 production, and further indicated that inflammatory signaling is of importance on palbociclib-induced apoptosis in LUSC.
Myc is amplified in up to 33% of NSCLC patients and a prognostic marker of early-stage tumors [29]. Our discovery that Myc mediated sensitivity of LUSC cells to apoptosis in response to palbociclib suggested that CDK inhibitors can selectively target tumor cells with specific genetic alterations. Previous works also identified that a form of synthetic-lethal interaction between Myc overexpression and CDK1 inhibition in engineered breast cancer cell lines [30]. Our research into the effects of CDK4/6 inhibitor on LUSC identified high Myc expression as a potential marker of sensitivity, which indicated that palbociclib have value in the treatment of human LUSC malignancies that over-express Myc.
Our work demonstrated that palbociclib promoted apoptosis in LUSC cell lines, and might be an effective alternative therapeutic strategy for LUSC patients. Specifically, we disclosed for the first time that palbociclib induced cell apoptosis via inhibition of Src/STAT3 signaling but independent on canonical CDK4/6-Rb signaling. Furthermore, blockage of IL-1β or IL-6 mediated palbociclib-inhibited Src/STAT3 signaling. Moreover, Myc can regulate LUSC cells apoptosis in response to palbociclib. Taken together, palbociclib can exert its anti-LUSC activities beyond simply enforcing cytostatic growth arrest, which might be useful in the treatment of human malignant progression of LUSC that over-express STAT3.