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Although multitargeted tyrosine kinase inhibitor sunitinib has been used as first-line therapeutic agent against metastatic renal cell carcinoma (mRCC), the molecular mechanism and functional role per se for its therapeutic performance remains obscure. Our present study revealed that sunitinib-treated RCC cells exhibit senescence characteristics including increased SA-β-gal activity, DcR2 and Dec1 expression, and senescence-associated secretary phenotype (SASP) such as proinflammatory cytokines interleukin (IL)-1α, IL-6 and IL-8 secretion. Moreover, sunitinib administration also led to cell growth inhibition, G1-S cell cycle arrest and DNA damage response in RCC cells, suggesting therapeutic significance of sunitinib-induced RCC cellular senescence. Mechanistic investigations indicated that therapy-induced senescence (TIS) following sunitinib treatment mainly attributed to p53/Dec1 signaling activation mediated by Raf-1/NF-κB inhibition in vitro. Importantly, in vivo study showed tumor growth inhibition and prolonged overall survival were associated with increased p53 and Dec1 expression, decreased Raf-1 and Ki67 staining, and upregulated SA-β-gal activity after sunitinib treatment. Immunohistochemistry analysis of tumor tissues from RCC patients receiving sunitinib neoadjuvant therapy confirmed the similar treating phenotype. Taken together, our findings suggested that sunitinib treatment performance could be attributable to TIS, depending on p53/Dec1 activation via inhibited Raf-1/nuclear factor (NF)-κB activity. These data indicated potential insights into therapeutic improvement with reinforcing TIS-related performance or overcoming SASP-induced resistance.
Renal cell carcinoma (RCC) accounts for 2–3% of all malignant diseases in adults, with an incidence of 64 770 new cases and 13 570 deaths in the USA in 2012. Clear cell RCC (ccRCC) is the major histologic subtype (about 80–90%). Approximately 25–30% of RCC patients are diagnosed due to symptoms associated with metastatic disease.
Despite partial and radical nephrectomy offering gold-standard treatments to cure localized ccRCC patients, 20–40% of them eventually relapse after surgery. mRCC, characterized by poor prognosis, is highly resistant to chemotherapy and radiotherapy. Immunotherapy with either interleukin (IL)-2 or interferon (IFN), which was developed into an alternative treatment for mRCC patients with limited overall response rates of 21.0–23.2% to high-dose IL-2 and 11–16% to IFN, respectively, failed to show any significant overall survival benefit along with substantial toxicities.[6-8]
Sunitinib is a multitargeted inhibitor of receptor tyrosine kinases (RTK) known to selectively inhibit several growth factor receptors, including vascular endothelial growth factor (VEGF) receptors (VEGFR1, VEGFR2 and VEGFR3), platelet-derived growth factor (PDGF) receptors (PDGFRα and PDGFRβ), FLT3, KIT, RET and CSF1R. Despite it being used mainly as a default antiangiogenic therapy against mRCC, the bona fide therapeutic target cell and underlying molecular mechanism of sunitinib treatment remain controversial.[10, 11] More recently, sunitinib has been shown to significantly increase progression-free survival and objective response rate compared with interferon in the first-line treatment of patients with mRCC.[12, 13] However, therapeutic response to sunitinib occurs merely in partial mRCC patients, with the vast majority eventually developing acquired therapeutic resistance and disease progression.[14, 15] Along with increasingly widespread sunitinib administration against mRCC, functional significance and molecular mechanism for sunitinib treatment performance as well as acquired therapeutic resistance await urgent elucidation for personalized molecular-targeted therapeutics to fortify efficacy and overcome resistance.
Cellular senescence, characterized by irreversible growth arrest, emerging as an intrinsic tumor suppressive mechanism. Rb and p53 pathways dominate the senescence program in response to oncogene activation, replicative limitation, and anticancer therapeutics. Therapy-induced senescence (TIS) occurs in various cancer cells undergoing genotoxic chemotherapy and radiotherapy, and produces limited toxicity-related side-effects and increased tumor-specific immune activity.[18, 19] Accumulating studies focus on the mechanisms, agents, and biomarkers of TIS in order to find efficient senescence-inducing drugs and other targeted approaches to TIS in the clinical treatment of cancer. As a potential therapeutic goal, TIS might contribute to developing a “pro-senescence” approach for cancer prevention and therapy.[20-22] Experimental evidence also suggests that TIS might function as an alternative course of action to therapy in cancer cells disabled in apoptotic pathways. Senescent cells exhibit several distinctive features, including a large and flat morphology and an increase in senescence-associated β-galactosidase (SA-β-gal) activity. Besides, senescent cells markedly secrete inflammatory cytokines including IL-1α, IL-6 and IL-8 known as senescence-associated secretory phenotype (SASP).[25, 26] Senescence-associated secretory phenotype acts as a double-edged sword in tumorgenesis through oversecreting a variety of cytokines to reinforce senescent phenotype on one hand and creating a gradient to promote cell proliferation, migration and invasion on the other hand.[27, 28]
In this study, we investigated functional relevance and molecular mechanism for TIS in RCC cells following sunitinib treatment. We discovered that sunitinib administration induced cellular senescence as well as SASP secretion of RCC cells dependent on p53/Dec1 activation in vitro and in vivo, illustrating a new potential mechanism for therapeutic performance and acquired resistance in RCC patients receiving sunitinib treatment.
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Molecular-targeted therapies against RCC were developed based on specific molecular pathways attributed to kidney tumorigenesis. As a small-molecule RTK inhibitor, sunitinib treatment performance in RCC patients through suppressing tumor angiogenesis has been well established.[9, 44] Besides, Xin et al. reported that sunitinib induced RCC cell apoptosis and reduced immunosuppressive cells by Stat3 suppression. All of these therapeutic effects of sunitinib need a high concentration above 5 μM far beyond pharmacologically and clinically relevant plasma drug concentration, which ignites our interest in investigating the functional role per se of sunitinib treatment in RCC tumorgenesis.
Although Huang et al. found that the primary action of sunitinib in RCC was through antiangiogenic rather than directly targeted on RCC cells at pharmacologically relevant concentrations, Gotink et al. reported that sunitinib inhibited tumor cell growth at clinically relevant concentrations in vitro, with IC50 values from 1.4 to 2.3 μM. These contradictory findings implicate that functional relevance of sunitinib treatment is still controversial. Our present study explored the pivotal therapeutic effect and potential molecular mechanism for sunitinib administration in RCC cells, suggesting that TIS dependent on p53/Dec1 activation mediated by Raf-1/NF-κB inhibition contributes to sunitinib treatment performance in vitro and in vivo.
Senescent cells occur with active changes in gene expression, that manifest characteristic senescent phenotype. In this study, sunitinib-treated RCC cells exhibit senescent phenotype characterized by increased SA-β-gal activity, DcR2 and Dec1 expression, as well as SASP secretion. Further functional analysis elucidates that RCC cell growth inhibition, cell cycle arrest and DNA damage response are involved in sunitinib administration, indicating the therapeutic relevance of sunitinib-induced RCC cellular senescence. With regarding to the molecular mechanism underlying TIS conferred by sunitinib treatment, our current data demonstrate that sunitinib-induced senescence depends on p53/Dec1 signal activation, which was mediated by Raf-1/NF-κB inhibition.
Consistent with previous study that RCC cells expressing wild-type p53 were deficient in transcriptional activation of target genes including p21Waf1/Cip, our data indicated that p53 activity failed to induce p21 expression in sunitinib-induced senescence. Qian et al. have demonstrated that Dec1 acted as one of the effectors downstream of p53 in DNA damage-induced senescence in human cancer. Our current investigation revealed that TIS under sunitinib administration was mediated by p53/Dec1 activation. Moreover, our results demonstrated that sunitinib treatment stimulated p53/Dec1 throughRaf-1/NF-κB pathway inhibition, suggesting inhibition of Raf-1 kinase activity by sunitinib treatment was involved in sunitinib-induced RCC cellular senescence. Contradictory to previous study indicating NF-κB activity acted as a master regulator of SASP, our current data showed that sunitinib treatment induces SASP accompanied with NF-κB inhibition. Since SASP induction could also be mediated by another crucial transcription factor C/EBPβ, the molecular mechanism of SASP induction under sunitinib treatment will be addressed in detail in our future studies.
In addition to RCC cell growth inhibition mediated by sunitinib treatment in vitro, our current research also showed sunitinib-induced senescence ameliorated RCC tumor formation and disease progression in vivo, indicating that TIS represents a novel functional target that may improve cancer therapy.[18, 48] Renal cell carcinoma is characterized by resistance to chemotherapy and radiotherapy with unknown apoptotic block mechanisms. Jing et al. showed that tumor models with apoptotic block signaling pathways responded to TIS, as an alternative, outcome-improving chemotherapeutic effector mechanism. Our present study suggested that TIS conferred by sunitinib administration might contribute to treatment performance in RCC patients.
Nevertheless, only 40% of mRCC patients show initial positive response to sunitinib treatment, with the majority exhibiting disease progression after 1-year of treatment.[14, 15] Thus, the potential biomarkers predicting response to sunitinib treatment remains an urgent investigation. Whether SASP proinflammatory cytokines presented by our current study could serve as serum markers to measure the response of sunitinib treatment or not merits further exploration. Besides, SASP could also mediate acquired therapeutic resistance to sunitinib indicated by a previous study that IL-8 is an important contributor to sunitinib resistance in ccRCC. Secreted factors from senescent cells may function to reinforce senescent phenotypes and promote immune response to clear senescent cells. Conversely, uncleared senescent cells may have potential to provoke tumorigenesis through the ability to stimulate proliferation of neighboring cells.[25, 28, 48] The therapeutic or vicious role of SASP in sunitinib-induced senescence remains the most interesting problem that awaits further extensive study, which might open a new avenue to reinforce sunitinib treatment performance or overcome acquired therapeutic resistance.