SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

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.[1] Clear cell RCC (ccRCC) is the major histologic subtype (about 80–90%).[2] Approximately 25–30% of RCC patients are diagnosed due to symptoms associated with metastatic disease.[3]

Despite partial and radical nephrectomy offering gold-standard treatments to cure localized ccRCC patients, 20–40% of them eventually relapse after surgery.[4] mRCC, characterized by poor prognosis, is highly resistant to chemotherapy and radiotherapy.[5] 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.[9] 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.[16]

Cellular senescence, characterized by irreversible growth arrest, emerging as an intrinsic tumor suppressive mechanism.[17] Rb and p53 pathways dominate the senescence program in response to oncogene activation, replicative limitation, and anticancer therapeutics.[18] 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.[23] Senescent cells exhibit several distinctive features, including a large and flat morphology and an increase in senescence-associated β-galactosidase (SA-β-gal) activity.[24] 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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Cell lines and human samples

Human RCC cell lines ACHN, 786-O, OS-RC-2, and murine RCC cell line Renca characterized by DNA fingerprinting analysis using STR (short tandem repeat) markers were obtained from Shanghai Cell Bank (Shanghai, China) and cultured in DMEM or RPMI 1640 supplemented with 10% FBS at 37°C in a humidified 5% CO2 incubator. Relevant information of human RCC tissues is described in detail in the Supporting Information.

Plasmids construction

The plasmids containing Ikkβ (pcDNA3.1-Ikkβ), p53 luciferase reporter (pGL3-p53) and nuclear factor (NF)-κB luciferase reporter (pGL3-NF-κB) were generated as previously described.[29, 30] The constitutive activation mutant Raf-1 (Δ1-306, Y340/341D) was constructed on the basis of the aforementioned Raf-1 expression plasmid.[31] All plasmid constructs were confirmed by DNA sequencing.

Plasmid transfection and RNA interference

Transient and stable transfections with various plasmids and corresponding empty vectors as control were performed as before,[23] and described in detail in the Supporting Information.[32] Two siRNAs against DEC1 gene Dec1 siRNA (h), corresponding control siRNA-A, one shRNA againstDEC1 gene Dec1shRNA (h) and control shRNA (h) lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for RNA interference as described previously.[33] The gene silencing effect was confirmed by Western blot and RT-PCR at 72 h post-transfection.

Western blot, qRT-PCR and ELISA

Western blot, qRT-PCR and ELISA were carried out as previously described.[20, 34] Primary antibodies used in western blot included those against p-p53 Ser15, p53, Dec1, Raf1, Ikkβ, GAPDH (Santa Cruz Biotechnology), and p21, p16 (BD Pharmingen, San Diego, CA, USA). Primer sequences used in qRT-PCR were listed in Table S1. Enzyme linked immunosorbent assay was performed with Human IL-1α, IL-6, and IL-8 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

SA-β-gal, immunofluorescence, flow cytometry analysis, cell proliferation, bromodeoxyuridine incorporation, colony formation assay, cell-cycle analysis, TUNEL, and luciferase activity assay

All abovementioned experiments were performed as before,[35, 36] and described in detail in the Supporting Information.

Tumor xenograft experiments and sunitinib (SU11248) treatment

Tumor xenograft experiments in nude mice were carried out as previously described[36] and are described in detail in the Supporting Information.

Immunohistochemistry

Tumor sections from subcutaneous tumor xenografted nude mice and patients with RCC were immunohistochemically analyzed as described previously.[36] Primary antibodies used here included those against p53, Dec1, Raf-1 (Santa Cruz Biotechnology) and Ki67 (Dako, Carpinteria, CA, USA).

Statistical analysis

Experimental data were presented as mean ± SD or SEM of at least three independent replicates analyzed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) and comparisons between different groups were assessed by the Student's t-test, one-way anova. Overall survival was defined as the time elapsed from the date of tumor cells injection to the date of death from any cause. Kaplan–Meier survival plots were generated and comparisons were made by using the log-rank sum statistic. Differences were considered significant at values of P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Sunitinib treatment induces cellular senescence markers and proliferation arrest in RCC cells

Despite the focus on achieving complete cure through tumor eradication, increasing information supports an approach that incorporates TIS for cancer therapy. To examine whether this phenomenon occurred in RCC cells following sunitinib intervention, SA-β-gal assay was performed in OS-RC-2 cells and 786-O cells after 48 h exposure to sunitinib. Interestingly, sunitinib treatment increased SA-β-gal activity in a dose-dependent manner in RCC cells (Fig. 1a). Furthermore, sunitinib administration also increased DcR2 expression, a well-known senescence biomarker,[35] in a dose-dependent manner (Fig. S1a). qRT-PCR and ELISA of known SASP cytokines confirmed the transcriptional and secretion levels of IL-1α, IL-6, and IL-8 were upregulated in RCC cells under sunitinib treatment in a dose- and time-dependent manner (Figs 1b,S1b–d). Protein levels of Dec1, another characteristic molecular marker for cellular senescence,[18] was also increased in sunitinib-treated OS-RC-2, ACHN, and Renca cells in a dose- and time-dependent manner (Figs 1c,S1e). Moreover, qRT-PCR showed that sunitinib treatment increased Dec1 transcription of OS-RC-2, ACHN, and Renca cells in a dose-dependent manner (Fig. S1f). Collectively, these results demonstrated that sunitinib treatment induced RCC cellular senescence accompanied with SASP induction, indicating that TIS might be involved in sunitinib therapeutic performance.

image

Figure 1. Sunitinib treatment endows renal cell carcinoma (RCC) cells with senescence phenotype. (a) SA-β-gal assay for OS-RC-2 and 786-O cells after sunitinib (0, 0.625, 1.25, 2.5, 5 μM) treatment for 48 h.*P < 0.05. (b) Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of senescence-associated secretary phenotype (SASP) factors (interleukin [IL]-1α, IL-6 and IL-8) relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for the abovementioned OS-RC-2 and 786-O cells. *P < 0.05. (c) Western bolt analysis of Dec1 relative to GAPDH for OS-RC-2, ACHN and Renca cells after sunitinib (0, 0.625, 1.25, 2.5, 5 μM) treatment for 48 h. *P < 0.05.

Download figure to PowerPoint

To further investigate functional relevance of sunitinib-induced RCC cellular senescence, we examined the effect of sunitinib administration on RCC cell growth. Cell proliferation assay confirmed that long-term exposure of OS-RC-2 and ACHN cells to sunitinib treatment induced significant growth inhibition in a dose-dependent manner (Fig. S2a). Furthermore, a dose-dependent inhibition of BrdU incorporation and colony formation was seen after the addition of sunitinib to both OS-RC-2 and ACHN cells (Fig. S2b,c). Moreover, cell cycle analysis indicated that sunitinib treatment induced G1-S cell cycle arrest in OS-RC-2 and ACHN cells in a dose-dependent manner (Fig. S2d). All of these data reinforced the argument that RCC cell growth inhibition might be involved in sunitinib treatment performance, partially due to sunitinib-induced senescence.

Dec1 dictates sunitinib-induced senescence in RCC cells

To further characterize the molecular mechanism underlying sunitinib-induced senescence, we analyzed two predominant signal pathways (p53 and Rb) arbitrating cellular senescence in RCC cells under sunitinib treatment. Our results excluded Rb signal activation in sunitinib-induced RCC cellular senescence (data not shown). Consistent with previous results that overexpression of Dec1 induced G1 cell cycle arrest and cellular senescence,[37] our aforementioned data showed sunitinib treatment increased Dec1 expression in RCC cells (Fig. 1c), proving the potential role of Dec1 in sunitinib-induced senescence. To testify this hypothesis, functional effects of siRNA- and shRNA-mediated Dec1 knockdown on sunitinib-induced senescence were performed in RCC cells. The efficiency of knockdown using two specific siRNAs on Dec1 expression were confirmed by Western bolt in OS-RC-2 cells (Fig. 2a). Notably, Sunitinib-induced upregulation of Dec1 protein levels could be partially reversed by Dec1 knockdown (Fig. 2b). More importantly, siRNA-mediated Dec1 knockdown caused a marked reduction of SA-β-gal activity under sunitinib treatment in OS-RC-2 cells (Fig. 2c). Meanwhile, flow cytometry analysis of DcR2 (Fig. 2d), qRT-PCR and ELISA of SASP factors (Figs 2e,S3a) showed sunitinib-induced senescence was rescued by siRNA-mediated Dec1 knockdown in OS-RC-2 cells. Moreover, BrdU incorporation assay (Fig. 2f) and cell cycle analysis (Fig. 2g) showed that sunitinib-induced proliferation inhibition and cell cycle arrest were blunted by Dec1 knockdown in OS-RC-2 cells. To further confirm the abovementioned hypothesis contingent with shRNA-mediated stable Dec1 knockdown, functional experiments under sunitinib treatment were carried out in ACHN cells with Dec1 shRNA transfection. ACHN cells with stable Dec1 knockdown showed alleviated Dec1 protein levels under sunitinib administration (Fig. S3b). Consistent with the aforementioned phenomenon in OS-RC-2 cells, SA-β-gal assay (Fig. S3c) and flow cytometry analysis of DcR2 (Fig. S3d) showed that sunitinib-mediated senescence were blunted by stable Dec1 knockdown in ACHN cells. Stable Dec1 knockdown also significantly inhibit BrdU incorporation (Fig. S3e) and colony formation (Fig. S3f) of ACHN cells. Taken together, these results indicate that Dec1 arbitrates sunitinib-induced senescence in RCC cells.

image

Figure 2. Dec1 arbitrates sunitinib-induced senescence in renal cell carcinoma (RCC) cells. (a) Western blot analysis of Dec1 relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for OS-RC-2 cells after transient Dec1 siRNAs transfection. (b) Western bolt analysis of Dec1 relative to GAPDH for OS-RC-2 cells after transient Dec1 siRNA transfection with sunitinib (0, 0.625 μM) treatment for 48 h. (c–d) SA-β-gal assay (c) and flow cytometry analysis of DcR2 (d) for abovementioned OS-RC-2 cells. *P < 0.05. (e, f) Quantitative reverse transcription-polymerase chain reaction(qRT-PCR) analysis of senescence-associated secretary phenotype (SASP) factors (interleukin [IL]-1α, IL-6 and IL-8) relative to GAPDH (e) and BrdU incorporation assay (f) for the abovementioned OS-RC-2 cells.*P < 0.05. (g) Cell cycle analysis for the abovementioned OS-RC-2 cells.

Download figure to PowerPoint

DNA damage response participates in sunitinib-induced senescence

Since DNA damage response constitutes the majority of TIS conferred by anticancer drugs,[18] we investigated potential DNA damage response to further elucidate the molecular mechanisms underlying sunitinib-induced senescence. Flow cytometry analysis of phosphorylated histone variant γ-H2AX, a marker of non-repaired DNA double-strand breaks (DSB),[20] showed that sunitinib treatment induced double-strand DNA breaks in OS-RC-2 cells in a dose-dependent manner (Fig. 3a). In addition, immunofluorescence analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG), a marker of oxidative DNA damage,[38] indicated that oxidative DNA damage participate in sunitinib-induced senescence in OS-RC-2 cells (Fig. 3b). Furthermore, TUNEL assay also implicated that sunitinib treatment increased DNA breaks of OS-RC-2 cells in a dose-dependent manner (Fig. 3c). In sum, these findings demonstrated that DNA damage response might participate in sunitinib-induced RCC cellular senescence.

image

Figure 3. DNA damage response involves in sunitinib-induced senescence. (a) Flow cytometry analysis of γ-H2AX for OS-RC-2 cells after sunitinib (0, 0.625, 1.25, 2.5, 5 μM) treatment for 48 h.*P < 0.05. (b) Immunofluorescence analysis of 8-oxo-dG for the abovementioned OS-RC-2 cells. *P < 0.05. (c) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays for OS-RC-2 cells after sunitinib (0, 0.625, 1.25, 2.5, 5 μM) or doxorubicin (400 ng/mL, as positive control) treatment for 48 h. *P < 0.05.

Download figure to PowerPoint

p53 activation orchestrates Dec1-dependent TIS under sunitinib treatment

To further interlink DNA damage response and TIS in sunitinib-treated RCC cells, we analyzed tumor suppressor p53 activation based on its crucial relevance in correlating DNA damage and cellular senescence partly due to Dec1 or p21 activation.[37] Consistent with previous studies, we could not detect p21 expression in RCC cells with or without sunitinib treatment (data not shown). Western blot analysis showed that sunitinib treatment stimulated p53 expression in a dose- and time-dependent fashion in OS-RC-2, ACHN, and Renca cells (Figs 4a,S4a). Moreover, qRT-PCR analysis confirmed sunitinib-induced p53 expression occurred at the transcription level in a dose-dependent manner (Fig. S4b). To examine the pivotal role of p53 activity in sunitinib-induced senescence, Decl expression and TIS under sunitinib treatment was analyzed based on p53 activity inhibition with two p53 inhibitors including pifithrin-α (PFTα),[39] and pifithrin-μ (PFTμ).[40] Western blot analysis showed that sunitinib-induced p53 and Dec1 accumulation could be reversed by incubation with PFTα and PFTμ in Renca and OS-RC-2 cells (Fig. 4b,c). Furthermore, SA-β-gal assay showed that sunitinib-induced senescence could also be rescued by incubation with PFTα and PFTμ in Renca and OS-RC-2 cells (Fig. 4d,e). These results suggested that p53-Dec1 activation orchestrated sunitinib-induced RCC cellular senescence.

image

Figure 4. p53 activation is essential for Dec1-dependent sunitinib-induced senescence. (a) Western bolt analysis of p53 relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for OS-RC-2, ACHN and Renca cells after sunitinib (0, 0.625, 1.25, 2.5, 5 μM) treatment for 48 h. *P < 0.05. (b, c) Western blot analysis of p53, Dec1 relative to GAPDH for Renca and OS-RC-2 cells after sunitinib (0, 0.625 μM) with or without PFTα (10 μM) (b) or PFTμ (10 μM) (c) treatment. (d, e) SA-β-gal assay for the abovementioned Renca and OS-RC-2 cells.*P < 0.05.

Download figure to PowerPoint

Raf-1/NF-κB inhibition mediates p53 activity in sunitinib-induced senescence

Despite mutation of p53 tumor suppressor gene occurs rarely in RCC patients, aberrant NF-κB signal could completely or partially repress wild-type p53 function during tumorigenesis.[30] We speculated that NF-κB signal might be functionally involved in sunitinib-induced p53 activation. Luciferase activity assay showed that increased p53 promoter transcription activity (Fig. 5a) and decreased NF-κB promoter transcription activity (Fig. 5b) of Renca and OS-RC-2 cells in a dose-dependent manner by sunitinib treatment. More importantly, enhanced NF-κB activation by transiently transfecting with Ikkβ, a subunit of IκB kinase (IKK) essential for NF-κB activation,[29] or stimulating with lipopolysaccharide (LPS) attenuated the sunitinib-increased p53 promoter transcription activity (Figs 5c,S5a) and SA-β-gal activity (Fig. S5b,c). Since tyrosine kinase inhibitors (TKIs) including sunitinib and sorafenib are potent MAPK pathway inhibitors,[9, 41] in which Raf-1 protein kinase stimulate NF-κB activation by dissociating the cytoplasmic IκB-NF-κB complex,[42] we hypothesized that sunitinib treatment increased p53 function via inhibiting Raf-1/NF-κB activation. To verify our hypothesis, luciferase activity assay of p53 and NF-κB promoter transcription activity in Renca and OS-RC-2 cells showed sunitinib-induced NF-κB inhibition and p53 activity could be reversed by constitutive activation mutant Raf-1 (CA-Raf1, Δ1-306,Y340/341D) transfection (Figs 5d,S5d). Western blot analysis of p53 phosphorylation level at the Ser-15, a marker for p53 transcriptional activity during DNA damage or replicative senescence,[43] and p53 target gene Dec1 showed that Ikkβ or CA-Raf1 transfection could repress p53 activity upon sunitinib treatment in Renca and OS-RC-2 cells (Fig. 5e,f). All of these data demonstrated that Raf-1/NF-κB inhibition mediates p53 activity in sunitinib-induced RCC cellular senescence.

image

Figure 5. Sunitinib increases p53 activity dependent on Raf-1/nuclear factor (NF)-κB inhibition. (a, b) Luciferase activity assay for p53 promoter transcription (a) and NF-κB promoter transcription (b) analysis in Renca and OS-RC-2 cells after sunitinib (0, 0.625, 5 μM) for 48 h. *P < 0.05. (c, d) Luciferase activity assay for p53 promoter transcription analysis in Renca and OS-RC-2 cells after sunitinib (0, 0.625 μM) with or without Ikkβ (c) or CA-Raf1 (d) transfection for 48 h. *P < 0.05. (e, f) Western bolt analysis of p53, P-p53 Ser15, Dec1, Ikkβ, Raf1 relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for Renca and OS-RC-2 cells after sunitinib (0, 0.625 μM) with or without Ikkβ (e) or CA-Raf1 (f) transfection for 48 h.

Download figure to PowerPoint

TIS ameliorates RCC tumor formation and disease progression in vivo

To elucidate translational significance of TIS under sunitinib treatment in RCC development and progression, RCC subcutaneous xenograft experiments in nude mice and immunohistochemistry of tumor tissues from the abovementioned mice and RCC patients were performed to analyze tumor formation and disease progression under sunitinib administration. As shown in Figure 6(a–c), sunitinib treatment significantly inhibited tumor formation of OS-RC-2 cells in nude mice in a dose-dependent manner. Survival analysis showed sunitinib treated mice had a significant improvement in life-span dependent on drug dosage (Fig. 6d). Interestingly, immunohistochemistry of tumor tissues from the abovementioned xenografted nude mice showed that sunitinib treatment increased p53, Dec1, and SA-β-gal staining as well as decreased Raf-1 and proliferative marker Ki67 staining depends on drug dosage (Fig. 6e). Consistent with the aforementioned results in nude mice, immunohistochemistry of tumor tissues from RCC patients with sunitinib neoadjuvant therapy confirmed the same treating phenotype (Fig. S6). All of these data indicated that sunitinib treatment performance in vivo could partly be attributed to p53/Dec1-mediated TIS via Raf-1 inhibition.

image

Figure 6. Therapy-induced senescence (TIS) involves in sunitinib treatment performance in vivo. (a–d) Subcutaneous tumors images (a), in vivo subcutaneous tumor growth curves (b, *P < 0.05), total tumor weight (c, *P < 0.05) and overall survival analysis (d, *P = 0.0094) of three groups of nude mice xenografted with OS-RC-2 cells after sunitinib (0, 20 and 40 mg/kg, = 4) treatment for 4 weeks were shown. (e) Immunohistochemistry staining with p53, Dec1, Raf-1 and Ki67 expression and SA-β-gal activity in renal cell carcinoma (RCC) tumor tissues from the abovementioned nude mice.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

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.[10] 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.[11] 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.[45] 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.[18] 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,[46] our data indicated that p53 activity failed to induce p21 expression in sunitinib-induced senescence. Qian et al.[37] 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,[47] 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,[26] 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.[49] 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.[14] 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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

This work was supported by grants from National Key Projects for Infectious Diseases of China (2012ZX10002-012), National Natural Science Foundation of China (31100629, 31270863), and Shanghai Rising-Star Program (13QA1400300).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cas12176-sup-0001-DataS1.docWord document47KData S1. Materials and methods.
cas12176-sup-0002-TableS1.docWord document38KTable S1. Quantitative reverse transcription-polymerase chain reaction(qRT-PCR) primers used in the study.
cas12176-sup-0004-FigS1.tifimage/tif3518KFig. S1. Sunitinib treatment sustains renal cell carcinoma (RCC) cells senescence phenotype.
cas12176-sup-0005-FigS2.tifimage/tif6641KFig. S2. Sunitinib treatment induces cell growth inhibition in renal cell carcinoma (RCC) cells.
cas12176-sup-0006-FigS3.tifimage/tif6469KFig. S3. Dec1 knockdown inhibits sunitinib-induced senescence.
cas12176-sup-0007-FigS4.tifimage/tif1260KFig. S4. p53 is upregulated in renal cell carcinoma (RCC)cells by sunitinib administration.
cas12176-sup-0008-FigS5.tifimage/tif5437KFig. S5. p53 activity is regulated by Raf1/NF-κB pathway.
cas12176-sup-0009-FigS6.tifimage/tif10658KFig. S6. Therapy-induced senescence (TIS) involves in sunitinib treatment performance in sunitinib neoadjuvant patients.
cas12176-sup-0003-FigureLegends.docWord document34K 

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.