Long non-coding RNA GAS5 sensitizes renal cell carcinoma to sorafenib via miR-21/SOX5 pathway

Although the use of sorafenib appears to increase the survival rate of renal cell carcinoma (RCC) patients, there is also a proportion of patients who exhibit a poor primary response to sorafenib treatment. Therefore, it is critical to elucidate the mechanisms underlying sorafenib resistance and find representative biomarkers for sorafenib treatment in RCC patients. Herein, we identified that a long noncoding RNA GAS5 was downregulated in sorafenib nonresponsive RCCs. GAS5 overexpression conferred sorafenib sensitive to nonresponsive RCC cells, whereas knockdown of GAS5 promoted responsive RCC cells resistant to sorafenib treatment in vitro and in vivo. Mechanistically, GAS5 functioned as competing endogenous RNA to repress miR-21, which controlled its down-stream target SOX5. We proposed that GAS5 was responsible for sorafenib resistance in RCC cells and GAS5 exerted its function through the miR-21/ SOX5 axis. Our findings suggested that GAS5 downregulation may be a new marker of poor response to sorafenib and GAS5 could be a potential therapeutic target for sorafenib treatment in RCC.     

GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines

Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.     

Chelerythrine induces apoptosis via ROS‐mediated endoplasmic reticulum stress and STAT3 pathways in human renal cell carcinoma

Renal cell carcinoma (RCC) is a heterogeneous histological disease and it is one of the most common kidney cancer. The treatment of RCC has been improved for the past few years, but its mortality still remains high. Chelerythrine (CHE) is a natural benzo[c]phenanthridine alkaloid and a widely used broad‐range protein kinase C inhibitor which has anti‐cancer effect on various types of human cancer cells. However, its effect on RCC has not been fully elucidated. In this study, we evaluated the effect and mechanism of CHE on RCC cells. Our study showed that CHE induced colony formation inhibition and G2/M cell cycle arrest in a dose‐dependent manner in RCC cells. In addition, CHE increased cellular ROS level, leading to endoplasmic reticulum (ER) stress, inactivating STAT3 activities and inducing apoptosis in RCC cells which were suppressed by NAC, a special ROS inhibitor. We further found that both knockdown of ATF4 protein and overexpression of STAT3 protein could reduce CHE‐induced apoptosis in Caki cells. These results demonstrated that the apoptosis induced by CHE was mediated by ROS‐caused ER stress and STAT3 inactivation. Collectively, our studies provided support for CHE as a potential new therapeutic agent for the management of RCC.     

Specific Inhibition of the Nuclear Exporter Exportin-1 Attenuates Kidney Cancer Growth

Purpose

Despite the advent of FDA-approved therapeutics to a limited number of available targets (kinases and mTOR), PFS of kidney cancer (RCC) has been extended only one to two years due to the development of drug resistance. Here, we evaluate a novel therapeutic for RCC which targets the exportin-1 (XPO1) inhibitor.

Materials and Methods

RCC cells were treated with the orally available XPO1 inhibitor, KPT-330, and cell viability and Annexin V (apoptosis) assays, and cell cycle analyses were performed to evaluate the efficacy of KPT-330 in two RCC cell lines. Immunoblotting and immunofluorescence analysis were performed to validate mechanisms of XPO1 inhibition. The efficacy and on-target effects of KPT-330 were further analyzed in vivo in RCC xenograft mice, and KPT-330-resistant cells were established to evaluate potential mechanisms of KPT-330 resistance.

Results

KPT-330 attenuated RCC viability through growth inhibition and apoptosis induction both in vitro and in vivo, a process in which increased nuclear localization of p21 by XPO1 inhibition played a major role. In addition, KPT-330 resistant cells remained sensitive to the currently approved for RCC multi-kinase inhibitors (sunitinib, sorafenib) and mTOR inhibitors (everolimus, temsirolimus), suggesting that these targeted therapeutics would remain useful as second line therapeutics following KPT-330 treatment.

Conclusion

The orally-available XPO1 inhibitor, KPT-330, represents a novel target for RCC whose in vivo efficacy approaches that of sunitinib. In addition, cells resistant to KPT-330 retain their ability to respond to available RCC therapeutics suggesting a novel approach for treatment in KPT-330-naïve as well as -resistant RCC patients.     

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.     

Alteration in the Balance of Prosurvival and Proapoptotic Signalling Pathways Leads to Sequence-Dependent Synergism Between Docetaxel and Sorafenib in Human Non-small Cell Lung Cancer Cell Lines

To examine the antiproliferative effect of the combination of docetaxel and sorafenib, applied to the representative non-small cell lung cancer cell line A549 cells either wild type or with acquired resistance to docetaxel (A549/D). The aim of this study is to evaluate the synergistic effect of combination treatment on cell growth inhibition and to elucidate the involved molecular mechanisms. A549 cells with acquired resistance to docetaxel were established by continuous exposure to docetaxel. We examined the effect of different combinatorial treatment on cell proliferation and cell cycle distribution. In addition, the effect of combinatorial treatments on proliferative and apoptotic signalling pathway were studied. Our results showed that the synergistic effect presented when A549 cells were treated with docetaxel followed by sorafenib or when A549/D cells were treated in reverse sequence. Furthermore, we suggested that synergistic effect in A549/D cells was caused by inhibiting P-gp function and altering in the balance of growth and apoptotic signalling pathways. Our data suggested a potential role of sorafenib in chemosensitizing docetaxel-resistant cancer cells. This study also provides molecular evidence for applying different therapeutic strategies for patients with different genetic and proteomic profile.     

Inhibition of Autophagy by Chloroquine Enhances the Antitumor Efficacy of Sorafenib in Glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive and common brain tumor in adults. Sorafenib, a multi-kinase inhibitor, has been shown to inhibit cell proliferation and induce apoptosis through inhibition of STAT3 signaling in glioblastoma cells and in intracranial gliomas. However, sorafenib also induces cell autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the therapeutic effect of sorafenib on glioblastoma is uncertain. Here, we combined sorafenib treatment in GBM cells (U373 and LN229) and tumors with the autophagy inhibitor chloroquine. We found that blockage of autophagy further inhibited cell proliferation and migration and induced cell apoptosis in vitro and in vivo. These findings suggest the possibility of combination treatment with sorafenib and autophagy inhibitors for GBM.     

TOPK/PBK is phosphorylated by ERK2 at serine 32, promotes tumorigenesis and is involved in sorafenib resistance in RCC

TOPK/PBK (T-LAK Cell-Originated Protein Kinase) is a serine/threonine kinase that is highly expressed in a variety of human tumors and is associated with poor prognosis in many types of human malignancies. Its activation mechanism is not yet fully understood. A bidirectional signal transduced between TOPK and ERK2 (extracellular signal-regulated kinase 2) has been reported, with ERK2 able to phosphorylate TOPK at the Thr9 residue. However, mutated TOPK at Thr9 cannot repress cellular transformation. In the present study, Ser32 was revealed to be a novel phosphorylated site on TOPK that could be activated by ERK2. Phospho-TOPK (S32) was found to be involved in the resistance of renal cell carcinoma (RCC) to sorafenib. Herein, combined a TOPK inhibitor with sorafenib could promoted the apoptosis of sorafenib-resistant RCC. High expression of HGF/c-met contributes to activation of p-TOPK (S32) during the development of sorafenib resistance in RCC. The current research presents a possible mechanism of sorafenib resistance in RCC and identifies a potential diagnostic marker for predicting sorafenib resistance in RCC, providing a valuable supplement for the clinically targeted treatment of advanced RCC.Subject terms: Predictive markers, Renal cancer     

TNF-α Induces Epithelial-Mesenchymal Transition of Renal Cell Carcinoma Cells via a GSK3β-Dependent Mechanism

TNF-α is a cytokine with antitumorigenic property. In contrast, low dose, chronic TNF-α production by tumor cells or stromal cells may promote tumor growth and metastasis. Serum levels of TNF-α are significantly elevated in renal cell carcinoma (RCC) patients. Here, we showed that TNF-α induced epithelial-mesenchymal transition (EMT) and promoted tumorigenicity of RCC by repressing E-cadherin, upregulating vimentin, activating MMP9, and invasion activities. In addition, TNF-α treatment inhibited glycogen synthase kinase 3β (GSK-3β) activity through serine-9 phosphorylation mediated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway in RCC cells. Inhibition of PI3K/AKT by LY294002 reactivated GSK-3β and suppressed the TNF-α-induced EMT of RCC cells. Inactivation of GSK-3β by LiCl significantly increased MMP9 activity and EMT of RCC cells. Activation of GSK-3β by transduction of constitutively active GSK-3β into RCC cells suppressed TNF-α-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient GSK-3β, in contrast, potentiated EMT, anchorage-independent growth and drastically enhanced tumorigenicity in vivo. Most importantly, a 15-fold inactivation of GSK-3β activity, 3-fold decrease of E-cadherin, and 2-fold increase of vimentin were observed in human RCC tumor tissues. These results indicated that inactivation of GSK-3β plays a pivotal role in the TNF-α-mediated tumorigenesis of RCC. Mol Cancer Res; 10(8); 1109-19. ?2012 AACR.     

Suppression of Extensive Neurofilament Phosphorylation Rescues α-Internexin/Peripherin-Overexpressing PC12 Cells from Neuronal Cell Death

Intermediate filament (IF) overproduction induces abnormal accumulation of neuronal IF, which is a pathological indicator of some neurodegenerative disorders. In our study, α-Internexin- and peripherin-overexpressing PC12 cells (pINT-EGFP and pEGFP-peripherin) were used as models to study neuropathological pathways responsible for neurodegenerative diseases. Microarray data revealed that Cdk5-related genes were downregulated and Cdk5 regulatory subunit-associated protein 3 (GSK-3α and GSK-3β) were upregulated in pINT-EGFP cells. Increased expression of phosphorylated neurofilament and aberrant activation of Cdk5 and GSK-3β were detected in both pEGFP-peripherin and pINT-EGFP cells by Western blotting. In addition, pharmacological approaches to retaining viability of pINT-EGFP and pEGFP-peripherin cells were examined. Treatment with Cdk5 inhibitor and GSK-3β inhibitor significantly suppressed neuronal death. Dynamic changes of disaggregation of EGFP-peripherin and decrease in green fluorescence intensity were observed in pEGFP-peripherin and pINT-EGFP cells by confocal microscopy after GSK-3β inhibitor treatment. We conclude that inhibition of Cdk5 and GSK-3β suppresses neurofilament phosphorylation, slows down the accumulation of neuronal IF in the cytoplasm, and subsequently avoids damages to cell organelles. The results suggest that suppression of extensive neurofilament phosphorylation may be a potential strategy for ameliorating neuron death. The suppression of hyperphosphorylation of neuronal cytoskeletons with kinase inhibitors could be one of potential therapeutic treatments for neurodegenerative diseases.     

BCRP/ABCG2 Inhibition Sensitizes Hepatocellular Carcinoma Cells to Sorafenib

The multikinase inhibitor, sorafenib (Nexavar®, BAY43-9006), which inhibits both the Raf/MEK/ERK pathway and several receptor tyrosine kinases (RTKs), has shown significantly therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond to sorafenib well and new therapeutic strategies to optimize the efficacy of sorafenib are urgently required. Overexpression of breast cancer resistance protein (BCRP/ABCG2) mediates the drug-efflux of several tyrosine kinase inhibitors (TKIs) to attenuate their efficacy. This study aimed to investigate the role of BCRP/ABCG2 in the sensitivity of HCC to sorafenib. Our data showed that BCRP/ABCG2 mediated the efflux of sorafenib. Co-treatment with a BCRP/ABCG2 inhibitor greatly augmented the cytotoxicity of sorafenib in HCC cells. Similar results were also achieved by the competitive inhibitor of BCRP/ABCG2, gefitinib, in combination with sorafenib. These results suggest not only that BCRP/ABCG2 is a potential predictor for the sorafenib sensitivity in HCC, but also that blockage of BCRP/ABCG2 may be a potential strategy to increase the response of HCC cells to sorafenib.     

Sensitization of glioma cells to tamoxifen-induced apoptosis by Pl3-kinase inhibitor through the GSK-3β/β-catenin signaling pathway

Malignant gliomas represent one of the most aggressive types of cancers and their recurrence is closely linked to acquired therapeutic resistance. A combination of chemotherapy is considered a promising therapeutic model in overcoming therapeutic resistance and enhancing treatment efficacy. Herein, we show by colony formation, Hochest 33342 and TUNEL staining, as well as by flow cytometric analysis, that LY294002, a specific phosphatidylinositide-3-kinase (PI3K) inhibitor, enhanced significantly the sensitization of a traditional cytotoxic chemotherapeutic agent, tamoxifen-induced apoptosis in C6 glioma cells. Activation of PI3K signaling pathway by IGF-1 protected U251 cells from apoptosis induced by combination treatment of LY294002 and tamoxifen. Interference of PI3K signaling pathway by PI3K subunit P85 siRNA enhanced the sensitization of U251 glioma cells to tamoxifen -induced apoptosis. By Western blotting, we found that combination treatment showed lower levels of phosphorylated Akt(Ser473) and GSK-3β(Ser9) than a single treatment of LY294002. Further, we showed a significant decrease of nuclear β-catenin by combination treatment. In response to the inhibition of β-catenin signaling, mRNA and protein levels of Survivin and the other three antiapoptotic genes Bcl-2, Bcl-xL, and Mcl-1 were significantly decreased by combination treatment. Our results indicated that the synergistic cytotoxic effect of LY294002 and tamoxifen is achieved by the inhibition of GSK-3β/β-catenin signaling pathway.     

Inhibition of pMAPK14 Overcomes Resistance to Sorafenib in Hepatoma Cells with Hepatitis B Virus

Hepatitis B virus (HBV) targets the liver and is a major driver for liver cancer. Clinical data suggest that HBV infection is associated with reduced response to treatment with the multi-kinase inhibitor sorafenib, the first available molecularly targeted anti-hepatocellular carcinoma (HCC) drug. Given that Raf is one of the major targets of sorafenib, we investigated the activation state of the Raf-Mek-Erk pathway in the presence of HBV and in response to sorafenib. Here we show that hepatoma cells with replicating HBV are less susceptible to sorafenib inhibitory effect as compared to cells in which HBV expression is suppressed. However, although HBV replication is associated with increased level of pErk, its blockade only modestly augments sorafenib effect. In contrast, the phosphorylated form of the pro-oncogenic Mitogen-Activated Protein Kinase 14 (pMAPK14), a protein kinase that was recently linked to sorafenib resistance, is induced in sorafenib-treated hepatoma cells in association with HBV X protein expression. Knocking down pMAPK14 results in augmentation of the therapeutic efficacy of sorafenib and largely alleviates resistance to sorafenib in the presence of HBV. Thus, this study suggests that HBV promotes HCC resistance to sorafenib. Combining pMAPK14 inhibitors with sorafenib may be beneficial in patients with HBV-associated HCC.     

The role of glycogen synthase kinase-3β in glioma cell apoptosis induced by remifentanil

The aim of malignant glioma treatment is to inhibit tumor cell proliferation and induce tumor cell apoptosis. Remifentanil is a clinical anesthetic drug that can activate the N-methyl-D-aspartate (NMDA) receptor. NMDA receptor signaling activates glycogen synthase kinase-3β (GSK-3β). Discovered some 32 years ago, GSK-3β was only recently considered as a therapeutic target in cancer treatment. The purpose of this study was to assess whether remifentanil can induce the apoptosis of C6 cells through GSK-3β activation. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was used to detect cell viability. Hoechst 33342 staining and flow cytometry were used to detect cell apoptosis. The effect of GSK-3β activation was detected using a GSK-3β activation assay kit and 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a potent and selective small molecule inhibitor of GSK-3β. The MTT assay indicated that remifentanil induced C6 cell death in a concentration- and time-dependent manner. Hoechst 33342 staining and flow cytometry showed that remifentanil significantly induced C6 cell apoptosis. The measurement of GSK-3β activation showed that remifentanil increased the cellular level of GSK-3β. All of these toxic effects can be attenuated by treatment with TDZD-8. These results suggest that remifentanil is able to induce C6 cell apoptosis through GSK-3β activation, which provides a basis for its potential use in the treatment of malignant gliomas.     

Glycogen synthase kinase-3 inhibition sensitizes pancreatic cancer cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines with little or no effect on normal cells. However, its effect is limited as some cancers including pancreatic cancer show de novo resistance to TRAIL induced apoptosis. In this study we report that GSK-3 inhibition using the pharmacologic agent AR-18, enhanced TRAIL sensitivity in a range of pancreatic and prostate cancer cell lines. This sensitization was found to be caspase-dependent, and both pharmacological and genetic knock-down of GSK-3 isoforms resulted in apoptotic features as shown by cleavage of PARP and caspase-3. Elevated levels of reactive oxygen intermediates and disturbance of mitochondrial membrane potential point to a mitochondrial amplification loop for TRAIL-induced apoptosis after GSK-3 inhibition. Consistent with this, overexpression of anti-apoptotic mitochondrial targets such as Bcl-XL, Mcl-1, and Bcl-2 rescued PANC-1 and PPC-1 cells from TRAIL sensitization. However, overexpression of the caspase-8 inhibitor CrmA also inhibited the sensitizing effects of GSK-3 inhibitor, suggesting an additional role for GSK-3 that inhibits death receptor signaling. Acute treatment of mice bearing PANC-1 xenografts with a combination of AR-18 and TRAIL also resulted in a significant increase in apoptosis, as measured by caspase-3 cleavage. Sensitization to TRAIL occurred despite an increase in β-catenin due to GSK-3 inhibition, suggesting that the approach might be effective even in cancers with dysregulated β-catenin. These results suggest that GSK-3 inhibitors might be effectively combined with TRAIL for the treatment of pancreatic cancer.     

Direct GSK-3β Inhibition Enhances Mesenchymal Stromal Cell Migration by Increasing Expression of Beta-PIX and CXCR4

Mesenchymal stromal cells (MSCs) are emerging as candidate cells for the treatment of neurological diseases because of their neural replacement, neuroprotective, and neurotrophic effects. However, the majority of MSCs transplanted by various routes fail to reach the site of injury, and they have demonstrated only minimal therapeutic benefit in clinical trials. Therefore, enhancing the migration of MSCs to target sites is essential for this therapeutic strategy to be effective. In this study, we assessed whether inhibition of glycogen synthase kinase-3β (GSK-3β) increases the migration capacity of MSCs during ex vivo expansion. Human bone marrow MSCs (hBM-MSCs) were cultured with various GSK-3β inhibitors (LiCl, SB-415286, and AR-A014418). Using a migration assay kit, we found that the motility of hBM-MSCs was significantly enhanced by GSK-3β inhibition. Western blot analysis revealed increased levels of migration-related signaling proteins such as phospho-GSK-3β, β-catenin, phospho-c-Raf, phospho-extracellular signal-regulated kinase (ERK), phospho-β-PAK-interacting exchange factor (PIX), and CXC chemokine receptor 4 (CXCR4). In addition, real-time polymerase chain reaction demonstrated increased expression of matrix metalloproteinase-2 (MMP-2), membrane-type MMP-1 (MT1-MMP), and β-PIX. In the reverse approach, treatment with β-PIX shRNA or CXCR4 inhibitor (AMD 3100) reduced hBM-MSC migration. These findings suggest that inhibition of GSK-3β during ex vivo expansion of hBM-MSCs may enhance their migration capacity by increasing expression of β-catenin, phospho-c-Raf, phospho-ERK, and β-PIX and the subsequent up-regulation of CXCR4. Enhancing the migration capacity of hBM-MSCs by treating these cells with GSK-3β inhibitors may increase their therapeutic potential.     

Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation

The in vivo regulation of hematopoietic stem cell (HSC) function is poorly understood. Here, we show that hematopoietic repopulation can be augmented by administration of a glycogen synthase kinase-3 (GSK-3) inhibitor to recipient mice transplanted with mouse or human HSCs. GSK-3 inhibitor treatment improved neutrophil and megakaryocyte recovery, recipient survival and resulted in enhanced sustained long-term repopulation. The output of primitive Lin(-)c-Kit(+)Sca-1(+) cells and progenitors from HSCs increased upon GSK-3 inhibitor treatment without altering secondary repopulating ability, suggesting that the HSC pool is maintained while overall hematopoietic reconstitution is increased. GSK-3 inhibitors were found to modulate gene targets of Wnt, Hedgehog and Notch pathways in cells comprising the primitive hematopoietic compartment without affecting mature cells. Our study establishes GSK-3 as a specific in vivo modulator of HSC activity, and suggests that administration of GSK-3 inhibitors may provide a clinical means to directly enhance the repopulating capacity of transplanted HSCs.     

Sorafenib-induced mitochondrial complex I inactivation and cell death in human neuroblastoma cells

Sorafenib is a multikinase inhibitor that is approved for use against renal cell and hepatocellular carcinoma. We found that sorafenib potently induced cell death in human neuroblastoma cells. To understand the molecular basis of sorafenib-mediated cell death in human SH-SY5Y cells, we performed a temporal quantitative proteome analysis. The results showed significant quantitative changes of 193 unique proteins. Bioinformatics-assisted pathway analysis of the regulated proteins revealed that mitochondrial proteins, especially components of the electron transport chain and the mitochondrial ribosomes, were significantly affected upon exposure to sorafenib. The observed down-regulation of the respiratory chain complex I (NADH dehydrogenase) was accompanied with loss of mitochondrial transmembrane potential (Δψm) and complete impairment of complex I enzyme activity. The destabilization of complex I subunits was consistent, rapid, and independent of caspase activation as well as Bcl-2 overexpression. This study provides an overview of the molecular machinery driving sorafenib-mediated cell death in neuroblastoma cells and suggests that sorafenib could be a potential therapeutic drug for the treatment of neuroblastoma.     

Targeting EZH2 as a novel therapeutic strategy for sorafenib‐resistant thyroid carcinoma

Thyroid carcinoma is the most common endocrine malignancy. Surgery, post‐operative selective iodine‐131 and thyroid hormone suppression were the most common methods for the therapy of thyroid carcinoma. Although most patients with differentiated thyroid carcinoma (DTC) showed positive response for these therapeutic methods, some patients still have to face the radioactive iodine (RAI)‐refractory problems. Sorafenib is an oral multikinase inhibitor for patients with advanced RAI refractory DTC. However, the side effects and drug resistance of sorafenib suggest us to develop novel drugs and strategies for the therapy of thyroid carcinoma. In this study, we firstly found that patients with sorafenib resistance showed no significant change in rapidly accelerated fibrosarcoma and VEGFR expression levels compared with sorafenib sensitive patients. Moreover, a further miRNAs screen by qRT‐PCR indicated that miR‐124‐3p and miR‐506‐3p (miR‐124/506) were remarkably reduced in sorafenib insensitive patients. With a bioinformatics prediction and functional assay validation, we revealed that enhancer of zeste homolog 2 (EZH2) was the direct target for miR‐124/506. Interestingly, we finally proved that the sorafenib resistant cells regained sensitivity for sorafenib by EZH2 intervention with miR‐124/506 overexpression or EZH2 inhibitor treatment in vitro and in vivo, which will lead to the decreased tri‐methylation at lysine 27 of histone H3 (H3K27me3) and increased acetylated lysine 27 of histone H3 (H3K27ac) levels. Therefore, we conclude that the suppression of EZH2 represents a potential target for thyroid carcinoma therapy.