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1.  Sorafenib enhances pemetrexed cytotoxicity through an autophagy- dependent mechanism in cancer cells 
Autophagy  2011;7(10):1261-1262.
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
doi:10.4161/auto.7.10.17029
PMCID: PMC3210312  PMID: 21814046
pemetrexed; sorafenib; autophagy; apoptosis; PDGFR; ZMP; AMP; thymidylate synthase
2.  Differential regulation of autophagy and cell viability by ceramide species 
Cancer Biology & Therapy  2015;16(5):733-742.
The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. Combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of early autophagosomes but not autolysosomes, which correlated with increased LC3II processing and increased p62 levels, suggestive of stalled autophagic flux. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing. Knock down of ceramide synthase 6 suppressed autophagosome production and cell killing whereas knock down of ceramide synthase 2 enhanced vesicle formation and facilitated death. Collectively our findings argue that pemetrexed and FTY720 could be a novel adjunct modality for breast cancer treatment.
doi:10.1080/15384047.2015.1026509
PMCID: PMC4623104  PMID: 25803131
FTY720; Gilenya; Pemetrexed; Alimta; ceramide; S1P; autophagy; CerS2; CerS6
3.  Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation 
Cancer biology & therapy  2008;7(10):1648-1662.
We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2α phosphorylation; suppression of eIF2α function abolished drug combination lethality. Cell killing was paralleled by PERK- and eIF2α-dependent lowering of c-FLIP-s protein levels and over-expression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRβ and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Over-expression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2α -↓c-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).
PMCID: PMC2674577  PMID: 18787411
Vorinostat; Sorafenib; CD95; c-FLIP-s; PDGFRβ; FLT3; autophagy; ceramide; cell death; ASMase
4.  Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer 
Molecular Cancer  2014;13:230.
Background
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed, a multi-target folate antagonist, has demonstrated efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS) expression. Among many other potential targets, histone deacetylase inhibitors (HDACi) modulate TS expression, potentially sensitizing to the cytotoxic action of anti-cancer drugs that target the folate pathway, such as pemetrexed. Since high levels of TS have been linked to clinical resistance to pemetrexed in NSCLC, herein we investigated the molecular and functional effects of combined pemetrexed and ITF2357, a pan-HDACi currently in clinical trials as an anti-cancer agent.
Results
In NSCLC cell lines, HDAC inhibition by ITF2357 induced histone and tubulin acetylation and downregulated TS expression at the mRNA and protein level. In combination experiments in vitro ITF2357 and pemetrexed demonstrated sequence-dependent synergistic growth-inhibitory effects, with the sequence pemetrexed followed by ITF2357 inducing a strikingly synergistic reduction in cell viability and induction of both apoptosis and autophagy in all cell line models tested, encompassing both adenocarcinoma and squamous cell carcinoma. Conversely, simultaneous administration of both drugs achieved frankly antagonistic effects, while the sequence of ITF2357 followed by pemetrexed had additive to slightly synergistic growth-inhibitory effects only in certain cell lines. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells (LCSC) exposed to pemetrexed followed by ITF2357. In terms of molecular mechanisms of interaction, the synergistic growth-inhibitory effects observed were only partially related to TS modulation by ITF2357, as genetic silencing of TS expression potentiated growth inhibition by either pemetrexed or ITF2357 and, to a lesser extent, by their sequential combination. Genetic and pharmacological approaches provided an interesting link between the autophagic and apoptotic pathways, and showed that sequential pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival.
Conclusions
Overall, these data provide a strong rationale for the clinical development of sequential schedules employing pemetrexed followed by HDACi in NSCLC.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-230) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-230
PMCID: PMC4198757  PMID: 25301686
HDAC inhibitors; ITF2357; Givinostat; Pemetrexed; Apoptosis; Autophagy; Synergism; NSCLC
5.  Sorafenib activates CD95 and promotes autophagy and cell death via Src family kinases in GI tumor cells 
Molecular cancer therapeutics  2010;9(8):2220-2231.
Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and the present studies have determined individually how sorafenib and vorinostat contribute to CD95 activation. Sorafenib (3-6 μM) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib (3 μM) –induced CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 μM), or sorafenib (3 μM) and vorinostat (500 nM) treatment promoted higher levels of CD95 phosphorylation that correlated with DISC formation, receptor surface localization and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to plasma membrane localize or induce autophagy. Knock down / knock out of Src family kinases abolished sorafenib –induced: CD95 tyrosine phosphorylation; DISC formation; and the induction of cell death and autophagy. Knock down of PDGFRβ enhanced Src Y416 and CD95 tyrosine phosphorylation that correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased ROS levels; and in a delayed NFκB-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95 however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by ~50%. Thus sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation whereas vorinostat contributes to CD95 activation via initial facilitation of ROS generation and subsequently of FAS-L expression.
doi:10.1158/1535-7163.MCT-10-0274
PMCID: PMC2933415  PMID: 20682655
Vorinostat; Sorafenib; CD95; c-FLIP-s; FAS-L; cell death; autophagy
6.  FTY720 (Fingolimod) sensitizes hepatocellular carcinoma cells to sorafenib-mediated cytotoxicity 
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. The multityrosine kinase inhibitor sorafenib is used in the therapy of advanced disease. However, the effects of sorafenib are limited, and combination treatments aiming at improved survival are encouraged. The sphingosine analog FTY720 (Fingolimod), which is approved for treatment of multiple sclerosis, has shown tumor suppressive effects in cell lines and animal models of HCC. In the present study, we combined sorafenib with FTY720 in order to sensitize the HCC cell lines Huh7 and HepG2 to sorafenib treatment. Using the XTT assay we show that noncytotoxic doses of FTY720 synergistically enhanced the decrease in viability caused by treatment of both cell lines with increasing doses of sorafenib. Further studies in Huh7 revealed that combined treatment with FTY720 and sorafenib resulted in G1 arrest and enhanced cell death measured using flow cytometry analysis of cells labeled with propidium iodide (PI)/Annexin-V and PI and 4′,6-diamidino-2-phenylindole-staining of nuclei. In addition, signs of both caspase-dependent and – independent apoptosis were observed, as cotreatment with FTY720 and sorafenib caused cytochrome c release and poly-ADP ribose polymerase-cleavage as well as translocation of Apoptosis-inducing factor into the cytosol. We also detected features of autophagy blockage, as the protein levels of LC3-II and p62 were affected by combined treatment with FTY720 and sorafenib. Together, our results suggest that FTY720 sensitizes HCC cells to cytotoxic effects induced by treatment with sorafenib alone. These findings warrant further investigations of combined treatment with sorafenib and FTY720 in vivo in order to develop more effective treatment of HCC.
doi:10.1002/prp2.171
PMCID: PMC4618642  PMID: 26516583
Cytotoxicity; FTY720; hepatocellular carcinoma; sensitization; sorafenib
7.  MiR-21 mediates sorafenib resistance of hepatocellular carcinoma cells by inhibiting autophagy via the PTEN/Akt pathway 
Oncotarget  2015;6(30):28867-28881.
Sorafenib resistance remains a major obstacle for the effective treatments of hepatocellular carcinoma (HCC). Recent studies indicate that activated Akt contributes to the acquired resistance to sorafenib, and miR-21 dysregulates phosphatase and tensin homolog (PTEN), which inhibits Akt activation. Sorafenib-resistant HCC cells were shown to be refractory to sorafenib-induced growth inhibition and apoptosis. Akt and its downstream factors were highly activated and/or upregulated in sorafenib-resistant cells. Inhibition of autophagy decreased the sensitivity of sorafenib-resistant cells to sorafenib, while its induction had the opposite effect. Differential screening of miRNAs showed higher levels of miR-21 in sorafenib-resistant HCC cells. Exposure of HCC cells to sorafenib led to an increase in miR-21 expression, a decrease in PTEN expression and sequential Akt activation. Transfection of miR-21 mimics in HCC cells restored sorafenib resistance by inhibiting autophagy. Anti-miR-21 oligonucleotides re-sensitized sorafenib-resistant cells by promoting autophagy. Inhibition of miR-21 enhances the efficacy of sorafenib in treating sorafenib-resistant HCC tumors in vivo. We conclude that miR-21 participates in the acquired resistance of sorafenib by suppresing autophagy through the Akt/PTEN pathway. MiR-21 could serve as a therapeutic target for overcoming sorafenib resistance in the treatment of HCC.
PMCID: PMC4745697  PMID: 26311740
sorafenib; hepatocellular carcinoma; miR-21; phosphatase and tensin homolog; Akt; autophagy
8.  Mcl-1-dependent activation of Beclin 1 mediates autophagic cell death induced by sorafenib and SC-59 in hepatocellular carcinoma cells 
Cell Death & Disease  2013;4(2):e485-.
We investigated the molecular mechanisms underlying the effect of sorafenib and SC-59, a novel sorafenib derivative, on hepatocellular carcinoma (HCC). Sorafenib activated autophagy in a dose- and time-dependent manner in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B. Sorafenib downregulated phospho-STAT3 (P-STAT3) and subsequently reduced the expression of myeloid cell leukemia-1 (Mcl-1). Inhibition of Mcl-1 by sorafenib resulted in disruption of the Beclin 1-Mcl-1 complex; however, sorafenib did not affect the amount of Beclin 1, suggesting that sorafenib treatment released Beclin 1 from binding with Mcl-1. Silencing of SHP-1 by small interference RNA (siRNA) reduced the effect of sorafenib on P-STAT3 and autophagy. Ectopic expression of Mcl-1 abolished the effect of sorafenib on autophagy. Knockdown of Beclin 1 by siRNA protected the cells from sorafenib-induced autophagy. Moreover, SC-59, a sorafenib derivative, had a more potent effect on cancer cell viability than sorafenib. SC-59 downregulated P-STAT3 and induced autophagy in all tested HCC cell lines. Furthermore, our in vivo data showed that both sorafenib and SC-59 inhibited tumor growth, downregulated P-STAT3, enhanced the activity of SHP-1 and induced autophagy in PLC5 tumors, suggesting that sorafenib and SC-59 activate autophagy in HCC. In conclusion, sorafenib and SC-59 induce autophagy in HCC through a SHP-1-STAT3-Mcl-1-Beclin 1 pathway.
doi:10.1038/cddis.2013.18
PMCID: PMC3734819  PMID: 23392173
SC-59; sorafenib; STAT3; HCC
9.  The Role of Sorafenib in the Treatment of Advanced Hepatocellular Carcinoma: An Update 
Sorafenib is the first and only orally administered drug currently approved to treat advanced hepatocellular carcinoma (HCC). However, concerns have been raised about sorafenib therapy, including acquired drug resistance. This review provides an overview of sorafenib in the treatment of HCC on the basis of data obtained in the laboratory and in clinical studies. Three underlying mechanisms have been found to support sorafenib therapy. First, sorafenib blocks HCC cell proliferation by inhibiting BRaf and Raf1/c-Raf serine/threonine kinase phosphorylation in the mitogen activated protein kinase (MAPK) pathway. Second, sorafenib induces apoptosis by reducing elF4E phosphorylation and down-regulating Mcl-1 levels in tumor cells. Third, sorafenib prevents tumor-associated angiogenesis by inactivating vascular endothelial growth factor receptors (VEGFR-2 and VEGFR-3) and the platelet-derived growth factor receptor-β (PDGFR-β). Clinical trials have demonstrated the effectiveness and relative safety of sorafenib, and thus the drug is used in un-resectable HCC. However, many patients may develop acquired resistance to sorafenib, so their response to sorafenib is eventually lost. Sorafenib may induce autophagy, which leads to apoptosis. However, autophagy can also cause drug resistance. Many studies have combined sorafenib with other treatments in an effort to increase its effects, reduce the necessary dosage, or overcome resistance. It is urgent to study the mechanisms underlying how sorafenib interacts with cellular molecules and other drugs to increase its efficacy and reduce resistance in HCC patients.
doi:10.1111/j.1872-034X.2012.01113.x
PMCID: PMC3574194  PMID: 23145926
Sorafenib; Liver Neoplasms; Carcinoma; Hepatocellular; raf Kinases; Angiogenesis inhibitors; Drug Resistance
10.  A phase I trial of sorafenib combined with cisplatin/etoposide or carboplatin/pemetrexed in refractory solid tumor patients 
Introduction
Sorafenib has demonstrated single agent activity in non-small cell (NSCLC) and small cell lung cancer (SCLC). Carboplatin/pemetrexed (CbP) and cisplatin/etoposide (PE) are commonly used in the treatment of these diseases.
Methods
A phase I trial escalating doses of sorafenib in combination with fixed doses of PE (Arm A) or CbP (Arm B) was performed using a 3-patient cohort design to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT); DLT were assessed in the first cycle. The trial was subsequently amended with closure of Arm B and to include Arm C with a reduced dose of carboplatin.
Results
Between 9/2007 and 9/2008, 20 pts were treated on the trial; median age 62 (range 42-73), male/female ratio 12/8, PS 0/1 ratio 6/14, and median number of prior therapies 2 (range 1-4). The most common tumor types were NSCLC and SCLC. On Arm A at dose level 0 (sorafenib 200 mg BID), 2 of 4 patients experienced DLT; 2 patients were enrolled at dose level -1 (sorafenib 200 mg QD) without DLT, but this arm was closed due to slow accrual. On Arm B, 2 of 3 patients experienced DLT at dose level 0 (sorafenib 200 mg BID). On Arm C at dose level 0 (sorafenib 200 mg BID), 1 of 6 patients experienced DLT, and at dose level +1 (sorafenib 400 mg BID) 2 of 5 patients experienced a DLT.
Conclusions
The MTD of sorafenib was 200 mg BID continuously in combination with carboplatin (AUC of 5) and pemetrexed 500 mg/m2 every 3 weeks. However, only 6 patients were treated at this dose level, and the results should be interpreted cautiously.
doi:10.1016/j.lungcan.2010.05.022
PMCID: PMC2978774  PMID: 20580118
Sorafenib; non-small cell lung cancer; small cell lung cancer; safety and toxicity; phase I
11.  Sorafenib and pemetrexed toxicity in cancer cells is mediated via SRC-ERK signaling 
Cancer Biology & Therapy  2012;13(9):793-803.
The present studies sought to further understand how the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib interact to kill tumor cells. Sorafenib activated SRC, and via SRC the drug combination activated ERK1/2. Expression of dominant negative SRC or dominant negative MEK1 abolished drug-induced ERK1/2 activation, together with drug-induced autophagy, acidic lysosome formation, and tumor cell killing. Protein phosphatase 2A is an important regulator of the ERK1/2 pathway. Fulvestrant resistant MCF7 cells expressed higher levels of the PP2A inhibitor SET/I2PP2A, had lower endogenous PP2A activity, and had elevated basal ERK1/2 activity compared with their estrogen dependent counterparts. Overexpression of I2PP2A blocked drug-induced activation of ERK1/2 and tumor cell killing. PP2A can be directly activated by ceramide and SET/I2PP2A can be inhibited by ceramide. Inhibition of the de novo ceramide synthase pathway blocked drug-induced ceramide generation, PP2A activation and tumor cell killing. Collectively these findings demonstrate that ERK1/2 plays an essential role downstream of SRC in pemetrexed and sorafenib lethality and that PP2A plays an important role in regulating this process.
doi:10.4161/cbt.20562
PMCID: PMC3679099  PMID: 22673740
ERK; I2PP2A; PP2A; SRC; autophagy; ceramide; pemetrexed; sorafenib
12.  Combined therapeutic efficacy of 188Re-liposomes and sorafenib in an experimental colorectal cancer liver metastasis model by intrasplenic injection of C26-luc murine colon cancer cells 
Molecular and Clinical Oncology  2014;2(3):380-384.
Rhenium-188 (188Re) displays abundant intermediate energy β emission and possesses a physical half-life of 16.9 h. Sorafenib is an orally available multikinase inhibitor that targets Raf kinases and vascular endothelial growth factor receptors (VEGFRs). Sorafenib has demonstrated preclinical and clinical activity against several types of tumors, such as renal cell and colorectal carcinoma. In this study, we investigated the efficacy of radiotherapeutics of 188Re-liposomes combined with sorafenib in a C26-luc metastatic colorectal liver tumour mouse model. Liver metastases were established by intrasplenic injection of C26-luc murine colon cancer cells. Based on the results of the toxicity assessment, an administration dose of 80% the maximum tolerated dose was selected. 188Re-liposomes were administered on day 1, when metastases of several hundred micrometers in diameter were observed. In the combination therapy group, 10 mg/kg sorafenib (co-developed and co-marketed by Bayer and Onyx Pharmaceuticals as Nexavar) was administered every other day for 1 week and the survival of mice was assessed. The tumor growth was more significantly inhibited in the 188Re-liposome plus sorafenib group compared with the 188Re-liposome alone, sorafenib alone and untreated normal saline groups (P=0.0000). Furthermore, 188Re-liposomes combined with sorafenib achieved higher survival rates compared with the 188Re-liposome alone, sorafenib alone and untreated normal saline groups (P=0.0000). These results support the use of combined radio-chemotherapy with 188Re-liposomes plus sorafenib as a viable treatment option in the adjuvant setting for liver metastases of colorectal cancer.
doi:10.3892/mco.2014.246
PMCID: PMC3999117  PMID: 24772304
liver metastasis; liposomes; rhenium-188; colon cancer; radiotherapy
13.  HAND-FOOT SKIN REACTION INCREASES WITH CUMULATIVE SORAFENIB DOSE AND WITH COMBINATION ANTI-VEGF THERAPY 
Purpose
Sorafenib, a vascular endothelial growth factor receptor (VEGFR)-2 and RAF-kinase inhibitor, commonly causes skin toxicity. We retrospectively analyzed dermatologic toxicity in patients receiving combined anti-angiogenic therapy sorafenib and bevacizumab.
Experimental Design
Castration-resistant prostate cancer and metastatic non-small cell lung cancer patients were accrued to phase II studies, receiving sorafenib400mg BID. A phase I study explored sorafenib 200–400mg BID with bevacizumab 5–10mg/kg every 2 weeks in patients with advanced solid tumors. Probability of development of maximum grade of dermatologic toxicity as a function of the cumulative dose of sorafenib was determined. Additional analyses compared extent of toxicity, pharmacokinetics, and patient risk factors.
Results
Ninety-six patients were enrolled: 54 pts received sorafenib, 42 received bevacizumab/sorafenib. HFSR (hand-foot skin reaction) was observed in 50/96(52%) patients. Grade 2–3 HFSR developed in 16/54(30%) sorafenib patients and 24/42(57%) bevacizumab/sorafenib patients (p=0.012) and was associated with cumulative sorafenib exposure (p=0.0008). 24/42 phase I patients randomized to start with bevacizumab had increased risk of grade 2–3 HFSR than those starting with sorafenib (p=0.013) after adjusting for association between HFSR risk and hypertension (p=0.01), which was the only toxicity associated with HFSR. There was no association between HFSR and baseline history of neuropathy, prior taxane/platinum treatment, or systemic sorafenib levels.
Conclusions
Sorafenib-related HFSR is associated with increasing cumulative sorafenib dose. HFSR is increased in patients treated with bevacizumab/sorafenib combination anti-VEGF therapy, and this finding is not explained by pharmacokinetic interaction between the two agents. Our results suggest that the pathophysiology of HFSR may be related to VEGF inhibition.
doi:10.1158/1078-0432.CCR-08-1141
PMCID: PMC2718558  PMID: 19228742
sorafenib; bevacizumab; hand-foot skin reaction; rash; angiogenesis inhibition
14.  Multi-kinase inhibition in ovarian cancer 
Cancer Biology & Therapy  2013;15(1):1-2.
Sorafenib (Nexavar) is a multi-kinase inhibitor that was developed as an inhibitor of RAF-1, in the ERK1/2 pathway, but which was subsequently shown to inhibit class III tyrosine kinase receptors.1 More recently regorafenib (Stivarga) has been developed, which is a further fluorinated version of sorafenib with greater bioavailability and similar inhibitory properties against RAF-1/class III RTKs.2 Some of the anti-tumor effects of sorafenib have been ascribed to anti-angiogenic actions of this agent on endothelial associated kinases such as VEGFR2. Other effects of sorafenib clearly have to be due to its effects on the inherent biology of the tumor cells themselves. For example, through various mechanisms sorafenib has been shown in the laboratory and the clinic to suppress expression of the protective protein MCL-1.3 Sorafenib has also been linked to inhibition of STAT3, NFκB, and activation of the death receptor CD95.4 Sorafenib is routinely dosed daily (400 mg BID) and 7 d after the start of dosing has a Cmax of ~21 μM with a nadir at 12 h of ~10 μM, and is a highly protein bound based on in vitro assays.5 Despite this in vitro binding data sorafenib has profound in vivo effects on tumor cells in renal carcinoma and hepatocellular carcinoma patients; cells which are not per se addicted to high activity oncogene signals that are targets of sorafenib/regorafenib. Thus the precise stable bioavailable level of sorafenib/regorafenib in patient plasma is not known.
doi:10.4161/cbt.26708
PMCID: PMC3938511  PMID: 24309512
sorafenib; ovarian clear cell carcinoma; progression-free survival
15.  Nutlin-3 Enhances Sorafenib Efficacy in Renal Cell Carcinoma 
Molecular carcinogenesis  2011;52(1):39-48.
The renal cell carcinoma (RCC) is one of the top ten cancers in USA. The renal tumors are highly angiogenic and are resistant to conventional interventions, particularly radiotherapy. The advent of multispecific tyrosine kinase inhibitor sorafenib has improved the progression-free survival in RCC, but overall survival in recurrent and metastatic RCC is still a concern that has lead to characterization of combinatorial regimens. Hence, we studied the effect of combination of nutlin-3, an MDM2 inhibitor which increases p53 levels, and sorafenib in RCC. Sorafenib along with nutlin-3 synergistically inhibited the cell survival and enhanced caspase-3 cleavage leading to apoptosis in RCC. Nutlin-3 and sorafenib were more effective in reducing the migration of RCC, in combination than as single agents. Sorafenib and nutlin-3 decreased the phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) and ERK along with inducing p53 activity. The sorafenib and nutlin-3 co-treatment lead to enhanced levels of p53, p-p53 and increase in the levels of p53 pro-apoptotic effector PUMA, Bax and decrease in the anti-apoptotic Bcl-2 levels. Importantly, our studies revealed that sorafenib alone can activate p53 in a concentration dependent manner. Thus, co-treatment of nutlin-3 with sorafenib leads to increased half-life of p53, which in turn can be activated by sorafenib, to induce downstream pro-apoptotic and anti-proliferative effects. This is the first report showing the synergistic effect of sorafenib and nutlin-3 while providing a strong clinical-translational rationale for further testing of sorafenib and nutlin-3 combinatorial regimen in human RCC.
doi:10.1002/mc.20875
PMCID: PMC4153352  PMID: 22006587
Renal Cell Carcinoma; Nutlin-3; Sorafenib; p53; Chemotherapy
16.  SC-2001 Overcomes STAT3-mediated Sorafenib Resistance through RFX-1/SHP-1 Activation in Hepatocellular Carcinoma☆☆☆ 
Neoplasia (New York, N.Y.)  2014;16(7):595-605.
Hepatocellular carcinoma is the fifth most common solid cancer worldwide. Sorafenib, a small multikinase inhibitor, is the only approved therapy for advanced HCC. The clinical benefit of sorafenib is offset by the acquisition of sorafenib resistance. Understanding of the molecular mechanism of STAT3 overexpression in sorafenib resistance is critical if the clinical benefits of this drug are to be improved. In this study, we explored our hypothesis that loss of RFX-1/SHP-1 and further increase of p-STAT3 as a result of sorafenib treatment induces sorafenib resistance as a cytoprotective response effect, thereby, limiting sorafenib sensitivity and efficiency. We found that knockdown of RFX-1 protected HCC cells against sorafenib-induced cell apoptosis and SHP-1 activity was required for the process. SC-2001, a molecule with similar structure to obatoclax, synergistically suppressed tumor growth when used in combination with sorafenib in vitro and overcame sorafenib resistance through up-regulating RFX-1 and SHP-1 resulting in tumor suppression and mediation of dephosphorylation of STAT3. In addition, sustained sorafenib treatment in HCC led to increased p-STAT3 which was a key mediator of sorafenib sensitivity. The combination of SC-2001 and sorafenib strongly inhibited tumor growth in both wild-type and sorafenib-resistant HCC cell bearing xenograft models. These results demonstrate that inactivation of RFX/SHP-1 induced by sustained sorafenib treatment confers sorafenib resistance to HCC through p-STAT3 up-regulation. These effects can be overcome by SC-2001 through RFX-1/SHP-1 dependent p-STAT3 suppression. In conclusion, the use of SC-2001 in combination with sorafenib may constitute a new strategy for HCC therapy.
doi:10.1016/j.neo.2014.06.005
PMCID: PMC4198826  PMID: 25047655
17.  Single-agent therapy with sorafenib or 5-FU is equally effective in human colorectal cancer xenograft—no benefit of combination therapy 
Background
We initiated this preclinical study in order to analyze the impact of sorafenib single treatment versus combination treatment in human colorectal cancer.
Methods
The effect of increasing sorafenib doses on proliferation, apoptosis, migration, and activation of signal cascades was analyzed in vitro. The effect of sorafenib single treatment versus 5-fluorouracil (5-FU) single treatment and combination therapy on in vivo proliferation and target cytokine receptor/ligand expression was analyzed in a human colon cancer xenograft mouse model using HT29 tumor cells.
Results
In vitro, SW480 and HT29 cell lines were sensitive to sorafenib, as compared to Caco2 and SW620 cell lines, independent of the mutation status of K-ras, Raf, PTEN, or PI3K. The effect on migration was marginal, but distinct differences in caspases activation were seen. Combination strategies were beneficial in some settings (sorafenib + 5-FU; irinotecan) and disadvantageous in others (sorafenib + oxaliplatin), depending on the chemotherapeutic drug and cell line chosen. Sensitive cell lines revealed a downregulation of AKT and had a weak expression level of GADD45β. In resistant cell lines, pp53 and GADD45β levels decreased upon sorafenib exposure. In vivo, the combination treatment of sorafenib and 5-FU was equally effective as the respective monotherapy concerning tumor proliferation. Interestingly, treatment with either sorafenib or 5-FU resulted in a significant decrease of VEGFR1 and PDGFRβ expression intensity.
Conclusions
In colorectal cancer, a sensitivity towards sorafenib exists, which seems similarly effective as a 5-FU monotherapy. A combination therapy, in contrast, does not show any additional effect.
doi:10.1007/s00384-012-1551-2
PMCID: PMC3587684  PMID: 22983756
Colorectal cancer; TKI; Sorafenib
18.  Metformin sensitizes sorafenib to inhibit postoperative recurrence and metastasis of hepatocellular carcinoma in orthotopic mouse models 
Background
Sorafenib is recognized as a standard treatment for advanced hepatocellular carcinoma (HCC). However, many patients have to adopt dose reduction or terminate the use of sorafenib because of side effects. In addition, a large number of patients are resistant to sorafenib. Thus, it is essential to investigate the underlying mechanisms of the resistance to sorafenib and seek potential strategy to enhance its efficacy.
Methods
The protein expression of hypoxia-inducible factors (HIF)-2α, 30-kDa HIV Tat-interacting protein (TIP30), E-cadherin, N-cadherin, and pAMPK was detected by Western blot. Cell viability assays were performed to study the influence of metformin and sorafenib on cell proliferation. Annexin V-FITC apoptosis assays were used to detect the influence of metformin and sorafenib on cell apoptosis. The relationship between HIF-2α and TIP30 was studied using gene silencing approach and chromatin immunoprecipitation assay. To investigate the effect of metformin and sorafenib on postoperative recurrence and lung metastasis of HCC in tumor-bearing mice, the mice were orally treated either with metformin or sorafenib once a day for continuous 37 days after the operation to remove the lobe where the tumor was implanted. CD31, Ki67, and TUNEL were examined by immunohistochemistry.
Results
Our study demonstrated that metformin synergized with sorafenib reduced HIF-2α expression as examined by Western blot. Gene silencing approach indicated TIP30 was upregulated after knocking-down of HIF-2α and chromatin immunoprecipitation assay revealed that HIF-2α could bind to TIP30 promoter under hypoxic condition. Cell Counting Kit-8 (CCK8) cell viability assay and Annexin V-FITC apoptosis assay showed that metformin in combination with sorafenib suppressed cell proliferation and promoted cell apoptosis. Besides, combined therapy suppressed epithelial-mesenchymal transition (EMT) process both in vitro and in vivo. Moreover, metformin in combination with sorafenib significantly minimized postoperative recurrence and lung metastasis of HCC in orthotopic mouse model. Combined therapy inhibited CD31 and Ki67 expression but promoted TUNEL expression.
Conclusions
Metformin may potentially enhance the effect of sorafenib to inhibit HCC recurrence and metastasis after liver resection by regulating the expression of HIF-2α and TIP30.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-016-0253-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13045-016-0253-6
PMCID: PMC4784359  PMID: 26957312
Sorafenib; Metformin; Hypoxia-inducible factors; TIP30; Hepatocellular carcinoma
19.  Enhancing mda-7/IL-24 therapy in renal carcinoma cells by inhibiting multiple protective signaling pathways using sorafenib and by Ad.5/3 gene delivery 
Cancer Biology & Therapy  2010;10(12):1290-1305.
We have determined whether an adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, more effectively infects and kills renal carcinoma cells (RCCs) compared to a serotype 5 virus, Ad.5-mda-7. RCCs are a tumor cell type that generally does not express the receptor for the type 5 adenovirus; the coxsakie and adenovirus receptor (CAR). Ad.5/3-mda-7 infected RCCs to a much greater degree than Ad.5-mda-7. MDA-7/IL-24 protein secreted from Ad.5/3-mda-7-infected RCCs induced MDA-7/IL-24 expression and promoted apoptosis in uninfected “bystander” RCCs. MDA-7/IL-24 killed both infected and bystander RCCs via CD95 activation. Knockdown of intracellular MDA-7/IL-24 in uninfected RCCs blocked the lethal effects of conditioned media. Infection of RCC tumors in one flank, with Ad.5/3-mda-7, suppressed growth of infected tumors and reduced the growth rate of uninfected tumors implanted on the opposite flank. The toxicity of the serotype 5/3 recombinant adenovirus to express MDA-7/IL-24 was enhanced by combined molecular or small molecule inhibition of MEK1/2 and PI3K; inhibition of mTOR, PI3K and MEK1/2; or use of the multi-kinase inhibitor sorafenib. In RCCs, combined inhibition of cytoprotective cell signaling pathways enhanced the MDA-7/IL-24-induction of CD95 activation, with greater mitochondrial dysfunction due to loss of MCL-1 and BCL-XL expression and tumor cell death. Treatment of RCC tumors in vivo with sorafenib also enhanced Ad.5/3-mda-7 toxicity and prolonged animal survival. Future combinations of these approaches hold promise for developing a more effective therapy for kidney cancer.
doi:10.4161/cbt.10.12.13497
PMCID: PMC3047088  PMID: 20948318
ERK; JNK; PI3K; AKT; MDA-7/IL-24; sorafenib; PERK; MAPK; interleukin; RCC; kidney
20.  Evaluation of in vivo responses of sorafenib therapy in a preclinical mouse model of PTEN-deficient of prostate cancer 
Background
Despite recent advances in the treatment for advanced prostate cancer, outcomes remain poor. This lack of efficacy has prompted the development of alternative treatment strategies. In the present study we investigate the effects of the multikinase inhibitor sorafenib in a genetically engineered mouse model of prostate cancer and explore the rational combination with the mTOR inhibitor everolimus.
Methods
Conditional prostate specific PTEN-deficient knockout mice were utilized to determine the pharmacodynamic and chemopreventive effects of sorafenib. This mouse model was also used to examine the therapeutic efficacy of sorafenib alone or in combination with everolimus. Preclinical efficacy was assessed by comparing the reduction of tumor burden, proliferation, angiogenesis and the induction of apoptosis. Molecular responses were assessed by immunohistochemical, TUNEL and western blot assays.
Results
Pharmacodynamic analysis revealed that a single dose of sorafenib decreased activation of the PI3K/AKT/mTOR signaling axis at doses of 30–60 mg/kg, but activated JAK/STAT3 signaling. Levels of cleaved casapase-3 increased in a dose dependent manner. Chemoprevention studies showed that chronic sorafenib administration was capable of inhibiting tumor progression through the reduction of cancer cell proliferation, angiogenesis and the induction of apoptosis. In intervention models of established castration-naïve and castration-resistant prostate cancer, treatment with sorafenib provided modest but statistically insignificant reduction in tumor burden. However, sorafenib significantly inhibited cancer cell proliferation and MVD but had minimal effects on the induction of apoptosis. Interestingly, the administration of sorafenib increased the expression levels of the androgen receptor, p-GSK3β and p-ERK1/2 in castration-resistant prostate cancers. In both intervention models, combination therapy demonstrated a clear tendency of enhanced antitumor effects over monotherapy. Notably, the treatment combination of sorafenib and everolimus overcame therapeutic escape from single agent therapy in castration-resistant prostate cancers.
Conclusions
In summary, we provide insights into the molecular responses of sorafenib therapy in a clinically relevant model of prostate cancer and present preclinical evidence for the development of targeted treatment strategies based on the use of multikinase inhibitors in combination with mTOR inhibitors for the treatment of advanced prostate cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0509-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12967-015-0509-x
PMCID: PMC4438623  PMID: 25953027
Prostate cancer; Castration-resistant prostate cancer; PTEN; Combination therapy; Sorafenib; Everolimus; Mouse model; Preclinical
21.  Sorafenib/Regorafenib and Lapatinib interact to kill CNS tumor cells 
Journal of cellular physiology  2015;230(1):131-139.
The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with the ERBB1/ERBB2 inhibitor lapatinib to kill CNS tumor cells. In multiple CNS tumor cell types sorafenib and lapatinib interacted in a greater than additive fashion to cause tumor cell death. Tumor cells lacking PTEN, and anoikis or lapatinib resistant cells were as sensitive to the drug combination as cells expressing PTEN or parental cells, respectively. Similar data were obtained using regorafenib. Treatment of brain cancer cells with [sorafenib + lapatinib] enhanced radiation toxicity. The drug combination increased the numbers of LC3-GFP vesicles; this correlated with a reduction in endogenous LC3II, and p62 and LAMP2 degradation. Knock down of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, BCL-XL or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 or activated mTOR was required to strongly suppress drug combination lethality. As both lapatinib and sorafenib are FDA approved agents, our data argue for further determination as to whether lapatinib and sorafenib is a useful glioblastoma therapy.
doi:10.1002/jcp.24689
PMCID: PMC4182138  PMID: 24911215
Sorafenib; Lapatinib; Autophagy; Glioma; AKT; ERK1/2; mTOR; PTEN; p70 S6K; Necrosis
22.  Rapamycin downregulates thymidylate synthase and potentiates the activity of pemetrexed in non-small cell lung cancer 
Oncotarget  2014;5(4):1062-1070.
Non-small cell lung cancer (NSCLC) accounts for 80–85% of lung cancer cases, and almost half of newly diagnosed patients have metastatic disease. Pemetrexed is a widely used drug for NSCLC and inhibits several folate-dependent enzymes including thymidylate synthase (TS). Increased expression of TS confers resistance to pemetrexed in vitro and predicts poor response to pemetrexed. Rapamycin is an mTOR inhibitor and suppresses cap-dependent synthesis of specific mRNA species. Here, we show that the combination of rapamycin and pemetrexed synergistically inhibits proliferation of NSCLC cells. Although pemetrexed as a single agent induced TS, pretreatment with rapamycin suppressed pemetrexed-induced TS expression. In vivo, the combination of rapamycin and pemetrexed inhibited growth of NSCLC xenografts, which correlated with decreased mTOR activity and suppression of pemetrexed-induced TS expression. The ability of rapamycin to enhance the efficacy of pemetrexed and prevent TS expression has implications for the design of Phase I and/or Phase II NSCLC clinical trials with mTOR inhibitors in combination with pemetrexed.
PMCID: PMC4011583  PMID: 24658085
Rapamycin; Pemetrexed; Drug synergy; mTOR; Thymidylate Synthase; Lung Cancer
23.  Pro-apoptotic Sorafenib signaling in murine hepatocytes depends on malignancy and is associated with PUMA expression in vitro and in vivo 
Cell Death & Disease  2014;5(1):e1030-.
The multi-kinase inhibitor Sorafenib increases the survival of patients with advanced hepatocellular carcinoma (HCC). Current data suggest that Sorafenib inhibits cellular proliferation and angiogenesis and promotes apoptosis. However, the underlying pro-apoptotic molecular mechanisms are incompletely understood. Here we compared the pro-apoptotic and anti-proliferative properties of Sorafenib in murine hepatoma cells and syngeneic healthy hepatocytes in vitro and in animal models of HCC and liver regeneration in vivo. In vitro, we demonstrate that cell cycle activity and expression of anti-apoptotic Bcl-2 like proteins are similarly downregulated by Sorafenib in Hepa1-6 hepatoma cells and in syngeneic primary hepatocytes. However, Sorafenib-mediated activation of caspase-3 and induction of apoptosis were exclusively found in hepatoma cells, but not in matching primary hepatocytes. We validated these findings in vivo by applying an isograft HCC transplantation model and partial hepatectomy (PH) in C57BL/6 mice. Sorafenib treatment activated caspase-3 and thus apoptosis selectively in small tumor foci that originated from implanted Hepa1-6 cells but not in surrounding healthy hepatocytes. Similarly, Sorafenib did not induce apoptosis after PH. However, Sorafenib treatment transiently inhibited cell cycle progression and resulted in mitotic catastrophe and enhanced non-apoptotic liver injury during regeneration. Importantly, Sorafenib-mediated apoptosis in hepatoma cells was associated with the expression of p53-upregulated-modulator-of-apoptosis (PUMA). In contrast, regenerating livers after PH revealed downregulation of PUMA and were completely protected from Sorafenib-mediated apoptosis. We conclude that Sorafenib induces apoptosis selectively in hepatoma cells but not in healthy hepatocytes and can additionally increase non-apoptotic hepatocyte injury in the regenerating liver.
doi:10.1038/cddis.2013.557
PMCID: PMC4040679  PMID: 24481444
hepatocellular carcinoma; syngeneic transplantation model; mitotic catastrophe; liver regeneration; partial hepatectomy
24.  Approval Summary: Pemetrexed Maintenance Therapy of Advanced/Metastatic Nonsquamous, Non-Small Cell Lung Cancer (NSCLC) 
The Oncologist  2010;15(12):1352-1358.
The study that led to U.S. Food and Drug Administration approval of pemetrexed injection for maintenance treatment of patients with locally advanced or metastatic nonsquamous non-small cell lung cancer whose disease has not progressed after four cycles of platinum-based doublet induction chemotherapy is reviewed.
Learning Objectives
After completing this course, the reader will be able to: Consider tumor histology when making treatment decisions for patients with NSCLC.Identify patients with NSCLC who may be appropriate candidates for maintenance therapy with pemetrexed.
This article is available for continuing medical education credit at CME.TheOncologist.com.
On July 2, 2009, the U.S. Food and Drug Administration approved pemetrexed injection (Alimta® Injection; Eli Lilly and Company, Indianapolis, IN) for maintenance treatment of patients with locally advanced or metastatic nonsquamous non-small cell lung cancer whose disease has not progressed after four cycles of platinum-based doublet induction chemotherapy.
A double-blind study of pemetrexed plus best supportive care versus placebo plus best supportive care was conducted. Pemetrexed, 500 mg/m2 i.v., was administered every 21 days until disease progression. Folic acid, vitamin B12, and a corticosteroid were given to all study patients.
There were 663 randomized patients (pemetrexed, 441; placebo, 222). Treatments were well balanced with respect to baseline disease characteristics and stratification factors.
The median overall survival (OS) time for intent-to-treat (ITT) patients was 13.4 months for patients receiving pemetrexed and 10.6 months for those receiving placebo (hazard ratio [HR] 0.79; 95% confidence interval [CI], 0.65–0.95; p = .012). Median OS times were 15.5 months versus 10.3 months for patients with nonsquamous histologies receiving pemetrexed and placebo, respectively (HR, 0.70; 95% CI, 0.56–0.88). The median OS time in patients with squamous histology receiving pemetrexed was 9.9 months, versus 10.8 months for those receiving placebo (HR, 1.07; 95% CI, 0.77–1.50). A significantly longer progression-free survival interval for both the ITT and nonsquamous patient populations receiving pemetrexed maintenance therapy was also observed.
The most common (>5%) adverse reactions in patients receiving pemetrexed were hematologic toxicity, an increase in hepatic enzymes, fatigue, gastrointestinal toxicity, sensory neuropathy, and skin rash.
doi:10.1634/theoncologist.2010-0224
PMCID: PMC3227931  PMID: 21148615
Pemetrexed; Non-small cell lung cancer; Maintenance treatment
25.  Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma 
Cancer immunology, immunotherapy : CII  2012;62(4):10.1007/s00262-012-1380-8.
Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25− effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determineimmune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings showthat sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.
doi:10.1007/s00262-012-1380-8
PMCID: PMC3863727  PMID: 23223899
Sorafenib; T cell; Regulatory T cells; Hepatocellular carcinoma; HCV

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