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1.  Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells 
Cancer Biology & Therapy  2013;14(11):1039-1049.
In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca2+. Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda-7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.
doi:10.4161/cbt.26110
PMCID: PMC3925659  PMID: 24025359
MDA-7/IL-24; HDACI; ceramide; apoptosis; bystander; cytokine; ROS; caspase; animal study
2.  OSU-03012 enhances Ad.mda-7-induced GBM cell killing via ER stress and autophagy and by decreasing expression of mitochondrial protective proteins 
Cancer biology & therapy  2010;9(7):526-536.
The present studies focused on determining whether the autophagy-inducing drug OSU-03012 (AR-12) could enhance the toxicity of recombinant adenoviral delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) in glioblastoma multiforme (GBM) cells. The toxicity of a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7) was enhanced by OSU-03012 in a diverse panel of primary human GBM cells. The enhanced toxicity correlated with reduced ERK1/2 phosphorylation and expression of MCL-1 and BCL-XL, and was blocked by molecular activation of ERK1/2 and by inhibition of the intrinsic, but not the extrinsic, apoptosis pathway. Both OSU-03012 and expression of MDA-7/IL-24 increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) that correlated with increased levels of autophagy and expression of dominant negative PERK blocked autophagy induction and tumor cell death. Knockdown of ATG5 or Beclin1 suppressed OSU-03012 enhanced MDA-7/IL-24-induced autophagy and blocked the lethal interaction between the two agents. Ad.mda-7-infected GBM cells secreted MDA-7/IL-24 into the growth media and this conditioned media induced expression of MDA-7/IL-24 in uninfected GBM cells. OSU-03012 interacted with conditioned media to kill GBM cells and knockdown of MDA-7/IL-24 in these cells suppressed tumor cell killing. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.
PMCID: PMC2888700  PMID: 20107314
ROS; caspase; ER stress; CD95; cell death
3.  Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) in combination with the Apogossypol derivative BI-97C1 (Sabutoclax) improves therapeutic efficacy in low CAR colorectal cancer cells 
Journal of Cellular Physiology  2012;227(5):2145-2153.
Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients.
doi:10.1002/jcp.22947
PMCID: PMC3228880  PMID: 21780116
Viral gene therapy; Mcl-1 inhibition; apoptosis induction; anti-tumor activity
4.  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
5.  Melanoma differentiation-associated gene-7, MDA-7/IL-24, selectively induces growth suppression, apoptosis in human hepatocellular carcinoma cell line HepG2 by replication-incompetent adenovirus vector 
AIM: To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma (HCC) cell line HepG2 and normal liver cell line L02.
METHODS: We constructed the recombinant replication-incompetent Ad.mda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the mRNA expressing in cells. by ELISA was used to detect MDA-7/IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.mda-7 was confirmed by Hoechst staining and flow cytometry assay with Annexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry.
RESULTS: Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RT-PCR showed that the Ad.mda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.mda-7-infected L02 and HepG2 cells, respectively. Ad.mda-7 infection obviously induced apoptosis (from 2.60±0.72% to 33.6±13.2%, P  = 0.00012) and growth suppression in HepG2 (inhibition ratio IR = 68%) and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle (from 6.44% to 32.29%, P < 0.01), but not in L02 cells.
CONCLUSION: These results confirm selectively induction of apoptosis and growth suppression by the mda-7/IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.
doi:10.3748/wjg.v12.i11.1774
PMCID: PMC4124357  PMID: 16586551
Cancer gene therapy; Hepatocellular carcinoma (HCC); Apoptosis; Growth suppression; MDA-7/IL-24
6.  Sorafenib attenuates p21 in kidney cancer cells and augments cell death in combination with DNA-damaging chemotherapy 
Cancer Biology & Therapy  2011;12(9):827-836.
There are few effective therapeutic options for metastatic renal cell carcinoma (RCC). Conventional chemotherapeutic agents are ineffective since these tumors are unusually resistant to DNA damage, likely due to an exuberant DNA repair response. Sorafenib, as one of the few available effective therapeutic options for metastatic RCC, has been shown to inhibit cell proliferation by inhibition of tyrosine kinases. We have recently shown that sorafenib inhibits soluble epoxide hydrolase, which catalyzes metabolism of the anti-inflammatory epoxyeicosatrienoic acids. Given previous work demonstrating the anti-apoptotic role of p21 in RCC as a potential mechanism for its drug resistance, we asked whether sorafenib signals through this pathway. We now show that sorafenib markedly decreases p21 levels in several RCC and hepatocellular carcinoma cells. Neither the MEK inhibitor PD98059 nor the sEH inhibitor t-AUCB, which represent known sorafenib-targeted signaling pathways, alter p21 levels, demonstrating that the p21 inhibitory effect of sorafenib is independent of these signaling cascades. In cells treated with doxorubicin to augment p21, sorafenib markedly decreases this protein, and the combinations of paclitaxel or doxorubicin with sorafenib show additive cytotoxicity as a function of the VHL status of the cells, suggesting that lower doses of each agent could be used in the clinical setting. In summary, we show a novel signaling pathway by which sorafenib exerts its salutary effects in RCC; future work will focus on the use of these drug combinations in the context of conventional therapeutics, and novel compounds and protocols targeting p21 in conjunction with sorafenib should be pursued.
doi:10.4161/cbt.12.9.17680
PMCID: PMC3225758  PMID: 21878748
sorafenib; p21; kidney cancer; apoptosis; DNA damage; soluble epoxide hydrolase
7.  Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7 
Molecular Cancer  2014;13(1):206.
Background
MicroRNAs are endogenous small noncoding RNAs that are functionally involved in numerous critical cellular processes including tumorigenesis. Data mining using a microRNA array database suggested that let-7d microRNA may be associated with renal cell carcinoma (RCC) malignant progression. Here, we performed further analyses to determine whether let-7d is functionally linked to RCC malignancy.
Methods
Quantitative real-time PCR was used to determine the level of mature let-7d in RCC clinical specimens and its correlation with clinicopathological data. Immunohistochemical staining was conducted to characterize the stroma of RCC. Let-7d overexpressing RCC cell lines combined with mouse models bearing cell-derived xenografts and patient-derived xenografts were used to assess the functional role of let-7d in vitro and in vivo.
Results
Downregulation of let-7d in clinical RCC samples was associated with advanced tumor grade and T stage and increased vascular invasion. An inverse relationship between let-7d expression and macrophage infiltration was found in clinical RCC samples. Functional studies indicated that ectopic expression of let-7d significantly inhibited RCC cell proliferation, migration, and peripheral blood monocyte (PBMC) recruitment in vitro, as well as tumor growth, metastasis, and tumor macrophage infiltration in vivo. In silico analysis and subsequent experimental validation confirmed collagen, type III, alpha 1 (COL3A1) and C-C subfamily chemokine member CCL7 as direct let-7d target genes. The addition of COL3A1 and CCL7 counteracted the inhibitory effects of let-7d on RCC cell proliferation, migration, and PBMC recruitment. The inhibition of let-7d increased cell proliferation, migration, and PBMC recruitment by the enhanced expression of COL3A1 and CCL7 genes in vitro. The mRNA levels of COL3A1 and CCL7 were inversely correlated with let-7d level in RCC clinical specimens.
Conclusions
These results suggest that let-7d may suppress RCC growth, metastasis, and tumor macrophage infiltration at least partially through targeting COL3A1 and CCL7.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-206) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-206
PMCID: PMC4168121  PMID: 25193015
Renal cell carcinoma; MicroRNA; Let-7
8.  Critical appraisal of pazopanib as treatment for patients with advanced metastatic renal cell carcinoma 
The management of renal cell carcinoma (RCC) has undergone significant changes during the past 10 years, with the treatment of metastatic RCC undergoing the most radical changes. These developments reflect an enhanced understanding of this tumor’s underlying biology, which was then translated into the development of a new treatment paradigm. Current therapeutic approaches for the management of patients with metastatic RCC utilize knowledge of histology, molecular abnormalities, clinical prognostic factors, the natural history of this malignancy, and the treatment efficacy and toxicity of available agents. The treatment options available for patients with metastatic RCC have changed dramatically over the past 6 years. Interferon-α and interleukin-2 were the previous mainstays of therapy, but since December 2005, six new agents have been approved in the US for the treatment of advanced RCC. Three are multi-targeted tyrosine kinase inhibitors (TKI) including sunitinib, sorafenib, and pazopanib, two target the mammalian target of rapamycin (temsirolimus and everolimus), and one is a humanized monoclonal antibody (bevacizumab in combination with interferon-α). The current review focuses on the newest TKI available to treat patients with metastatic RCC, pazopanib. The development of this agent both preclinically and clinically is reviewed. The efficacy and safety data from the pivotal clinical trials are discussed, and the potential role of pazopanib in the treatment of patients with metastatic RCC in comparison to other treatment alternatives is critically appraised. This agent has a favorable overall risk benefit, and the available data demonstrate efficacy in patients with metastatic RCC who are either treatment-naïve or cytokine refractory. It therefore represents another alternative for treatment of metastatic RCC patients.
doi:10.2147/CMR.S15557
PMCID: PMC3173017  PMID: 21931501
renal cell carcinoma; metastatic; pazopanib
9.  Inhibition of angiogenic and non-angiogenic targets by sorafenib in renal cell carcinoma (RCC) in a RCC xenograft model 
British Journal of Cancer  2011;104(6):941-947.
Background:
It is widely recognised that sorafenib inhibits a range of molecular targets in renal cell carcinoma (RCC). In this study, we aim to use patient-derived RCC xenografts to delineate the angiogenic and non-angiogenic molecular targets of sorafenib therapy for advanced RCC (aRCC).
Methods:
We successfully generated three patient RCC-derived xenografts in severe combined immunodeficient mice, consisting of three different RCC histological subtypes: conventional clear cell, poorly differentiated clear cell RCC with sarcomatoid changes, and papillary RCC. This study also used clear cell RCC cells (786-0/EV) harbouring mutant VHL to investigate the clonogenic survival of cells transfected with survivin sense and antisense oligonucleotides.
Results:
All three xenografts retain their original histological characteristics. We reported that sorafenib inhibited all three RCC xenograft lines regardless of histological subtypes in a dose-dependant manner. Sorafenib-induced growth suppression was associated with not only inhibition of angiogenic targets p-PDGFR-β, p-VEGFR-2, and their downstream signalling pathways p-Akt and p-ERK, cell cycle, and anti-apoptotic proteins that include cyclin D1, cyclin B1, and survivin but also upregulation of proapoptotic Bim. Survivin knockdown by survivin-specific antisense-oligonucleotides inhibited colony formation and induced cell death in clear cell RCC cells.
Conclusion:
This study has shed light on the molecular mechanisms of sorafenib in RCC. Inhibition of non-angiogenic molecules by sorafenib could contribute in part to its anti-tumour activities observed in vivo, in addition to its anti-angiogenic effects.
doi:10.1038/bjc.2011.55
PMCID: PMC3065286  PMID: 21407223
renal cell carcinoma; sorafenib; molecular targeted therapy; xenograft; survivin
10.  Novel mechanism of MDA-7/IL-24 cancer-specific apoptosis through SARI induction 
Cancer research  2013;74(2):563-574.
Subtraction-hybridization combined with induction of cancer cell terminal differentiation in human melanoma cells identified melanoma differentiation associated gene-7 (mda-7/IL-24) and SARI (Suppressor of AP-1, induced by IFN) that display potent antitumor activity. These genes are not constitutively expressed in cancer cells and forced expression of mda-7/IL-24 (Ad.mda-7) or SARI(Ad.SARI) promotes cancer-specific cell death. Ectopic expression of mda-7/IL-24 induces SARI mRNA and protein in a panel of different cancer cells leading to cell death, without harming corresponding normal cells. Simultaneous inhibition of K-ras downstream extracellular regulated kinase 1/2 (ERK1/2) signaling in pancreatic cancer cells reverses the translational block of MDA-7/IL-24 and induces SARI expression and cell death. Using SARI-antisense-based approaches we demonstrate that SARI expression is necessary for mda-7/IL-24 antitumor effects. Secreted MDA-7/IL-24 protein induces antitumor ‘bystander’ effects by promoting its own expression. Recombinant MDA-7/IL-24 (His-MDA-7) induces SARI expression, supporting the involvement of SARI in the MDA-7/IL-24-driven autocrine loop culminating in antitumor effects. Moreover, His-MDA-7 after binding to its cognate receptors (IL-20R1/IL-20R2 or IL-22R/IL-20R2) induces intracellular signaling by phosphorylation of p38 MAPK leading to transcription of a family of growth arrest and DNA damage inducible (GADD) genes, culminating in apoptosis. Inhibition of p38 MAPK fails to induce SARI following Ad.mda-7 infection. These findings reveal the significance of the mda-7/IL-24-SARI axis in cancer-specific killing, and provide a potential strategy for treating both local and metastatic disease.
doi:10.1158/0008-5472.CAN-13-1062
PMCID: PMC3915776  PMID: 24282278
SARI; MDA-7/IL-24; apoptosis; IL-20/IL-22 receptors
11.  High dose intermittent sorafenib shows improved efficacy over conventional continuous dose in renal cell carcinoma 
Background
Renal cell carcinoma (RCC) responds to agents that inhibit vascular endothelial growth factor (VEGF) pathway. Sorafenib, a multikinase inhibitor of VEGF receptor, is effective at producing tumor responses and delaying median progression free survival in patients with cytokine refractory RCC. However, resistance to therapy develops at a median of 5 months. In an effort to increase efficacy, we studied the effects of increased sorafenib dose and intermittent scheduling in a murine RCC xenograft model.
Methods
Mice bearing xenografts derived from the 786-O RCC cell line were treated with sorafenib according to multiple doses and schedules: 1) Conventional dose (CD) continuous therapy; 2) high dose (HD) intermittent therapy, 3) CD intermittent therapy and 4) HD continuous therapy. Tumor diameter was measured daily. Microvessel density was assessed after 3 days to determine the early effects of therapy, and tumor perfusion was assessed serially by arterial spin labeled (ASL) MRI at day 0, 3, 7 and 10.
Results
Tumors that were treated with HD sorafenib exhibited slowed tumor growth as compared to CD using either schedule. HD intermittent therapy was superior to CD continous therapy, even though the total dose of sorafenib was essentially equivalent, and not significantly different than HD continuous therapy. The tumors exposed to HD sorafenib had lower microvessel density than the untreated or the CD groups. ASL MRI showed that tumor perfusion was reduced to a greater extent with the HD sorafenib at day 3 and at all time points thereafter relative to CD therapy. Further the intermittent schedule appeared to maintain RCC sensitivity to sorafenib as determined by changes in tumor perfusion.
Conclusions
A modification of the sorafenib dosing schedule involving higher dose intermittent treatment appeared to improve its efficacy in this xenograft model relative to conventional dosing. MRI perfusion imaging and histologic analysis suggest that this benefit is related to enhanced and protracted antiangiogenic activity. Thus, better understanding of dosing and schedule issues may lead to improved therapeutic effectiveness of VEGF directed therapy in RCC and possibly other tumors.
doi:10.1186/1479-5876-9-220
PMCID: PMC3258225  PMID: 22188900
Renal cell carcinoma; anti-angiogenic therapy; arterial spin labeled magnetic resonance imaging
12.  Gene expression pattern of the epidermal growth factor receptor family and LRIG1 in renal cell carcinoma 
BMC Research Notes  2012;5:216.
Background
Previous studies have revealed altered expression of epidermal growth factor receptor (EGFR)-family members and their endogenous inhibitor leucine-rich and immunoglobulin-like domains 1 (LRIG1) in renal cell carcinoma (RCC). In this study, we analyzed the gene expression levels of EGFR-family members and LRIG1, and their possible associations with clinical parameters in various types of RCC.
Methods
Gene expression levels of EGFR–family members and LRIG1 were analyzed in 104 RCC samples, including 81 clear cell RCC (ccRCC), 15 papillary RCC (pRCC), and 7 chromophobe RCC (chRCC) by quantitative real-time RT-PCR. Associations between gene expression levels and clinical data, including tumor grade, stage, and patient survival were statistically assessed.
Results
Compared to kidney cortex, EGFR was up-regulated in ccRCC and pRCC, LRIG1 and ERBB2 were down-regulated in ccRCC, and ERBB4 was strongly down-regulated in all RCC types. ERBB3 expression did not differ between RCC types or between RCC and the kidney cortex. The expression of the analyzed genes did not correlate with patient outcome.
Conclusions
This study revealed that the previously described up-regulation of EGFR and down-regulation of ERBB4 occurred in all analyzed RCC types, whereas down-regulation of ERBB2 and LRIG1 was only present in ccRCC. These observations illustrate the need to evaluate the different RCC types individually when analyzing molecules of interest and potential biological markers.
doi:10.1186/1756-0500-5-216
PMCID: PMC3419632  PMID: 22554477
Renal cell carcinoma; EGFR; ERBB2; ERBB3; ERRB4; LRIG1; Survival
13.  mda-7/IL-24: Multifunctional cancer-specific apoptosis-inducing cytokine 
Pharmacology & therapeutics  2006;111(3):596-628.
“Differentiation therapy” provides a unique and potentially effective, less toxic treatment paradigm for cancer. Moreover, combining “differentiation therapy” with molecular approaches presents an unparalleled opportunity to identify and clone genes mediating cancer growth control, differentiation, senescence, and programmed cell death (apoptosis). Subtraction hybridization applied to human melanoma cells induced to terminally differentiate by treatment with fibroblast interferon (IFN-β) plus mezerein (MEZ) permitted cloning of melanoma differentiation associated (mda) genes. Founded on its novel properties, one particular mda gene, mda-7, now classified as a member of the interleukin (IL)-10 gene family (IL-24) because of conserved structure, chromosomal location, and cytokine-like properties has become the focus of attention of multiple laboratories. When administered by transfection or adenovirus-transduction into a spectrum of tumor cell types, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) induces apoptosis, whereas no toxicity is apparent in normal cells. mda-7/IL-24 displays potent “bystander antitumor” activity and also has the capacity to enhance radiation lethality, to induce immune-regulatory activities, and to inhibit tumor angiogenesis. Based on these remarkable attributes and effective antitumor therapy in animal models, this cytokine has taken the important step of entering the clinic. In a Phase I clinical trial, intratumoral injections of adenovirus-administered mda-7/IL-24 (Ad.mda-7) was safe, elicited tumor-regulatory and immune-activating processes, and provided clinically significant activity. This review highlights our current understanding of the diverse activities and properties of this novel cytokine, with potential to become a prominent gene therapy for cancer.
doi:10.1016/j.pharmthera.2005.11.005
PMCID: PMC1781515  PMID: 16464504
mda-7/IL-24; Differentiation therapy of cancer; Programmed cell death; Antitumor bystander activity; Radiosensitization; Angiogenesis; Cell signaling; Phase I clinical trial
14.  TRAIL and IFNα act synergistically to induce renal cell carcinoma apoptosis 
The Journal of urology  2010;184(3):1166-1174.
Introduction
Despite modern targeted therapy, metastatic renal cell carcinoma (RCC) remains a deadly disease. Interferon alpha (IFNα) is currently used to treat this condition, mainly in combination with the targeted anti-VEGF antibody, bevacizumab. Apo2 ligand/Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a novel anti-neoplastic agent now in early phase clinical trials. IFNα and TRAIL can act synergistically to kill cancer cells, but this has never been tested in the context of RCC. We hypothesized that TRAIL and IFNα can synergistically induce apoptosis in RCC cells.
Methods
RCC cell lines were treated with recombinant TRAIL and/or IFNα. Viability and apoptosis were assessed by MTS assay, flow cytometery, and western blot. Synergy was confirmed by isobologram analysis. IFNα induced changes in signaling by RCC cells were assessed by western blot, flow cytometry, and ELISA.
Results
TRAIL and IFNα act synergistically to increase apoptotic cell death in RCC cells. Treatment with IFNα alters these cells ability to activate ERK and inhibiting ERK with UO126 abrogates the apoptotic synergy between TRAIL and IFNα. IFNα does not induce changes in TRAIL or death receptor expression, nor does it change other known mediators of the intrinsic and extrinsic apoptotic cascade in RCC cells.
Conclusion
TRAIL plus IFNα synergistically induces apoptosis in RCC cells. The mechanism is due at least in part to IFNα mediated changes in ERK activation. Combination therapy with TRAIL and IFNα may be a novel approach to systemically treat advanced RCC and warrants further testing in vivo.
doi:10.1016/j.juro.2010.04.064
PMCID: PMC2963111  PMID: 20663526
Apo2 ligand/Tumor necrosis factor related apoptosis inducing ligand (TRAIL); kidney cancer; immunotherapy; apoptosis; ERK
15.  A phase I/II trial of sorafenib and infliximab in advanced renal cell carcinoma 
British Journal of Cancer  2010;103(8):1149-1153.
There is clinical evidence to suggest that tumour necrosis factor-α (TNF-α) may be a therapeutic target in renal cell carcinoma (RCC). Multi-targeted kinase inhibitors, such as sorafenib and sunitinib, have become standard of care in advanced RCC. The anti-TNF-α monoclonal antibody infliximab and sorafenib have differing cellular mechanisms of action. We conducted a phase I/II trial to determine the safety and efficacy of infliximab in combination with sorafenib in patients with advanced RCC.
Eligible patients were systemic treatment-naive or had received previous cytokine therapy only. Sorafenib and infliximab were administered according to standard schedules. The study had two phases: in phase I, the safety and toxicity of the combination of full-dose sorafenib and two dose levels of infliximab were evaluated in three and three patients, respectively, and in phase II, further safety, toxicity and efficacy data were collected in an expanded patient population.
Acceptable safety was reported for the first three patients (infliximab 5 mg kg−1) in phase 1. Sorafenib 400 mg twice daily and infliximab 10 mg kg−1 were administered to a total of 13 patients (three in phase 1 and 10 in phase 2). Adverse events included grade 3 hand–foot syndrome (31%), rash (25%), fatigue (19%) and infection (19%). Although manageable, toxicity resulted in 75% of the patients requiring at least one dose reduction and 81% requiring at least one dose delay of sorafenib. Four patients were progression-free at 6 months (PFS6 31%); median PFS and overall survival were 6 and 14 months, respectively.
Sorafenib and infliximab can be administered in combination, but a significant increase in the numbers of adverse events requiring dose adjustments of sorafenib was observed. There was no evidence of increased efficacy compared with sorafenib alone in advanced RCC. The combination of sorafenib and infliximab does not warrant further evaluation in patients with advanced RCC.
doi:10.1038/sj.bjc.6605889
PMCID: PMC2967062  PMID: 20842130
renal cell carcinoma; sorafenib; infliximab
16.  Possible Prognostic and Therapeutic Significance of c-Kit Expression, Mast Cell Count and Microvessel Density in Renal Cell Carcinoma 
Renal cell carcinoma (RCC) is the most frequent renal tumor and its incidence is increasing worldwide. Tumor angiogenesis is known to play a crucial role in the etiopathogenesis of RCC and over the last few years an even deeper knowledge of its contribution in metastatic RCC development has led to the development of numerous molecular targeting agents (such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, and dovitinib). The above agents are principally directed against vascular endothelial growth factor receptor (VEGFR) members and also against c-Kit receptor (c-KitR). The role of c-kitR inhibition on clear cell RCC (ccRCC), the main RCC subtype, is less well established. Whether c-kitR activation through its ligand, stem cell factor (SCF) contributes significantly to the effects of tyrosine kinase inhibitors (TKIs) treatment remains to be established. It is important to underscore that the c-KitR is expressed on mast cells (MCs) and cancer cells. After an examination of the c-KitR/SCF pathway, we review here the principal studies that have evaluated c-Kit expression in RCC. Moreover, we summarize some investigations that have observed the distribution of MCs in primary renal cancer and in adjacent normal tissue with appropriate histological immunohistochemical techniques. We also focus on few studies that have evaluated the correlation between RCC proliferation, MC count and microvessel density (MVD), as hallmarks of tumor angiogenesis. Thus, the aim of this review of the literature is to clarify if c-KitR expression, MC count and MVD could have prognostic significance and the possible predictive therapeutic implications in RCC.
doi:10.3390/ijms150713060
PMCID: PMC4139891  PMID: 25056544
renal cell carcinoma; c-Kit receptor; stem cell factor; mast cells; microvessel density; molecular targeting agents
17.  Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma 
PLoS ONE  2010;5(5):e10696.
Background
Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. The purpose of this study is to define a biological pathway signature and a cellular differentiation program in ccRCC.
Methodology
We performed gene expression profiling of early-stage ccRCC and patient-matched normal renal tissue using Affymetrix HG-U133a and HG-U133b GeneChips combined with a comprehensive bioinformatic analyses, including pathway analysis. The results were validated by real time PCR and IHC on two independent sample sets. Cellular differentiation experiments were performed on ccRCC cell lines and their matched normal renal epithelial cells, in differentiation media, to determine their mesenchymal differentiation potential.
Principal Findings
We identified a unique pathway signature with three major biological alterations—loss of normal renal function, down-regulated metabolism, and immune activation–which revealed an adipogenic gene expression signature linked to the hallmark lipid-laden clear cell morphology of ccRCC. Culturing normal renal and ccRCC cells in differentiation media showed that only ccRCC cells were induced to undergo adipogenic and, surprisingly, osteogenic differentiation. A gene expression signature consistent with epithelial mesenchymal transition (EMT) was identified for ccRCC. We revealed significant down-regulation of four developmental transcription factors (GATA3, TFCP2L1, TFAP2B, DMRT2) that are important for normal renal development.
Conclusions
ccRCC is characterized by a lack of epithelial differentiation, mesenchymal/adipogenic transdifferentiation, and pluripotent mesenchymal stem cell-like differentiation capacity in vitro. We suggest that down-regulation of developmental transcription factors may mediate the aberrant differentiation in ccRCC. We propose a model in which normal renal epithelial cells undergo dedifferentiation, EMT, and adipogenic transdifferentiation, resulting in ccRCC. Because ccRCC cells grown in adipogenic media regain the characteristic ccRCC phenotype, we have indentified a new in vitro ccRCC cell model more resembling ccRCC tumor morphology.
doi:10.1371/journal.pone.0010696
PMCID: PMC2872663  PMID: 20502531
18.  The Relevance of Testing the Efficacy of Anti-Angiogenesis Treatments on Cells Derived from Primary Tumors: A New Method for the Personalized Treatment of Renal Cell Carcinoma 
PLoS ONE  2014;9(3):e89449.
Despite the numerous available drugs, the most appropriate treatments for patients affected by common or rare renal cell carcinomas (RCC), like those associated with the Xp11.2 translocation/transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) gene fusion (TFE3 RCC), are not clearly defined. We aimed to make a parallel between the sensitivity to targeted therapies on living patients and on cells derived from the initial tumor. Three patients diagnosed with a metastatic RCC (one clear cell RCC [ccRCC], two TFE3 RCC) were treated with anti-angiogenesis drugs. The concentrations of the different drugs giving 50% inhibition of cell proliferation (IC50) were determined with the Thiazolyl Blue Tetrazolium Bromide (MTT) assay on cells from the primary tumors and a reference sensitive RCC cell line (786-O). We considered the cells to be sensitive if the IC50 was lower or equal to that in 786-O cells, and insensitive if the IC50 was higher to that in 786-O cells (IC 50 of 6±1 µM for sunitinib, 10±1 µM for everolimus and 6±1 µM for sorafenib). Based on this standard, the response in patients and in cells was equivalent. The efficacy of anti-angiogenesis therapies was also tested in cells obtained from five patients with non-metastatic ccRCC, and untreated as recommended by clinical practice in order to determine the best treatment in case of progression toward a metastatic grade. In vitro experiments may represent a method for evaluating the best first-line treatment for personalized management of ccRCC during the period following surgery.
doi:10.1371/journal.pone.0089449
PMCID: PMC3968004  PMID: 24676409
19.  Axitinib for the Management of Metastatic Renal Cell Carcinoma 
Drugs in R&d  2012;11(2):113-126.
In recent years, targeted agents have changed the treatment landscape for patients with advanced renal cell carcinoma (RCC), greatly improving treatment outcomes. Several targeted agents are now licensed for the treatment of metastatic RCC (mRCC), and a number of new agents are under investigation. Axitinib, a small molecule indazole derivative is an oral, potent multitargeted tyrosine kinase receptor inhibitor, which selectively inhibits vascular endothelial growth factor receptors (VEGFR)-1, -2, and -3 at subnanomolar concentrations, in vitro. In various nonclinical models, axitinib has demonstrated in vivo target modulation and antiangiogenesis. In pharmacokinetic studies, axitinib administered orally with food at the proposed regimen of 5mg twice daily continuous daily dosing, is rapidly absorbed, reaching peak concentrations within 2–6 hours. Axitinib is metabolized primarily in the liver via the cytochrome P450 (CYP) system with less than 1% of the administered drug passing unchanged in the urine. The pharmacokinetics of axitinib do not appear to be altered by coadministered chemotherapies, and antacids do not have a clinically significant effect. However, coadministration with CYP3A4 and 1A2 inducers is contraindicated. In addition, proton pump inhibitors reduce the rate of axitinib absorption. Increased axitinib exposure is associated with higher efficacy indicated by decreased tumor perfusion and volume. In three phase II clinical trials in patients with advancedRCCpreviously treated with cytokines, chemotherapy or targeted agents, axitinib has demonstrated antitumor activity with a favorable noncumulative toxicity profile. In one study of Western patients with cytokine-refractory mRCC, an objective response rate (ORR) of 44.2% (95% CI 30.5, 58.7) was achieved. The median time to progression was 15.7 months (95%CI 8.4, 23.4) and the median overall survival (OS) was 29.9 months (95%CI 20.3, not estimable). In the second study of patients with sorafenib-refractory mRCC, ORR was 22.6% (95% CI 12.9, 35.0). The median progression-free survival (PFS) was 7.4 months (95% CI 6.7, 11.0) and a median OS of 13.6 months (95% CI 8.4, 18.8) was achieved. Results from the third study in Japanese patients with cytokine-refractory mRCC reported an ORR of 55% and median PFS of 12.9 months (95% CI 9.8, 15.6).
In the three studies, themost common adverse events reported were fatigue, hypertension, hand-foot syndrome (HFS), and gastrointestinal toxicity, which were generally manageable with standard medical intervention. Of note, the incidence of HFS and proteinuria in the Japanese study was higher than that reported in the Western study in cytokine-refractory mRCC patients.
An observed association between diastolic blood pressure ≥90 mmHg and increased efficacy suggests potential use as a prognostic biomarker. However, this requires further investigation. Two randomized phase III clinical trials are ongoing to determine the efficacy of axitinib in patients with mRCC in the first- and second-line setting. These results will help to determine the place of axitinib in the mRCC treatment algorithm.
doi:10.2165/11591240-000000000-00000
PMCID: PMC3585900  PMID: 21679004
20.  Axitinib for the Management of Metastatic Renal Cell Carcinoma 
Drugs in R&D  2012;11(2):113-126.
In recent years, targeted agents have changed the treatment landscape for patients with advanced renal cell carcinoma (RCC), greatly improving treatment outcomes. Several targeted agents are now licensed for the treatment of metastatic RCC (mRCC), and a number of new agents are under investigation. Axitinib, a small molecule indazole derivative is an oral, potent multitargeted tyrosine kinase receptor inhibitor, which selectively inhibits vascular endothelial growth factor receptors (VEGFR)-1, -2, and -3 at subnanomolar concentrations, in vitro. In various nonclinical models, axitinib has demonstrated in vivo target modulation and antiangiogenesis. In pharmacokinetic studies, axitinib administered orally with food at the proposed regimen of 5mg twice daily continuous daily dosing, is rapidly absorbed, reaching peak concentrations within 2–6 hours. Axitinib is metabolized primarily in the liver via the cytochrome P450 (CYP) system with less than 1% of the administered drug passing unchanged in the urine. The pharmacokinetics of axitinib do not appear to be altered by coadministered chemotherapies, and antacids do not have a clinically significant effect. However, coadministration with CYP3A4 and 1A2 inducers is contraindicated. In addition, proton pump inhibitors reduce the rate of axitinib absorption. Increased axitinib exposure is associated with higher efficacy indicated by decreased tumor perfusion and volume. In three phase II clinical trials in patients with advancedRCCpreviously treated with cytokines, chemotherapy or targeted agents, axitinib has demonstrated antitumor activity with a favorable noncumulative toxicity profile. In one study of Western patients with cytokine-refractory mRCC, an objective response rate (ORR) of 44.2% (95% CI 30.5, 58.7) was achieved. The median time to progression was 15.7 months (95%CI 8.4, 23.4) and the median overall survival (OS) was 29.9 months (95%CI 20.3, not estimable). In the second study of patients with sorafenib-refractory mRCC, ORR was 22.6% (95% CI 12.9, 35.0). The median progression-free survival (PFS) was 7.4 months (95% CI 6.7, 11.0) and a median OS of 13.6 months (95% CI 8.4, 18.8) was achieved. Results from the third study in Japanese patients with cytokine-refractory mRCC reported an ORR of 55% and median PFS of 12.9 months (95% CI 9.8, 15.6).
In the three studies, themost common adverse events reported were fatigue, hypertension, hand-foot syndrome (HFS), and gastrointestinal toxicity, which were generally manageable with standard medical intervention. Of note, the incidence of HFS and proteinuria in the Japanese study was higher than that reported in the Western study in cytokine-refractory mRCC patients.
An observed association between diastolic blood pressure ≥90 mmHg and increased efficacy suggests potential use as a prognostic biomarker. However, this requires further investigation. Two randomized phase III clinical trials are ongoing to determine the efficacy of axitinib in patients with mRCC in the first- and second-line setting. These results will help to determine the place of axitinib in the mRCC treatment algorithm.
doi:10.2165/11591240-000000000-00000
PMCID: PMC3585900  PMID: 21679004
21.  Prolyl hydroxylase 2 dependent and Von-Hippel-Lindau independent degradation of Hypoxia-inducible factor 1 and 2 alpha by selenium in clear cell renal cell carcinoma leads to tumor growth inhibition 
BMC Cancer  2012;12:293.
Background
Clear cell renal cell carcinoma (ccRCC) accounts for more than 80% of the cases of renal cell carcinoma. In ccRCC deactivation of Von-Hippel-Lindau (VHL) gene contributes to the constitutive expression of hypoxia inducible factors 1 and 2 alpha (HIF-α), transcriptional regulators of several genes involved in tumor angiogenesis, glycolysis and drug resistance. We have demonstrated inhibition of HIF-1α by Se-Methylselenocysteine (MSC) via stabilization of prolyl hydroxylases 2 and 3 (PHDs) and a significant therapeutic synergy when combined with chemotherapy. This study was initiated to investigate the expression of PHDs, HIF-α, and VEGF-A in selected solid cancers, the mechanism of HIF-α inhibition by MSC, and to document antitumor activity of MSC against human ccRCC xenografts.
Methods
Tissue microarrays of primary human cancer specimens (ccRCC, head & neck and colon) were utilized to determine the incidence of PHD2/3, HIF-α, and VEGF-A by immunohistochemical methods. To investigate the mechanism(s) of HIF-α inhibition by MSC, VHL mutated ccRCC cells RC2 (HIF-1α positive), 786–0 (HIF-2α positive) and VHL wild type head & neck cancer cells FaDu (HIF-1α) were utilized. PHD2 and VHL gene specific siRNA knockdown and inhibitors of PHD2 and proteasome were used to determine their role in the degradation of HIF-1α by MSC.
Results
We have demonstrated that ccRCC cells express low incidence of PHD2 (32%), undetectable PHD3, high incidence of HIF-α (92%), and low incidence of VEGF-A compared to head & neck and colon cancers. This laboratory was the first to identify MSC as a highly effective inhibitor of constitutively expressed HIF-α in ccRCC tumors. MSC did not inhibit HIF-1α protein synthesis, but facilitated its degradation. The use of gene knockdown and specific inhibitors confirmed that the inhibition of HIF-1α was PHD2 and proteasome dependent and VHL independent. The effects of MSC treatment on HIF-α were associated with significant antitumor activity against ccRCC xenograft.
Conclusions
Our results show the role of PHD2/3 in stable expression of HIF-α in human ccRCC. Furthermore, HIF-1α degradation by MSC is achieved through PHD2 dependent and VHL independent pathway which is unique for HIF-α regulation. These data provide the basis for combining MSC with currently used agents for ccRCC.
doi:10.1186/1471-2407-12-293
PMCID: PMC3466155  PMID: 22804960
Prolyl hydroxylases; Hypoxia-inducible factor; Clear cell renal cell carcinoma; Selenium
22.  Recombinant adenovirus vector-mediated human MDA-7 gene transfection suppresses hepatocellular carcinoma growth in a mouse xenograft model☆ 
Journal of Biomedical Research  2012;26(1):53-58.
Hepatocellular carcinoma is one of the most common tumors in the world. The purpose of the present study was to investigate the inhibitory effects of adenoviral transduction of human melanoma differentiation-associated gene-7 (MDA-7) gene on hepatocellular carcinoma, so as to provide a theoretical basis for gene therapy of the disease. The human MDA-7 gene was cloned into replication-defective adenovirus specific to HepG2 cells using recombinant virus technology. RT-PCR and Western blotting assays were used to determine the expression of human MDA-7 mRNA and MDA-7 protein in HepG2 cells in vitro. Induction of apoptosis by overexpression of the human MDA-7 gene was determined by flow cytometry. In-vivo efficacy of adenoviral delivery of the human MDA-7 gene was assessed in nude mice bearing HepG2 cell lines in vivo by determining inhibition of tumor growth, VEGF and CD34 expression, and microvascular density (MVD). The results showed that AdGFP/MDA-7 induced apoptosis of HepG2 cells in vitro and significantly inhibited tumor growth in vivo (P < 0.05). The intratumoral MVD decreased significantly in the treated tumors (P < 0.05). We conclude the recombination adenovirus AdGFP/MDA-7 can effectively express biologically active human MDA-7, which leads to inhibition of hepatocellular carcinoma growth.
doi:10.1016/S1674-8301(12)60007-4
PMCID: PMC3596080  PMID: 23554730
MDA-7; adenovirus; hepatocellular carcinoma; gene therapy; angiogenesis
23.  Critical appraisal of sorafenib in the treatment of Chinese patients with renal cell carcinoma 
OncoTargets and therapy  2014;7:925-935.
Renal cell carcinoma (RCC) accounts for 3% of all malignancies, and is the most aggressive cancer of the genitourinary system. Metastatic RCC is naturally resistant to chemotherapy and radiotherapy, and immunotherapy is of little benefit. In recent years, the emergence of molecular-targeted therapies has largely changed the therapeutic approach to metastatic RCC. These novel multikinase inhibitors have now become first-choice therapy because of their activity in inhibiting both cell proliferation and tumor angiogenesis. Sorafenib is the first tyrosine kinase inhibitor found to be effective in treating patients with metastatic RCC. Due to its good efficacy and safety, this agent is recommended as both first-line and second-line therapy for metastatic RCC in the People’s Republic of China. Sorafenib seems to be more effective in patients of Chinese ethnicity than in western patients, and is well tolerated with a manageable toxicity profile, even at higher dosages and when used in combination with other anticancer agents. Novel biomarkers for predicting the efficacy of sorafenib have potential clinical value for guiding individualized targeted therapy.
doi:10.2147/OTT.S41828
PMCID: PMC4057324  PMID: 24944516
kidney cancer; renal cell carcinoma; sorafenib; tyrosine kinase inhibitor
24.  5-fluorouracil enhances the antitumor effect of sorafenib and sunitinib in a xenograft model of human renal cell carcinoma 
Oncology Letters  2012;3(6):1195-1202.
Sorafenib and sunitinib are multi-kinase inhibitors with antitumor activity in patients with advanced renal cell carcinoma (RCC). Several studies have evaluated the effect of sorafenib/sunitinib in combination with chemotherapeutic agents in different types of tumor. However, few studies have addressed the activity of fluorinated pyrimidine in combination with sorafenib/sunitinib. In this study, we examined the potential of combination therapy with 5FU and sorafenib/sunitinib in human RCC cell lines. Three human RCC cell lines, ACHN, Caki-1 and Caki-2, were used to assess sensitivity to 5-fluorouracil (5FU), sorafenib and sunitinib alone or in combination using an in vitro cell survival assay. Caki-2 cells demonstrated significantly higher resistance to 5FU and sorafenib as compared to ACHN and Caki-1. Additive antitumor effects of the combination therapy were observed in the in vitro study. There was a tendency for a positive correlation between the sensitivity to sunitinib and platelet-derived growth factor β (PDGFR-β) expression levels, which were examined by reverse transcription polymerase chain reaction. Caki-1 xenograft models were prepared by inoculating cells subcutaneously into nude mice, which were divided randomly into six groups: control, 5FU (8 mg/kg/day, intraperitoneally), sorafenib (15 mg/kg/day, orally), sunitinib (20 mg/kg/day, orally), and 5FU with sorafenib or sunitinib. The treatments were administered on 5 days each week, and tumor growth was monitored for 42 days following inoculation of cells. Synergistic antitumor effects of the combination therapy were observed in an in vivo study. The resected tumors were evaluated using the Ki-67 labeling index and microvessel density. Both the Ki-67 labeling index and microvessel density were decreased in tumors treated with the combination therapy compared to those treated with sorafenib/sunitinib alone. These findings suggest that the combination therapy of 5FU with sorafenib/sunitinib may be an effective therapeutic modality for advanced RCC patients.
doi:10.3892/ol.2012.662
PMCID: PMC3392575  PMID: 22783417
renal cell carcinoma; 5-fluorouracil; sorafenib; sunitinib; angiogenesis
25.  Candidate tumor suppressor and pVHL partner Jade-1 binds and inhibits AKT in renal cell carcinoma 
Cancer research  2013;73(17):5371-5380.
The von Hippel-Lindau tumor suppressor pVHL (VHL) is lost in the majority of clear-cell renal cell carcinomas (RCCs). Activation of the PI3K/AKT/mTOR pathway is also common in RCC, with PTEN loss occurring in ~30% of the cases, but other mechanisms responsible for activating AKT at a wider level in this setting are undefined. Plant homeodomain protein Jade-1 (PHF17) is a candidate renal tumor suppressor stabilized by pVHL. Here using kinase arrays we identified phospho-AKT1 as an important target of Jade-1. Overexpressing or silencing Jade-1 in RCC cells increased or decreased levels of endogenous phospho-AKT/AKT1. Further, reintroducing pVHL into RCC cells increased endogenous Jade-1 and suppressed endogenous levels of phospho-AKT, which colocalized with and bound to Jade-1. The N-terminus of Jade-1 bound both the catalytic domain and the C-terminal regulatory tail of AKT, suggesting a mechanism through which Jade-1 inhibited AKT kinase activity. Intriguingly, RCC precursor cells where Jade-1 was silenced exhibited an increased capacity for AKT-dependent anchorage-independent growth, in support of a tumor suppressor function for Jade-1 in RCC. In support of this concept, an in silico expression analysis suggested that reduced Jade-1 expression is a poor prognostic factor in clear-cell RCC that is associated with activation of an AKT1 target gene signature. Taken together, our results identify two mechanisms for Jade-1 fine control of AKT/AKT1 in RCC, through loss of pVHL, which decreases Jade-1 protein, or through attenuation in Jade-1 expression. These findings help explain the pathologic cooperativity in clear-cell RCC between PTEN inactivation and pVHL loss, which leads to decreased Jade-1 levels that superactivate AKT. Additionally, they prompt further investigation of Jade-1 as a candidate biomarker and tumor suppressor in clear-cell RCC.
doi:10.1158/0008-5472.CAN-12-4707
PMCID: PMC3766477  PMID: 23824745
Jade-1; AKT; pVHL; renal tumor suppressor; anchorage-independent growth

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