Search

Metastatic renal cell carcinoma (RCC) remains refractory to therapies. The nuclear factor-κB (NF-κB) transcription factor is involved in cell growth, cell motility, and vascularization. We evaluated whether targeting NF-κB could be of therapeutic and prognostic values in human RCC. The activation of the NF-κB pathway in human RCC cells and tumors was investigated by Western blot. In vitro, the effects of BAY 11-7085 and sulfasalazine, two NF-κB inhibitors, on tumor cell growth were investigated by cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, and fluorescence-activated cell sorting. Their specificity toward NF-κB was analyzed by Western blot, confocal microscopy, NF-κB small interfering RNA, and NF-κB transcription assay. In vivo, the effects of BAY 11-7085 on the growth of human RCC tumors were investigated in nude mice. A tissue microarray (TMA) containing 241 cases of human RCC with 12 to 22 years of clinical follow-up and corresponding normal tissues was built up to assess prognostic significance of activated NF-κB. NF-κB is constitutively activated in cultured cells expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as a consequence of Akt kinase activation and in tumors. In vitro and in vivo NF-κB inhibition blocked tumor cell growth by inducing cell apoptosis. On the TMA, NF-κB activation was correlated with tumor dimension but was not found to be an independent prognostic factor for patient survival. This report provides strong evidence that the mechanisms responsible for the intrinsic resistance of RCC cells to apoptosis converge on NF-κB independently of VHL expression and that targeting this pathway has great anticancer potential. [Cancer Res 2007;67(24):11668–76]

Supplementary Legends 1-6 from Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Supplementary Figure 4 from Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Supplementary Figure 6 from Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Supplementary Figure 3 from Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Supplementary Figure 2 from Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Supplementary Figure 1 from Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Supplementary Figure 5 from Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Supplementary Tables 1-2 from Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Background: Today the objective for efficient Rheumatoid Arthritis (RA) therapy is not restricted to reduced synovial inflammation but to induce immunological and clinicaly lasting remission. Despite improvement in therapy of RA, anti-TNF alpha or anti-IL-6 (Tocilizumab) biologicals provide less than 20% of clinical remission. Interestingly, the complete remission can last up to 12 months in about 40% of these patients despite discontinuation of the above biologicals. In order to set up optimized strategy to obtain persistent clinical remission, new predictive markers of complete remission are needed. Objectives: By using Firalis’ BIOPRED panel, an innovative targeted gene sequencing panel of 2155 mRNA targets associated with immune-inflammatory pathways, we identified candidate biomarkers that have the potential to stratify patients for personalized therapy. Methods: Paxgene RNA samples obtained from 18 RA patients treated with Etanercept (n=11) and Tocilizumab (n=7) who had achieved remission (DAS28 Results: In total, 37 mRNA targets are found to be significantly up- or down-regulated in Etanercept groups as compared to Tocilizumab group (p-value 2 (p-value Conclusion: Our preliminary analysis shows that a panel of candidate predictive markers for biological specific (Etanercept) remission has the potential to stratify patients for personalized therapy in RA patients. Longitudinal studies are required with RA patients treated with all biologicals to validate this signature and explore further dimensions. Disclosure of Interests: None declared

International audience; Metastatic clear cell renal cell carcinoma (CCC) remains incurable despite advances in the development of anti-angiogenic targeted therapies and the emergence of immune checkpoint inhibitors. We have previously shown that the sonic hedgehog-Gli signaling pathway is oncogenic in CCC allowing us to identify the developmental Lim1 transcription factor as a Gli target and as a new oncogene in CCC regulating cell proliferation and apoptosis, and promoting tumor growth. In this previous study, preliminary in vitro results also suggested that Lim1 may be implicated in metastatic spread. Here we investigated the potential pro-metastatic role of Lim1 in advanced CCC (1) in vitro using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene either naturally or by gene transfer and (2) ex vivo in 30 CCC metastatic tissues, including lymph nodes, lung, skin, bone, and adrenal metastases, and (3) in vivo, using a metastatic model by intravenous injection of siRNA-transfected cells into Balb/c nude. Our in vitro results reveal that Lim1 knockdown time-dependently decreased CCC cell motility, migration, invasion, and clonogenicity by up to 50% regardless of their VHL status. Investigating the molecular machinery involved in these processes, we identified a large panel of Lim1 targets known to be involved in cell adhesion (paxillin and fibronectin), epithelial-mesenchymal transition (Twist1/2 and snail), invasion (MMP1/2/3/8/9), and metastatic progression (CXCR4, SDF-1, and ANG-1). Importantly, Lim1 was found constitutively expressed in all metastatic tissues. The H-score in metastatic tissues being significantly superior to the score in the corresponding primary tumor tissues (P value = 0.009). Furthermore, we showed that Lim1 silencing decreases pulmonary metastasis development in terms of number and size in the in vivo metastatic model of human CCC. Taken together, these experiments strengthen the potential therapeutic value of Lim1 targeting as a promising novel approach for treating metastatic human CCC.

International audience; Proliferative glomerulonephritis is characterized by local inflammation and mesangial cell deterioration, followed by mesangial proliferation and glomerular healing. Parathyroid hormone-related peptide (PTHrP) is a mesangial cytokine-like growth factor implicated in mesangial proliferation and survival. No data are available about its role in glomerulonephritis. Herein, we analyzed the expression and role of PTHrP in glomerular inflammation and healing in an experimental model of glomerulonephritis induced by i.v. injection of Habu snake venom in mice. The temporal analysis showed marked renal damage in the first days after venom injection and the beginning of recovery within 7 days. Glomerular expression of PTHrP (transcript and protein) was observed in the early phase after venom injection (from day 1 to day 3), along with an inflammatory environment. The inactivation of secreted PTHrP with PTHrP-neutralizing antibody (PTH2E11; 120 μg i.p. daily) reduced the markers of local inflammation (expression of macrophage chemotactic protein-1; regulated upon activation, normal T cell expressed and secreted; cyclooxygenase 2; IL-6; and macrophage infiltration) and abolished the expression of PTHrP itself. Moreover, the glomerular cell proliferation was hampered, and the healing process was prevented on day 7 after venom injection. These results show that PTHrP has antinomic actions in glomerulonephritis, participating in both the proinflammatory condition and the healing process. Our work reveals the essential role of PTHrP in early glomerular repair in an experimental model of glomerulonephritis.

Polydiacetylenic nanofibers (PDA-Nfs) obtained by photopolymerization of surfactant 1 were optimized for intracellular delivery of small interfering RNAs (siRNAs). PDA-Nfs/siRNA complexes efficiently silenced the oncogene Lim-1 in the renal cancer cells 786-O in vitro. Intraperitoneal injection of PDA-Nfs/siLim1 downregulated Lim-1 in subcutaneous tumor xenografts obtained with 786-O cells in nude mice. Thus, PDA-Nfs represent an innovative system for in vivo delivery of siRNAs.

Injury of mesangial cells (MC) is a prominent feature of glomerulonephritis. Activated MC secrete inflammatory mediators that induce cell apoptosis. Parathyroid hormone-related peptide (PTHrP) is a locally active cytokine that enhances cell survival and is upregulated by proinflammatory factors in many cell types. The aim of this study was to analyze the regulation of PTHrP expression by inflammatory cytokines and to evaluate whether PTHrP itself acts as a proinflammatory and/or survival factor on male murine MC in primary culture. Our results showed that IL-1β (10 ng/ml) and TNF-α (10 ng/ml) rapidly and transiently upregulated PTHrP expression in MC. The effects of IL-1β were both transcriptional and posttranscriptional, with stabilization of the PTHrP mRNA by human antigen R (HuR). Proteome profiler arrays showed that PTHrP itself enhanced cytokines within 2 h in cell lysates, mainly IL-17, IL-16, IL-1α, and IL-6. PTHrP also stimulated sustained expression (2–4 h) of chemokines, mainly regulated upon activation normal T cell expressed and secreted (RANTES)/C-C motif chemokine 5 (CCL5) and macrophage inflammatory protein-2 (MIP-2)/C-X-C motif chemokine 2 (CXCL2), thymus and activation-regulated chemokine (TARC)/CCL17, and interferon-inducible T cell α-chemoattractant (I-TAC)/CXCL11. Moreover, PTHrP markedly enhanced cyclooxygenase-2 (COX-2) expression and elicited its autoinduction through the activation of the NF-κB pathway. PTHrP induced MC survival via the COX-2 products, and PTHrP overexpression in MC blunted the apoptotic effects of IL-1β and TNF-α. Altogether, these findings suggest that PTHrP functions as a booster of glomerular inflammatory processes and may be a negative feedback loop preserving MC survival.

Human conventional renal cell carcinoma (CCC) remains resistant to current therapies. Focal Adhesion Kinase (FAK) is upregulated in many epithelial tumors and clearly implicated in nearly all facets of cancer. However, only few reports have assessed whether FAK may be associated with renal tumorigenesis. In this study, we investigated the potential role of FAK in the growth of human CCC using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as normal/tumoral renal tissue pairs. FAK was found constitutively expressed in human CCC both in culture cells and freshly harvested tumors obtained from patients. We showed that CCC cell growth was dramatically reduced in FAK-depleted cells or after FAK inhibition with various inhibitors and this effect was obtained through inhibition of cell proliferation and induction of cell apoptosis. Additionally, our results indicated that FAK knockdown decreased CCC cell migration and invasion. More importantly, depletion or pharmacological inhibition of FAK substantially inhibited tumor growth in vivo. Interestingly, investigations of the molecular mechanism revealed loss of FAK phosphorylation during renal tumorigenesis impacting multiple signaling pathways. Taken together, our findings reveal a previously uncharacterized role of FAK in CCC whereby FAK exerts oncogenic properties through a non canonical signaling pathway involving its scaffolding kinase-independent properties. Therefore, targeting the FAK scaffold may represent a promising approach for developing innovative and highly specific therapies in human CCC.

Background Anti-TNF alpha biologicals are an important breakthrough in the treatment of Rheumatoid Arthritis (RA) patients. However, 30–40% of RA patients do not respond to these therapies. Therefore, there is an unmet need for a tool to predict treatment response that would help clinicians choose an optimal treatment for RA patients. Objectives Under the framework of Horizone2020 SME Instrument of European Commission, Firalis has identified and developed a panel of 2159 mRNA genes which can predict non-response to anti-TNF alpha therapy using the HTG EdgeSeq platform, an innovative combination of a nuclease protection assay & next generation sequencing (NGS). Methods RABIOPRED assay is a proprietary panel of Firalis signatures, which also includes targets selected by the BTCure consortium , to predict treatment response of anti-TNF alpha biologicals. In total 2175 targets were selected for the development and 2159 are successfully included in the panel. Each oligonucleotide is a 100-mer comprising a 25-mer “wing” at the 59 end and 39 end, and a 50-mer sequence in between that is complementary to the target mRNA. QC is checked for secondary structure and absence of homology with other sequences. Analytical parameters are assessed and repeatability of the RABIOPRED assay is validated on both Paxgene and purified RNA samples. Sample input is set at 32 μl for Paxgene RNA blood and 25 ng for extracted RNA. Results Mean correlation factor for 12 samples on 8 replicates for Paxgene and RNA samples are R 2 >0.97 and R 2 >0.99 respectively. First analysis and predictive modelling shows an AUC over 0.95 for the prediction of non-response to anti-TNF alpha. In the present work, we disclose the performance of the CE-IVD RABIOPRED assay based on more than 200 samples obtained from the prospective clinical studies, PRINT and RA-TNF. The algorithm will be further validated within the ongoing RABIOPRED Proof-of-Performance study (ClinicalTrials.gov Identifier: NCT03016260) in 720 patients treated by anti-TNF alpha biologicals (5 originators and 3 biosimilars) launched in December 2016. First version of the CE-IVD RABIOPRED assay will be available during Q2 2017 and open for testing. Conclusions We are showing that we can accurately measure mRNA expression with RABIOPRED assay using HTG-EdgeSeq NGS platform. Preliminary performance of the assay shows that it can efficiently predict treatment response to anti-TNF alpha biologicals. The algorithm will be later on validated in a multi-centric proof-of-performance clinical study. Disclosure of Interest None declared

Myeloid-derived suppressor cells (MDSCs), identified as Gr1(+)CD11b(+) cells in mice, expand during cancer and promote tumor growth, recurrence and burden. However, little is known about their role in bone metastases. We hypothesized that MDSCs may contribute to tumor-induced bone disease, and inoculated breast cancer cells into the left cardiac ventricle of nude mice. Disease progression was monitored weekly by X-ray and fluorescence imaging and MDSCs expansion by fluorescence-activated cell sorting. To explore the contribution of MDSCs to bone metastasis, we co-injected mice with tumor cells or PBS into the left cardiac ventricle and Gr1(+)CD11b(+) cells isolated from healthy or tumor-bearing mice into the left tibia. MDSCs didn't induce bone resorption in normal mice, but increased resorption and tumor burden significantly in tumor-bearing mice. In vitro experiments showed that Gr1(+)CD11b(+) cells isolated from normal and tumor-bearing mice differentiate into osteoclasts when cultured with RANK ligand and macrophage colony-stimulating factor, and that MDSCs from tumor-bearing mice upregulate parathyroid hormone-related protein (PTHrP) mRNA levels in cancer cells. PTHrP upregulation is likely due to the 2-fold increase in transforming growth factor β expression that we observed in MDSCs isolated from tumor-bearing mice. Importantly, using MDSCs isolated from GFP-expressing animals, we found that MDSCs differentiate into osteoclast-like cells in tumor-bearing mice as evidenced by the presence of GFP(+)TRAP(+) cells. These results demonstrate that MDSCs expand in breast cancer bone metastases and induce bone destruction. Furthermore, our data strongly suggest that MDSCs are able to differentiate into osteoclasts in vivo and that this is stimulated in the presence of tumors.

We have shown that parathyroid hormone-related protein (PTHrP) is a survival factor for human renal cell carcinoma (RCC) and that its expression is negatively regulated by the von Hippel-Lindau (VHL) tumor suppressor gene at the level of messenger RNA (mRNA) stability, as observed for tumor growth factors (TGFs). Our goals were to analyze the alternative splicing of PTHrP mRNA in human RCC and from these results to identify VHL/hypoxia-induced factor (HIF) system-regulated mRNA-binding proteins involved in PTHrP mRNA stability. We used: (i) a panel of human RCC cells expressing or not VHL; (ii) VHL-deficient 786-0 cells transfected with active or inactive VHL and (iii) human RCC samples and corresponding normal tissues. By quantitative real-time reverse transcription-polymerase chain reaction analysis, the 141 PTHrP mRNA isoform was found to be predominant in all cells and tumors (80%). In cells transfected with VHL, the expressions of all isoforms were decreased by 50%. Eight proteins with molecular weights ranging from 20 to 75 kDa were found to bind to biotinylated transcripts spanning the 141 PTHrP mRNA AU-rich 3'-untranslated region whose abundancy was dependent on VHL expression. The protein having an apparent molecular weight of 30 kDa was identified by western blot as HuR, a RNA-binding protein with stabilizing functions on various mRNA coding for proteins important in malignant transformation including vascular endothelial growth factor and TGF-beta. PTHrP expression studies confirmed the involvement of HuR in PTHrP upregulation in this disease. Common mRNA-binding proteins regulated by the VHL/HIF system may constitute new therapeutic opportunities against human RCC that remains refractory to therapies.

We have recently shown that parathyroid hormone-related protein (PTHrP), a cytokine-like polyprotein, is critical for human renal cell carcinoma (RCC) growth by inhibiting tumor cell apoptosis. Here, we have explored mechanisms by which PTHrP controls tumor cell survival. Using specific inhibitors of phosphoinositide 3-kinase (PI3K) and depletion of Akt kinase by RNA interference, we established that PTHrP is one of the main factor involved in the constitutive activation of this pathway in human RCC, independently of von Hippel-Lindau (VHL) tumor suppressor gene expression. Interestingly, PTHrP induced phosphorylation of Akt at S473 but had no influence on phosphorylation at T308. Through transfection with integrin-linked kinase (ILK) constructs and RNA interference, we provide evidence that ILK is involved in human RCC cell survival. PTHrP activates ILK which then acts as a phosphoinositide-dependent kinase (PDK)-2 or a facilitator protein to phosphorylate Akt at S473. Among other kinases tested, only ILK was shown to exert this function in RCC. Using specific inhibitors, western blot and transcription assay, we identified nuclear factor kappa B (NF-kappaB) as the downstream Akt target regulated by PTHrP. Since RCC remains refractory to current therapies, our results establish that the PI3K/ILK/Akt/NF-kappaB axis is a promising target for therapeutic intervention.

// Herve Lang 1 , Claire Beraud 2 , Audrey Bethry 2 , Sabrina Danilin 3 , Veronique Lindner 4 , Catherine Coquard 3 , Sylvie Rothhut 3 , Thierry Massfelder 3 1 Department of Urology, Hopitaux Universitaires de Strasbourg, Nouvel Hopital Civil, Strasbourg, 67091 France 2 UROLEAD SAS, School of Medicine, Strasbourg, 67085 France 3 INSERM U1113, Section of Cell Signalisation and Communication in Kidney and Prostate Cancer, University of Strasbourg, School of Medicine, Federation de Medecine Translationnelle de Strasbourg (FMTS), Strasbourg, 67085 France 4 Department of Pathology, Hopitaux Universitaires de Strasbourg, Hopital de Strasbourg-Hautepierre, Strasbourg, 67200 France Correspondence to: Thierry Massfelder, email: massfeld@unistra.fr Keywords: renal cell carcinoma, human tumors, patient-derived xenograft models Abbreviations: CCC, clear cell renal cell carcinoma; PDX, patient-derived tumor xenograft; VHL, von Hippel-Lindau tumor suppressor gene Received: September 08, 2015 Accepted: July 05, 2016 Published: July 18, 2016 ABSTRACT The objective of the present work was to establish a large panel of preclinical models of human renal cell carcinoma (RCC) directly from patients, faithfully reproducing the biological features of the original tumor. RCC tissues (all stages/subtypes) were collected for 8 years from 336 patients undergoing surgery, xenografted subcutaneously in nude mice, and serially passaged into new mice up to 13 passages. Tissue samples from the primary tumor and tumors grown in mice through passages were analyzed for biological tissue stability by histopathology, mRNA profiling, von Hippel-Lindau gene sequencing, STR fingerprinting, growth characteristics and response to current therapies. Metastatic models were also established by orthotopic implantation and analyzed by imagery. We established a large panel of 30 RCC models (passage > 3, 8.9% success rate). High tumor take rate was associated with high stage and grade. Histopathologic, molecular and genetic characteristics were preserved between original tumors and case-matched xenografts. The models reproduced the sensitivity to targeted therapies observed in the clinic. Overall, these models constitute an invaluable tool for the clinical design of efficient therapies, the identification of predictive biomarkers and translational research.

Metastatic renal cell carcinoma is resistant to current therapies. The phosphoinositide 3-kinase (PI3K)/Akt signaling cascade induces cell growth, cell transformation, and neovascularization. We evaluated whether targeting this pathway could be of therapeutic value against human renal cell carcinoma. The activation of the PI3K/Akt pathway and its role in renal cell carcinoma progression was evaluated in vitro in seven human cell lines by Western blot, cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, terminal deoxyribonucleotide transferase–mediated nick-end labeling assays, and fluorescence-activated cell sorting analysis, using two PI3K inhibitors, LY294002 and wortmannin, as well as by transfection with various Akt constructs and through Akt knockdown by small interfering RNA (siRNA). In vivo nude mice bearing human renal cell carcinoma tumor xenografts were treated with LY294002 (75 mg/kg/wk, 4 weeks, i.p.). Tumor growth was measured and tumors were subjected to Western blot and immunohistochemical analysis. Akt was constitutively activated in all cell lines. Constitutive phosphorylation of glycogen synthase kinase-3 (GSK-3) was observed in all cell lines, whereas forkhead transcription factor and mammalian target of rapamycin, although expressed, were not constitutively phosphorylated. Exposure to LY294002 or wortmannin decreased Akt activation and GSK-3 phosphorylation and reduced cell growth by up to 70% through induction of cell apoptosis. These effects were confirmed by transfection experiments with Akt constructs or Akt siRNA. Importantly, LY294002 induced up to 50% tumor regression in mice through tumor cell apoptosis. Tumor neovascularization was significantly increased by LY294002 treatment. Blood chemistries showed no adverse effects of the treatment. Our results suggest an important role of PI3K/Akt inhibitors as a potentially useful treatment for patients with renal cell carcinoma. (Cancer Res 2006; 66(10): 5130-42)

Human conventional renal cell carcinoma (CCC) remains resistant to current therapies. Focal Adhesion Kinase (FAK) is upregulated in many epithelial tumors and clearly implicated in nearly all facets of cancer. However, only few reports have assessed whether FAK may be associated with renal tumorigenesis. In this study, we investigated the potential role of FAK in the growth of human CCC using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as normal/tumoral renal tissue pairs. FAK was found constitutively expressed in human CCC both in culture cells and freshly harvested tumors obtained from patients. We showed that CCC cell growth was dramatically reduced in FAK-depleted cells or after FAK inhibition with various inhibitors and this effect was obtained through inhibition of cell proliferation and induction of cell apoptosis. Additionally, our results indicated that FAK knockdown decreased CCC cell migration and invasion. More importantly, depletion or pharmacological inhibition of FAK substantially inhibited tumor growth in vivo. Interestingly, investigations of the molecular mechanism revealed loss of FAK phosphorylation during renal tumorigenesis impacting multiple signaling pathways. Taken together, our findings reveal a previously uncharacterized role of FAK in CCC whereby FAK exerts oncogenic properties through a non canonical signaling pathway involving its scaffolding kinase-independent properties. Therefore, targeting the FAK scaffold may represent a promising approach for developing innovative and highly specific therapies in human CCC.

Prostate, bladder and kidney cancers represent 1 900 000 new cases and 620 000 deaths per year worldwide with an incidence increasing by 1-10% each year. Surgery is curative at localized stages; however, current therapies are inefficient at advanced stages. One of the major needs in new drugs development is the availability of clinically pertinent models faithfully reproducing the heterogeneity of patient tumors. Patient-derived tumor xenografts (PDX) are thus developed as essential tools for drug testing and identification of predictive biomarkers for a better clinical response, the first step for personalized medicine. Prostate, bladder and kidney tumors were obtained from patients undergoing surgery, subcutaneously (and some orthopically) xenografted in nude mice and serially passaged up to passage (P) 12. Normal corresponding tissues were also harvested. Tissues were conserved at all passages for characterization and grafting. For all patients, informed consent and clinical history are available. For the 3 cancers, primary tumor and tumors grown in mice were characterized for growth behavior, histopathology, genetic stability (short tandem repeat fingerprinting), mRNA expression profiling and response to current therapies. For each cancer type, we also investigated more specific features: expression of the androgen receptor, PSA and pan-cytokeratin for prostate PDXs, expression and status of hotspot mutations including FGFR3, PIK3CA, HRAS, RXRa and p53 for bladder PDXs, and von Hippel-Lindau gene mutation status for kidney PDXs. Metastatic models were followed by infrared imagery (IR780 dye). So far and since 9 years, we have collected 230 prostate tumors, 130 bladder tumors and 336 kidney tumors at all stages, and established 5, 25 and 31 models (> P3 in mice) respectively (8.8% success rate). Tumor take rate was positively correlated to advanced stage and high grade and for kidney cancer, to sarcomatoid component. Tumor growth evaluation reveals that PDXs were stable from mouse to mouse and throughout passages. Histopathologic and genetic characteristics were preserved between original tumors and case-matched PDXs. Molecular characteristics were also stable with less than 5% of genes differentially expressed between the primary tumors and the PDXs. In bladder PDXs, this analysis and mutation status of the selected genes allowed to define molecular subtypes. The comparison with patient therapeutic response, when available, showed the clinical predictivity of the models. Orthotopic models developed metastases at classical sites. In conclusion, we developed here a unique platform of preclinical PDXs models for urologic cancers with stable biological characteristics and clinically predictive. This is an invaluable tool for the clinical design of efficient therapies, the identification of predictive biomarkers and translational research. Citation Format: Hervé Lang, Claire Béraud, Audrey Bethry, Sabrina Danilin, Véronique Lindner, Catherine Coquard, Sylvie Rothhut, Thierry Massfelder. Establishment of a large panel of patient-derived tumor xenograft models of prostate, bladder and kidney cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 635.

Background Millions of patients suffering Rheumatoid Arthritis (RA) are treated with TNF-α inhibiting agents, however, response rate is low (30 to 40%) and no tool exists to predict the treatment response. Objectives Using the HTG-Edgeseq platform, an innovative combination of nuclease protection assay and next generation sequencing, we identified sets of miRNAs that discriminate responders from non-responders to anti TNF-α therapy. Methods Sixty-seven patients diagnosed with RA, eligible for treatment with 1st line anti-TNFα and for whom DMARD therapy had failed were enrolled in the study. Twelve to 14 weeks after anti-TNFα therapy, patients were categorized as responders or non-responders based on DAS28 index. Patients9 miRNA profile was established from 15μl of plasma using HTG-Edgeseq Whole Transcriptome Assay (WTA) miRNA panel (2256 miRNA). Results were normalized based on the median of the sample and Random forest was used as the classification model. For 8 patients, miRNA were also analyzed with the qPCR Exiqon miRNA panel V4 to determine HTG-Edgeseq accuracy. Method reproductibility was assessed by analyzing 4 times an independent sample on different sites, with different instruments/days/operators. Results Results obtained from both HTG-Edgeseq and qPCR methods showed an overall correlation of 0.63 for the 341 miRNA common between those 2 kits, and correlations factors between the 4 independent experiments ranged from 0.993 to 0.999. Statistical analysis of patients9 miRNA profile identified 2 panels of 6 and 52 miRNAs with significant predictive power to discriminate responders from non-responders (sensitivity was 0.898 and 0.918, and AUC 0.773 and 0.824 respectively). Conclusions In conclusion, miRNA profiling in RA patients using HTG-EdgeSeq allowed us to build 2 predictive models for response to anti-TNF-α drugs. Moreover, we showed that HTG-Edgeseq platform offers accurate and sensitive miRNA expression measurement. Its low sample input requirement and compatibility with all biological material makes it an invaluable tool for biomarkers discovery. Disclosure of Interest None declared

Breast cancer cells frequently metastasize to bone, where they up-regulate their expression of the transcription factor GLI2 and the downstream osteolytic factor parathyroid hormone-related protein (PTHrP). The guanosine nucleotide 6-thioguanine (6-TG) inhibits PTHrP expression and blocks osteolytic bone destruction in mice inoculated with bone metastatic cells; however, the mechanism by which 6-TG inhibits PTHrP remains unclear. We hypothesized that 6-TG inhibition of PTHrP is mediated through GLI2 signaling.Human MDA-MB-231 breast cancer cells and RWGT2 squamous-cell lung carcinoma cells were treated with 100 μM 6-TG and examined for GLI2 mRNA expression and stability by Q-PCR, promoter activity by luciferase assay, and protein expression by Western blot.6-TG significantly blocked GLI2 mRNA and protein expression, but did not affect stability. Additionally, 6-TG directly inhibited GLI2 promoter activity, and when cells were transfected with constitutively expressed GLI2, the inhibitory effect of 6-TG on PTHrP expression was abolished.Taken together, these data indicate that 6-TG regulates PTHrP in part through GLI2 transcription, and therefore the clinical use of 6-TG or other guanosine nucleotides may be a viable therapeutic option in tumor types expressing elevated levels of GLI proteins.

Human conventional renal cell carcinoma (CRCC) remains resistant to therapy. The RNA-binding protein HuR regulates the stability and/or translation of multiple messenger RNAs involved in malignant transformation. In this study, we aimed to evaluate the potential role of HuR in this pathology. Using seven human CRCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as 15 normal/renal cell carcinoma tumor pairs, we showed that HuR is overexpressed in all tumors independently of the VHL status. Futhermore, HuR cytoplasmic presence appears to be more common in early tumor stages, suggesting a role in tumor promotion. We then assessed the effect of HuR knockdown using small interfering RNA in cultured cell and in tumor-bearing mice. Both in vitro and in vivo, we observed that cell growth was inhibited by 60% and that this effect was obtained through an inhibition of cell proliferation and an induction of cell apoptosis. Finally, we found that expression of vascular endothelium growth factor, tumor growth factor-beta and of the hypoxia-induced transcription factor-2alpha as well as the constitutive activation of the oncogenic phosphoinositide 3-kinase/Akt, nuclear factor-kappaB and mitogen-activated protein kinase pathways were decreased in HuR-depleted cells and tumors. All these results suggest a pivotal role for HuR in human CRCC.

Background Human clear cell renal cell carcinoma (CRCC) remains resistant to therapies. Recent advances in Hypoxia Inducible Factors (HIF) molecular network led to targeted therapies, but unfortunately with only limited clinical significance. Elucidating the molecular processes involved in kidney tumorigenesis and resistance is central to the development of improved therapies, not only for kidney cancer but for many, if not all, cancer types. The oncogenic PI3K/Akt, NF-kB and MAPK pathways are critical for tumorigenesis. The sonic hedgehog (SHH) signaling pathway is crucial to normal development. Results By quantitative RT-PCR and immunoblot, we report that the SHH signaling pathway is constitutively reactivated in tumors independently of the von Hippel-Lindau (VHL) tumor suppressor gene expression which is inactivated in the majority of CRCC. The inhibition of the SHH signaling pathway by the specific inhibitor cyclopamine abolished CRCC cell growth as assessed by cell counting, BrdU incorporation studies, fluorescence-activated cell sorting and β-galactosidase staining. Importantly, inhibition of the SHH pathway induced tumor regression in nude mice through inhibition of cell proliferation and neo-vascularization, and induction of apoptosis but not senescence assessed by in vivo studies, immunoblot and immunohistochemistry. Gli1, cyclin D1, Pax2, Lim1, VEGF, and TGF-β were exclusively expressed in tumors and were shown to be regulated by SHH, as evidenced by immunoblot after SHH inhibition. Using specific inhibitors and immunoblot, the activation of the oncogenic PI3K/Akt, NF-kB and MAPK pathways was decreased by SHH inhibition. Conclusions These findings support targeting SHH for the treatment of CRCC and pave the way for innovative and additional investigations in a broad range of cancers.

Metastatic renal cell carcinoma (RCC) remains refractory to therapies. The nuclear factor-κB (NF-κB) transcription factor is involved in cell growth, cell motility, and vascularization. We evaluated whether targeting NF-κB could be of therapeutic and prognostic values in human RCC. The activation of the NF-κB pathway in human RCC cells and tumors was investigated by Western blot. In vitro, the effects of BAY 11-7085 and sulfasalazine, two NF-κB inhibitors, on tumor cell growth were investigated by cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, and fluorescence-activated cell sorting. Their specificity toward NF-κB was analyzed by Western blot, confocal microscopy, NF-κB small interfering RNA, and NF-κB transcription assay. In vivo, the effects of BAY 11-7085 on the growth of human RCC tumors were investigated in nude mice. A tissue microarray (TMA) containing 241 cases of human RCC with 12 to 22 years of clinical follow-up and corresponding normal tissues was built up to assess prognostic significance of activated NF-κB. NF-κB is constitutively activated in cultured cells expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as a consequence of Akt kinase activation and in tumors. In vitro and in vivo NF-κB inhibition blocked tumor cell growth by inducing cell apoptosis. On the TMA, NF-κB activation was correlated with tumor dimension but was not found to be an independent prognostic factor for patient survival. This report provides strong evidence that the mechanisms responsible for the intrinsic resistance of RCC cells to apoptosis converge on NF-κB independently of VHL expression and that targeting this pathway has great anticancer potential. [Cancer Res 2007;67(24):11668–76]

Kidney, bladder and prostate cancers are the 3 prevalent urological cancers. They represent each year 1 500 000 new cases and 500 000 deaths worldwide with an incidence increasing steadily by 2-10% per year. At advanced stages, current first-line therapies are inefficient. One of the major needs in new drugs development is the availability of a large panel of clinically pertinent models representing patients’ heterogeneity. In addition, our ability to tailor specific drugs to patients is restricted by the lack of validated biomarkers and might be considered as the most important barrier for a better clinical response. Kidney, bladder and prostate tumor tissues at all stages were collected from patients undergoing surgery, xenografted in nude mice, and serially passaged into new mice. All tumors were implanted subcutaneously and some of them orthotopically. Informed consent and clinical history were obtained for all patients. For all 3 cancer types, tissue samples from the patients’ primary tumor and tumors grown in mice through passages were analyzed for primary tumor-models stability by histopathology, mRNA expression profiling, growth behavior and response to first-line therapies (sunitinib/sorafenib/everolimus, cisplatin and docetaxel for kidney, bladder and prostate cancer respectively). In addition, for each cancer type and models derived from it, we also analyzed more specific tumor characteristics for their stability: the VHL status and the genetic stability by short tandem repeat fingerprinting for kidney cancer; the expression of interest genes including FGFR3, HRAS or p53 genes for bladder cancer, and the expression/mutations of the androgen receptor and the expression of prostate specific antigen and cytokeratin for prostate cancer. Orthotopic models were followed by infrared imaging after intravenous injection of the IR780 dye. So far and since 2007, we have xenografted 380 kidney tumors, 80 bladder tumors and 150 prostate tumors, out of which we have established 31, 11 and 3 models (passage above 3 and up to 12 in mice), respectively. The tumor take rate in mice (7.4% overall success) was significantly correlated with tumor stages, but not with any other tumor features. Importantly, tumors were stable through passages at the histopathologic, molecular and genetic levels. In addition, the responsiveness of tumors grown in mice to current therapies compounds was similar to patients’ therapeutic responses, also highlighting the clinical predictivity of our models. Orthotopic models developed metastases at classical secondary sites. We developed a unique platform of well characterized, stable and clinically predictive mouse xenograft urological cancer models. This is an invaluable tool for the clinical design of efficient therapies and for the identification of predictive biomarkers. Citation Format: Thierry Massfelder, Claire Beraud, Audrey Bethry, Sabrina Danilin, Véronique Lindner, Didier Jacqmin, Hervé Lang. Development and characterization of a large panel of tumorgraft-based clinically relevant models derived from patients with kidney, bladder or prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2773. doi:10.1158/1538-7445.AM2013-2773

INTRODUCTION AND OBJECTIVE: Human renal cell carcinoma (RCC) remains resistant to therapy. Clear cell RCC (CCC) is the major sub-type (>75%). The RNA-binding protein HuR is crucial for stabilization and/or translation of ARE-containing labile mRNAs such as those encoding VEGF orTGF, that are involved in malignant transformation. The potential role of HuR in human CCC growth is unknown and was evaluated in vitro and in vivo. METHODS: We used 7 human CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene, as well as RCC tumors (7 pT1 and 8 pT3) and corresponding normal tissues. The total and subcellular expression of HuR was assessed by western blot. In vitro, the effect of HuR on cell proliferation and death was analyzed by cell counting, BrdU incorporation studies, and FACS in cells transfected with HuR siRNA (siHuR) or with control siRNA (siCtl). In vivo in nude mice bearing human 786-0 RCC tumors, the effects of HuR silencing on tumor growth was measured after intratumoral injection of siHuR or siCtl (10 mice/group, 10 nmol/kg, 2 times/week, 3 weeks). Tumor cell proliferation and death and vascularization in treated mice were analyzed by Ki67, TUNEL and factor VIII stainings. The expression of VEGF, TGF, and HIF-2 , as well as the activation of the survival pathways PI3K/Akt, NFkB and MAPK in cells and xenografted tumors treated with siHuR or siCtl was quantified by ELISA or western blot. RESULTS: The analysis of HuR expression in cells and tumorsnonmalignant tissues pairs showed that HuR is overexpressed in tumors and that the cytoplasmic presence of HuR is more common in early stages. No differences in HuR expression and localization were observed depending on the VHL status. Cell growth was inhibited by more than 50% when HuR was down-regulated. This effect was obtained through inhibition of cell proliferation and induction of apoptosis. Injection of siHuR in nude mice inhibited tumor growth by 60% compared to tumors injected with siCtl, at least through decrease in cell proliferation and increase in cell apoptosis. The effect on vascularization is under investigation. As a consequence of HuR down-regulation, the expression of VEGF and TGFwas decreased by 50% in both cells and tumors. Very interestingly, the expression of HIF-2 as well as the activaction of PI3K/Akt, NFB and MAPK pathways were also decreased in HuR-depleted cells and tumors. CONCLUSIONS: These results argue for an important role for HuR in human CCC growth and may lead to the design of new therapeutics.