Claire Béraud

Vitamin D3 triggers antitumor activity through targeting hedgehog signaling in human renal cell carcinoma

Human clear cell renal cell carcinoma (CCC) remains resistant to treatments despite the progress in targeted therapies. Several signaling pathways acting during renal development are reactivated during kidney tumorigenesis; this is the case of the sonic hedgehog (SHH)-Gli. Interestingly, the precursor of active vitamin D3 (VD3), cholecalciferol, has been demonstrated to be a strong inhibitor of SHH-Gli signaling. Here, we show the preclinical efficacy of cholecalciferol in CCC both in vitro and in vivo. A panel of CCC cell lines, tumors and normal corresponding tissues from CCC patients were used to evaluate the expression of the VD3 receptor and metabolizing enzymes and the effects of cholecalciferol treatment. Subsequently, xenografted mice were treated with cholecalciferol in a prophylactic or therapeutic manner; their response and the adverse effects were evaluated on the basis of weekly monitoring, followed by blood collection procedures and X-ray micro-computed tomography. VD3 receptor and metabolizing enzymes are dramatically decreased in human cell lines and tumors. Cholecalciferol decreases cell proliferation and increases cell death by inhibition of the SHH-Gli pathway. Xenografted mice treated with cholecalciferol exhibit absence of tumor development or substantial growth inhibition. The treatment was shown to be safe; it did not induce calcification or calcium reabsorption. These findings establish that, although VD3 receptors and metabolizing enzymes are absent in CCC, cholecalciferol supplementation is a strong tool to block the reactivation of SHH-Gli pathway in this pathology, leading ultimately to tumor regression. Cholecalciferol may have highly therapeutic potential in CCC.

LIM-class homeobox gene Lim1 , a novel oncogene in human renal cell carcinoma

Human clear cell renal cell carcinoma (CCC) remains resistant to therapies. The transcription factor LIM-class homeobox gene Lim1 is required for normal organogenesis, including nephrogenesis, by regulating cell movements, differentiation and growth. Its expression is controlled partly by the sonic hedgehog-Gli signaling pathway, which we have recently shown to be reactivated in human CCC. So far, no study has assessed whether Lim1 may be associated with tumorigenesis. Using a panel of human CCC cell lines expressing or not the von Hippel-Lindau tumor suppressor gene and 44 tumor/normal tissues pairs, we found that Lim1 is constitutively and exclusively reexpressed in tumors. Through Lim1 silencing or overexpressing, we show that Lim1 is a growth and survival factor in human CCC, at least through the activation of oncogenic pathways including the phosphoinositide kinase-3/Akt and nuclear factor-kappaB pathways. More importantly, in nude mice bearing human CCC tumors, Lim1 silencing abolished tumor growth through the same mechanism as in vitro. In Lim1-depleted cells and tumors, cell movements were substantially impaired because of the inhibition of expression of various proteins involved in metastatic spread, such as paxillin or tenascin-C. These findings establish that the developmental marker Lim1 acts as an oncogene in cancer cells and targeting Lim1 may constitute an innovative therapeutic intervention in human CCC.

Establishment of a large panel of patient-derived preclinical models of human renal cell carcinoma

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.

Targeting FAK scaffold functions inhibits human renal cell carcinoma growth.

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.

Parathyroid hormone-related protein modulates mice mesangial cells inflammation and blunts apoptosis by enhancing Cox-2 expression

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.

The Lim1 oncogene as a new therapeutic target for metastatic human renal cell carcinoma

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.

An FGFR3/MYC positive feedback loop provides new opportunities for targeted therapies in bladder cancers

FGFR3 alterations (mutations or translocation) are among the most frequent genetic events in bladder carcinoma. They lead to an aberrant activation of FGFR3 signaling, conferring an oncogenic dependence, which we studied here. We discovered a positive feedback loop, in which the activation of p38 and AKT downstream from the altered FGFR3 upregulates MYC mRNA levels and stabilizes MYC protein, respectively, leading to the accumulation of MYC, which directly upregulates FGFR3 expression by binding to active enhancers upstream from FGFR3. Disruption of this FGFR3/MYC loop in bladder cancer cell lines by treatment with FGFR3, p38, AKT, or BET bromodomain inhibitors (JQ1) preventing MYC transcription decreased cell viability in vitro and tumor growth in vivo. A relevance of this loop to human bladder tumors was supported by the positive correlation between FGFR3 and MYC levels in tumors bearing FGFR3 mutations, and the decrease in FGFR3 and MYC levels following anti‐FGFR treatment in a PDX model bearing an FGFR3 mutation. These findings open up new possibilities for the treatment of bladder tumors displaying aberrant FGFR3 activation.

MP87-08 ROLE OF THE C-MYC TARGET DNPH1, A NEW N-HYDROLASE, IN KIDNEY AND PROSTATE CANCERS

contrast, in PC3 cells, there was only a significant (3-fold) upregulation of ProL1 after 24h of hypoxia, with other markers of the hypoxic response (Hif1a and VEGF) significantly activated after 5h. CONCLUSIONS: For the first time we report up-regulation of the opiorphin gene (ProL1) in PrCa. Opiorphin is a mediator of the hypoxic response in certain cell types. We demonstrate hypoxic upregulation of ProL1 in two PrCa cell lines. Early upregulation in LnCaP cells, prior to other known markers of the hypoxic response (Hif1a and VEGF) suggests in certain PrCa cells (androgen dependent) ProL1may play a role in regulating downstream modulators of the hypoxic response. Taken together, these results suggest opiorphin may play a role in overcoming the hypoxic environment in certain PrCa tumors and might therefore represent a novel therapeutic for PrCa.

MP73-07 INVOLVEMENT OF ANGIOGENIN IN SUNITINIB RESISTANCE IN HUMAN RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Clear cell renal cell carcinoma (CCC), the main subtype of kidney cancer, represents 250 000 cases and 110 000 deaths worldwide/year. In 75% of cases there is a loss of the von Hippel-Lindau (VHL) tumor suppressor gene that is involved in hypoxia inducible factors (HIF) degradation. Current targeted therapies for advanced CCC arise from the knowledge of the VHL/HIF system. These include sunitinib, sorafenib and everolimus. Their efficiency is however limited due to acquired resistance. Since the classical mechanisms of resistance are not involved in CCC, we hypothesized that signalling pathways and/or markers are involved in such resistance. We focused here on sunitinib. METHODS: We used a panel of CCC cell lines expressing VHL (Caki-1, Caki-2, ACHN) or not (786-0, A498) and tumor/normal tissues pairs from 50 CCC patients. We analyzed tumor growth in response to sunitinib both in vitro using Cristal Violet assay and in vivo using the xenografted nude mice tumor model (40 mg/kg, 3 times/week, 4 weeks, per os). We analyzed protein expression and signaling pathways by RTqPCR, Western blot, proteome arrays specific for phosphokinases, apoptosis and angiogenesis and with the HTG EdgeSeq oncology biomarker panel assay (2560 genes of 24 signaling pathways). RESULTS: In vitro, sunitinib inhibited cell growth of all cell lines tested by up to 85%. In vivo, however, xenografted 786-0 and A498 tumors were resistant to sunitinib. By analyzing A498 tumors we identified, among others, angiogenin (Ang), a pro-angiogenic factor, as being stimulated more than 10 fold by sunitinib. The stimulation of Ang by sunitinib was also observed in all cell lines. Interestingly, in tissue pairs, the expression of Ang was found to be deregulated in 75% of tumors, regardless of tumor stage and grade. Ang stimulates rRNA transcription after nuclear translocation and activates oncogenic signaling pathways (Akt, JunK, MAPK) through a uncharacterized transmembrane receptor. Ang expression and activity has never been functionally linked to sunitinib. In 786-0 tumors-bearing nude mice, tumoral growth was inhibited by 30% by neamine, an inhibitor of Ang nuclear translocation, but it had no effect on sunitinib resistance. Akt and GSK-3 were also activated in vitro and in vivo by sunitinib. We are now performing in vivo studies using siRNA targeting Ang allowing to also inhibit its effect on oncogenic pathways, alone and in combination with sunitinib. CONCLUSIONS: Taken together, these results strongly suggest that Ang is involved in sunitinib resistance in human CCC, opening new therapeutic option for this refractory disease.

MP60-09 LIM1 ONCOGENE AS A NEW THERAPEUTIC TARGET IN ADVANCED HUMAN RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Clear cell renal cell carcinoma (CCC) is resistant to therapies. We and others have shown the oncogenicity of various signaling pathways/markers including the PI3K/Akt, NF-kB, MAPK, sonic hedgehog (SHH)-Gli and Notch pathways and Pax2 transcription factor. These are also pathways/markers involved in nephrogenesis leading us to hypothesize that tumor cells hijack developmental signaling pathways/markers for their own growth. Among Gli targets, we have identified the developmental Lim1 transcription factor as a new oncogene in CCC, regulating tumor growth. Preliminary results also suggested a role in metastasis development. Here, we subsequently investigated whether Lim1 has a role in advanced CCC. METHODS: Human 786-0, A498 (VHL-) and Caki2, ACHN (VHL+) cells were used. No chemical inhibitor of Lim1 is available. We thus investigated its role in tumor invasion using siRNA and Lim1 expressing vector. In vitro, Lim1 effect on cell motility, migration and invasion was studied in cells transiently transfected with Lim1 siRNAs for 24-96h, by wound healing assay, and using uncoated and Matrigelcoated Boyden chamber respectively. We assessed the expression of various proteins involve in cell movements after Lim1 silencing by Western blot and PCR. We also analyzed Lim1 expression in 8 metastatic samples (lymph node and adrenal metastases). To study the impact of Lim1 in vivo, we developed a model that we calibrated for metastasis spread qualitatively and quantitatively through injection of 50 000 tumor cells into the tail vein of nude mice. Untransfected cells and cells transfected with a vector expressing Lim1 or Lim1 siRNA were used. 10 days after cell injection mice were euthanized and organs were harvested for metastases analysis and molecular studies using the HTG EdgeSeq Oncology Biomarker Panel Assay (2560 genes). RESULTS: Lim1 expression was downregulated by > 95% after siRNAs transfection. In all cell lines, the depletion of Lim1 inhibited not only cell movements by up to 50 % in a time-dependent manner, but also the expression of various proteins including Fibronectin, MMP8/9, Paxillin, and CXCR4. Lim1 was found in all metastatic samples and the corresponding primary tumor. As expected, in vivo, lung and liver metastasis developed within 10 days post-injection. We are currently analyzing organs in terms of size and number of metastases. First results suggest that Lim1 is involved in metastasis development. Upcoming experiments will define the molecular mechanisms of such effects. CONCLUSIONS: These results show that targeting Lim1 has therapeutic potential in this refractory disease.