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.

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.

Role of the RNA-binding protein HuR in human renal cell carcinoma.

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.

The sonic hedgehog signaling pathway is reactivated in human renal cell carcinoma and plays orchestral role in tumor growth.

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 beta-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-beta 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.

von Hippel-Lindau tumor suppressor gene-dependent mRNA stabilization of the survival factor parathyroid hormone-related protein in human renal cell carcinoma by the RNA-binding protein HuR.

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.

Targeting the nuclear factor-kappaB rescue pathway has promising future in human renal cell carcinoma therapy.

Metastatic renal cell carcinoma (RCC) remains refractory to therapies. The nuclear factor-kappaB (NF-kappaB) transcription factor is involved in cell growth, cell motility, and vascularization. We evaluated whether targeting NF-kappaB could be of therapeutic and prognostic values in human RCC. The activation of the NF-kappaB pathway in human RCC cells and tumors was investigated by Western blot. In vitro, the effects of BAY 11-7085 and sulfasalazine, two NF-kappaB 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-kappaB was analyzed by Western blot, confocal microscopy, NF-kappaB small interfering RNA, and NF-kappaB 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-kappaB. NF-kappaB 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-kappaB inhibition blocked tumor cell growth by inducing cell apoptosis. On the TMA, NF-kappaB 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-kappaB independently of VHL expression and that targeting this pathway has great anticancer potential.

Parathyroid hormone-related protein induces cell survival in human renal cell carcinoma through the PI3K Akt pathway: evidence for a critical role for integrin-linked kinase and nuclear factor kappa B.

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.

The phosphoinositide 3-kinase/Akt pathway: a new target in human renal cell carcinoma therapy.

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.

Antitumor effect of parathyroid hormone-related protein neutralizing antibody in human renal cell carcinoma in vitro and in vivo.

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in 40-80% of human conventional renal cell carcinomas (RCCs). We showed recently that VHL-deficient RCCs expressed large amounts of parathyroid hormone-related protein (PTHrP), and that PTHrP, acting through the PTH1 receptor (PTH1R), plays an essential role in tumor growth. We also showed that PTHrP expression is negatively regulated by the VHL gene products (pVHL). Our goal was to determine whether blocking the PTHrP/PTH1R system might be of therapeutic value against RCC, independent of VHL status and PTHrP expression levels. The antitumor activity of PTHrP neutralizing antibody and of PTH1R antagonist were evaluated in vitro and in vivo in a panel of human RCC lines expressing or not pVHL. PTHrP is upregulated compared with normal tubular cells. In vitro, tumor cell growth and viability was decreased by up to 80% by the antibody in all cell lines. These effects resulted from apoptosis. Exogenously added PTHrP had no effect on cell growth and viability, but reversed the inhibitory effects of the antibody. The growth inhibition was reproduced by a specific PTH1R antagonist in all cell lines. In vivo, the treatment of nude mice bearing the Caki-1 RCC tumor with the PTHrP antibody inhibited tumor growth by 80%, by inducing apoptosis. Proliferation and neovascularization were not affected by the antiserum. Anti-PTHrP treatment induced no side effects as assessed by animal weight and blood chemistries. Current therapeutic strategies are only marginally effective against metastatic RCC, and adverse effects are common. This study provides a rationale for evaluating the blockade of PTHrP signaling as therapy for human RCC in a clinical setting.

Role of parathyroid hormone-related protein in the regulation of stretch-induced renal vascular smooth muscle cell proliferation.

In vivo, vascular smooth muscle cells (VSMC) are continuously exposed to mechanical cyclic stretch as a result of the pulsatile blood flow from the cardiac contractile cycle. Stretch is altered in pathologic conditions and contributes to vascular remodeling by modulating VSMC proliferation and death. Parathyroid hormone-related protein (PTHrP) is a locally produced poly-protein that regulates cell growth. It was shown previously that PTHrP inhibits VSMC proliferation through the auto/paracrine pathway by interacting with its receptor, the PTH1R, but stimulates VSMC proliferation through the intracrine pathway by translocating into the nucleus. In the current study, VSMC that were isolated from both resistance and compliance vessels were used to study the role of PTHrP in VSMC proliferation under experimental stretch. It is shown that PTHrP gene expression is upregulated by stretch and that PTHrP opposes the inhibitory effect induced by stretch on VSMC proliferation through the intracrine pathway. In addition, it is demonstrated that PTHrP expression is controlled at the post-transcriptional level by stretch. Taken together, these results strongly suggest that PTHrP plays a critical role in the modulation of VSMC proliferation in response to stretch. Thus, in conditions in which stretch is increased, such as in hypertension or in restenosis after angioplasty, PTHrP may contribute to vessel hyperplasia.

Intravenous delivery of PTH/PTHrP type 1 receptor cDNA to rats decreases heart rate, blood pressure, renal tone, renin angiotensin system, and stress-induced cardiovascular responses.

While parathyroid hormone type 1 receptor (PTH1R)-mediated vasodilatory, cardiac stimulatory, and renin-activating effects of exogenous PTH/PTH-related protein (PTHrP) are acknowledged, interactions of endogenous PTHrP with these systems remain unclear, mainly because the unavailability of viable PTHrP/PTH1R knockout mice. Transgenic mice overexpressing PTH1R in smooth muscle strongly have supported the PTHrP/PTH1R system as a cardiovascular system (CVS) regulator, but the consequences on renovascular (RVS) and renin-angiotensin systems (RAS) have not been explored in these studies. The aim was to develop a model in which one could study the consequences on CVS, RVS, and RAS of generalized PTH1R overexpression. Systemic PTH1R cDNA plasmid delivery was used in adult rats, a system that is amenable to studies in isolated perfused kidneys and that minimizes development-induced compensatory mechanisms. Intravenous administration of hPTH1R or green fluorescence protein-tagged hPTH1R in pcDNA3 resulted 3 wk later, in generalized expression of hPTH1R (mRNA and protein), especially in vessels, liver, heart, kidney, and central nervous system, where it is expressed physiologically. As expected, PTH1R overexpression decreased BP and renal tone. Unexpected, however, PTH1R overexpression decreased heart rate. These studies also revealed that endogenous PTHrP actually inhibits renin release and that hPTH1R overexpression tends to increase that effect. Striking, liver production and circulatory level of angiotensinogen and hence plasma renin activity were markedly reduced. Thus, abrupt PTH1R overexpression in adult rats profoundly alters the CVS, RVS, and RAS, strongly supporting the PTH/PTHrP/PTH1R system as crucial for heart and vascular tone regulation. In addition, these results revealed that PTH1R-mediated mechanisms might have protective effects against cardiovascular stress-induced responses, including stimulations in heart rate and RAS.

Parathyroid hormone-related protein is an essential growth factor for human clear cell renal carcinoma and a target for the von Hippel-Lindau tumor suppressor gene.

Clear cell renal carcinoma (CCRC) is responsible for 2% of cancer-related deaths worldwide and is resistant to virtually all therapies, indicating the importance of a search for new therapeutic targets. Parathyroid hormone-related protein (PTHrP) is a polyprotein derived from normal and malignant cells that regulates cell growth. In the current study, we show that blocking PTHrP with antibodies or antagonizing the common parathyroid hormone (PTH)/PTHrP receptor, the PTH1 receptor, dramatically blunts the expansion of human CCRC in vitro by promoting cell death. Importantly, in nude mice, anti-PTHrP antibodies induced complete regression of 70% of the implanted tumors by inducing cell death. In addition, we demonstrate that the von Hippel-Lindau tumor suppressor protein, which functions as a gatekeeper for CCRC, negatively regulates PTHrP expression at the post-transcriptional level. These studies indicate that PTHrP is an essential growth factor for CCRC and is a novel target for the von Hippel-Lindau tumor suppressor protein. Taken together, these results strongly suggest that targeting the PTHrP/PTH1 receptor system may provide a new avenue for the treatment of this aggressive cancer in humans.