A randomized, double-blind phase 1b study evaluating the safety, tolerability, pharmacokinetics and pharmacodynamics of the NLRP3 inhibitor selnoflast in patients with moderate to severe active ulcerative colitis.

BACKGROUND: The NLRP3 inflammasome drives release of pro-inflammatory cytokines including interleukin (IL)-1ß and IL-18 and is a potential target for ulcerative colitis (UC). Selnoflast (RO7486967) is an orally active, potent, selective and reversible small molecule NLRP3 inhibitor. We conducted a randomized, placebo-controlled Phase 1b study to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of selnoflast. METHODS: Nineteen adults with previous diagnosis of UC and current active moderate to severe disease were randomized 2:1 to selnoflast or placebo for 7 days. A dose of 450 mg QD (once daily) was selected to achieve 90% IL-1ß inhibition in plasma and colon tissue. Consecutive blood, sigmoid colon biopsies and stool samples were analyzed for a variety of PD markers. Safety and PK were also evaluated. RESULTS: Selnoflast was well-tolerated. Plasma concentrations increased rapidly after oral administration, reaching Tmax 1 h post-dose. Mean plasma concentrations stayed above the IL-1ß IC90 level throughout the dosing interval (mean Ctrough on Day 1 and Day 5: 2.55 µg/mL and 2.66 µg/mL, respectively). At steady state, post-dose selnoflast concentrations in sigmoid colon (5-20 µg/g) were above the IC90 . Production of IL-1ß was reduced in whole blood following ex vivo stimulation with lipopolysaccharide (LPS) (in the selnoflast arm). No changes were observed in plasma IL-18 levels. There were no meaningful differences in the expression of an IL-1-related gene signature in sigmoid colon tissue, and no differences in the expression of stool biomarkers. CONCLUSIONS: Selnoflast was safe and well-tolerated. Selnoflast 450 mg QD achieved plasma and tissue exposure predicted to maintain IL-1ß IC90 over the dosing interval. However, PD biomarker results showed no robust differences between treatment arms, suggesting no major therapeutic effects are to be expected in UC. The limitations of this study are its small sample size and indirect assessment of the effect on IL-1ß in tissue. TRIAL REGISTRATION: ISRCTN16847938.

Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies.

PURPOSE: Target-dependent TCB activity can result in the strong and systemic release of cytokines that may develop into cytokine release syndrome (CRS), highlighting the need to understand and prevent this complex clinical syndrome. EXPERIMENTAL DESIGN: We explored the cellular and molecular players involved in TCB-mediated cytokine release by single-cell RNA-sequencing of whole blood treated with CD20-TCB together with bulk RNA-sequencing of endothelial cells exposed to TCB-induced cytokine release. We used the in vitro whole blood assay and an in vivo DLBCL model in immunocompetent humanized mice to assess the effects of dexamethasone, anti-TNFα, anti-IL6R, anti-IL1R, and inflammasome inhibition, on TCB-mediated cytokine release and antitumor activity. RESULTS: Activated T cells release TNFα, IFNγ, IL2, IL8, and MIP-1ß, which rapidly activate monocytes, neutrophils, DCs, and NKs along with surrounding T cells to amplify the cascade further, leading to TNFα, IL8, IL6, IL1ß, MCP-1, MIP-1α, MIP-1ß, and IP-10 release. Endothelial cells contribute to IL6 and IL1ß release and at the same time release several chemokines (MCP-1, IP-10, MIP-1α, and MIP-1ß). Dexamethasone and TNFα blockade efficiently reduced CD20-TCB-mediated cytokine release whereas IL6R blockade, inflammasome inhibition, and IL1R blockade induced a less pronounced effect. Dexamethasone, IL6R blockade, IL1R blockade, and the inflammasome inhibitor did not interfere with CD20-TCB activity, in contrast to TNFα blockade, which partially inhibited antitumor activity. CONCLUSIONS: Our work sheds new light on the cellular and molecular players involved in cytokine release driven by TCBs and provides a rationale for the prevention of CRS in patients treated with TCBs. See related commentary by Luri-Rey et al., p. 4320.

Dissecting the mechanism of cytokine release induced by T-cell engagers highlights the contribution of neutrophils.

T cell engagers represent a novel promising class of cancer-immunotherapies redirecting T cells to tumor cells and have some promising outcomes in the clinic. These molecules can be associated with a mode-of-action related risk of cytokine release syndrome (CRS) in patients. CRS is characterized by the rapid release of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-6 and IL-1ß and immune cell activation eliciting clinical symptoms of fever, hypoxia and hypotension. In this work, we investigated the biological mechanisms triggering and amplifying cytokine release after treatment with T cell bispecific antibodies (TCBs) employing an in vitro co-culture assay of human PBMCs or total leukocytes (PBMCs + neutrophils) and corresponding target antigen-expressing cells with four different TCBs. We identified T cells as the triggers of the TCB-mediated cytokine cascade and monocytes and neutrophils as downstream amplifier cells. Furthermore, we assessed the chronology of events by neutralization of T-cell derived cytokines. For the first time, we demonstrate the contribution of neutrophils to TCB-mediated cytokine release and confirm these findings by single-cell RNA sequencing of human whole blood incubated with a B-cell depleting TCB. This work could contribute to the construction of mechanistic models of cytokine release and definition of more specific molecular and cellular biomarkers of CRS in the context of treatment with T-cell engagers. In addition, it provides insight for the elaboration of prophylactic mitigation strategies that can reduce the occurrence of CRS and increase the therapeutic index of TCBs.

Catalyzing Transcriptomics Research in Cardiovascular Disease: The CardioRNA COST Action CA17129.

Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field.COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).

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.

Parathyroid Hormone-Related Protein Contributes to Early Healing of Habu Snake Venom-Induced Glomerulonephritis in Mice.

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.

Parathyroid hormone-related protein modulates inflammation in mouse mesangial cells 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-1ß (10 ng/ml) and TNF-α (10 ng/ml) rapidly and transiently upregulated PTHrP expression in MC. The effects of IL-1ß 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-1ß 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.

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.

Myeloid-derived suppressor cells expand during breast cancer progression and promote tumor-induced bone destruction.

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 ß 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.

6-Thioguanine inhibition of parathyroid hormone-related protein expression is mediated by GLI2.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.