Mutation of the proline P81 into a serine modifies the tumour suppressor function of the von Hippel-Lindau gene in the ccRCC.

BACKGROUND: The von Hippel-Lindau disease is an autosomal dominant syndrome associated with tumour formation in various tissues, such as retina, central nervous system, kidney, and adrenal glands. VHL gene deletion or mutations support the development of various cancers. Unclassified VHL variants also referred as "of unknown significance" result from gene mutations that have an unknown or unclear effect on protein functions. The P81S mutation has been linked to low penetrance Type 1 disease but its pathogenic function was not clearly determined. METHODS: We established a stable cell line expressing the pVHL213 (c.241C>T, P81S) mutant. Using biochemical and physiological approaches, we herein analysed pVHL folding, stability and function in the context of this VHL single missense mutation. RESULTS: The P81S mutation mostly affects the non-canonical function of the pVHL protein. The cells expressing the pVHL213P81S acquire invasive properties in relation with modified architecture network. CONCLUSION: We demonstrated the pathogenic role of this mutation in tumour development in vhl patients and confirm a medical follow up of family carrying the c.241C>T, P81S.

The prefoldin complex stabilizes the von Hippel-Lindau protein against aggregation and degradation.

Loss of von Hippel-Lindau protein pVHL function promotes VHL diseases, including sporadic and inherited clear cell Renal Cell Carcinoma (ccRCC). Mechanisms controlling pVHL function and regulation, including folding and stability, remain elusive. Here, we have identified the conserved cochaperone prefoldin complex in a screen for pVHL interactors. The prefoldin complex delivers non-native proteins to the chaperonin T-complex-protein-1-ring (TRiC) or Cytosolic Chaperonin containing TCP-1 (CCT) to assist folding of newly synthesized polypeptides. The pVHL-prefoldin interaction was confirmed in human cells and prefoldin knock-down reduced pVHL expression levels. Furthermore, when pVHL was expressed in Schizosaccharomyces pombe, all prefoldin mutants promoted its aggregation. We mapped the interaction of prefoldin with pVHL at the exon2-exon3 junction encoded region. Low levels of the PFDN3 prefoldin subunit were associated with poor survival in ccRCC patients harboring VHL mutations. Our results link the prefoldin complex with pVHL folding and this may impact VHL diseases progression.

Liposomes Containing Nickel-Bis(dithiolene) Complexes for Photothermal Theranostics.

New thermosensitive liposomes with a phase transition at 42 °C, containing nickel-bis(dithiolene) complexes as efficient and stable photothermal agents, have been formulated and characterized. These liposomes are highly stable and keep their contents at 37 °C for more than 30 days. On the contrary, the mild hyperthermia generated by the nickel-bis(dithiolene) complex under 940 nm NIR irradiation allows for the fine controlled release of the liposome contents, making such liposomes highly suitable for on-demand drug delivery in the human body under NIR laser irradiation. These liposomes can also be directly used, as shown here, as nanoagents for photothermal therapy. In fact, strong cell death can be generated under laser irradiation in the presence of these photothermally active nanocargos containing less than 10% w/w of metal complex. We also demonstrate, for the first time, that nickel-bis(dithiolene) complexes are good photoacoustic agents, generating easily detectable ultrasonic signals directly proportional to the concentration of complexes and the used laser power.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

Biocompatible nanoparticles containing hydrophobic nickel-bis(dithiolene) complexes for NIR-mediated doxorubicin release and photothermal therapy.

Biocompatible nanoparticles (NPs) constituted by amphiphilic poly(ethylene glycol)-block-poly(benzyl malate), PEG-b-PMLABe, have been designed for site-specific PhotoThermal Controlled Release (PTCR) of drugs thanks to the presence of a near infra-red (NIR) photothermally active nickel-bis(dithiolene) complex in the inner core of the NPs, together with doxorubicin (Dox). A nanoprecipitation technique was used to prepare well-defined nickel-bis(dithiolene) and nickel-bis(dithiolene)/Dox loaded NPs, which were characterized by dynamic light scattering (DLS), zeta-potential measurements and Transmission Electron Microscopy (TEM). We have shown that the Dox release was effectively controlled by NIR irradiation (long or pulsed NIR laser irradiation). Cytotoxicity experiments on HeLa and MDA-MB-231 cells have shown that the incorporation of more than 10 w% of nickel-bis(dithiolene) complexes does not increase the intrinsic toxicity of the polymer nanoparticles. Finally, the viability of MDA-MB-231 cells was assessed after their incubation, for 24 hours, with empty NPs, Ni4C12 loaded NPs, Dox loaded NPs or Ni4C12/Dox loaded NPs, without or with NIR irradiation. Above all, the results have highlighted that the Ni4C12 loaded NPs after 5 min NIR laser irradiation can induce strong cell death up to 80% at 50 µg mL-1. These results demonstrate that these NPs are good candidates for photothermal therapy.

The pVHL172 isoform is not a tumor suppressor and up-regulates a subset of pro-tumorigenic genes including TGFB1 and MMP13.

The von Hippel-Lindau (VHL) tumor suppressor gene is often deleted or mutated in ccRCC (clear cell renal cell carcinoma) producing a non-functional protein. The gene encodes two mRNA, and three protein isoforms (pVHL213, pVHL160 and pVHL172). The pVHL protein is part of an E3 ligase complex involved in the ubiquitination and proteasomal degradation of different proteins, particularly hypoxia inducible factors (HIF) that drive the transcription of genes involved in the regulation of cell proliferation, angiogenesis or extracellular matrix remodelling. Other non-canonical (HIF-independent) pVHL functions have been described. A recent work reported the expression of the uncharacterized protein isoform pVHL172 which is translated from the variant 2 by alternative splicing of the exon 2. This splice variant is sometimes enriched in the ccRCCs and the protein has been identified in the respective samples of ccRCCs and different renal cell lines. Functional studies on pVHL have only concerned the pVHL213 and pVHL160 isoforms, but no function was assigned to pVHL172. Here we show that pVHL172 stable expression in renal cancer cells does not regulate the level of HIF, exacerbates tumorigenicity when 786-O-pVHL172 cells were xenografted in mice. The pVHL172-induced tumors developed a sarcomatoid phenotype. Moreover, pVHL172 expression was shown to up regulate a subset of pro-tumorigenic genes including TGFB1, MMP1 and MMP13. In summary we identified that pVHL172 is not a tumor suppressor. Furthermore our findings suggest an antagonistic function of this pVHL isoform in the HIF-independent aggressiveness of renal tumors compared to pVHL213.

Fine and Clean Photothermally Controlled NIR Drug Delivery from Biocompatible Nickel-bis(dithiolene)-Containing Liposomes.

This work demonstrates that metal-bis(dithiolene) complexes can be efficiently incorporated inside organic nanocarriers and, that under near-infrared (NIR) irradiation, their high photothermal activity can be finely used to release encapsulated drugs on demand. In contrast to gold nanoparticles and other organic NIR dyes, nickel-bis(dithiolene) complexes do not produce singlet oxygen under irradiation, a highly desirable characteristic to preserve the chemical integrity and activity of the loaded drug during the NIR-triggered release from the nanocarriers. Finally, cytotoxicity experiments performed on various cell lines have shown that the incorporation of such metal complexes do not increase the toxicity of the final liposomal formulation. These results offer great promise for the development of innovative biocompatible drug nanocargos that are able to safely deliver their content on demand under NIR laser irradiation. Moreover, this work demonstrates that metal-bis(dithiolene) complexes, owing to their versatility of functionalization and metal complexation, are attractive photothermal agents for the development of original NIR-responsive materials for application not only in biotechnology but also in materials science.

Expression and prognostic value of the cell polarity PAR complex members in thyroid cancer.

Establishment and maintenance of the apical-basal cell polarity, required for proper replication, migration, specialized functions and tissue morphogenesis, relies on three evolutionary conserved complexes: PAR, CRUMBS and SCRIBBLE. Loss of cell polarity/cohesiveness (LOP/C) is implicated in cancer progression, and members of the polarity complex have been described as either oncogenes or oncosuppressors. However, no information on their role in thyroid cancer (TC) progression is available. In the present study, we evaluated the gene expression of the PAR complex members aPKCι, PARD3α/ß and PARD6α/ß/γ in 95 papillary TC (PTC), compared to their normal matched tissues and in 12 anaplastic TC (ATC). The mRNA and protein levels of investigated genes were altered in the majority of PTC and ATC tissues. In PTC, univariate analysis showed that reduced expression of aPKCι, PARD3ß and PARD6γ mRNAs is associated with increased tumor size, and the reduced expression of PARD3ß mRNA is associated also with recurrences. Multivariate analysis demonstrated that the presence of lymph node metastasis at diagnosis and the reduced expression of PARD3ß are independent risk factors for recurrences, with hazard ratio, respectively, of 8.21 (p=0.006) and 3.04 (p=0.029). The latter result was confirmed by the Kaplan-Meier analysis, which evidenced the association between decreased PARD3ß mRNA levels and shorter disease-free interval. In conclusion, we demonstrated that the expression of PAR complex components is deregulated in the majority of PTC and there is a general trend towards their reduction in ATC tissues. Moreover, a prognostic value for the PARD3ß gene in PTCs is suggested.

Deregulated expression of VHL mRNA variants in papillary thyroid cancer.

Recent findings demonstrated that a subset of papillary thyroid cancers (PTCs) is characterized by reduced expression of the von Hippel-Lindau (VHL) tumor suppressor gene, and that lowest levels associated with more aggressive PTCs. In the present study, the levels of the two VHL mRNA splicing variants, VHL-213 (V1) and VHL-172 (V2), were measured in a series of 96 PTC and corresponding normal matched tissues by means of quantitative RT-PCR. Variations in the mRNA levels were correlated with patients' clinicopathological parameters and disease-free interval (DFI). The analysis of VHL mRNA in tumor tissues, compared to normal matched tissues, revealed that its expression was either up- or down-regulated in the majority of PTC. In particular, V1 and V2 mRNA levels were altered, respectively, in 78 (81.3%) and 65 (67.7%) out of the 96 PTCs analyzed. A significant positive correlation between the two mRNA variants was observed (p < 0.001). Univariate analysis documented the lack of association between each variant and clinicopathological parameters such as age, tumor size, histology, TNM stage, lymph node metastases, and BRAF mutational status. However, a strong correlation was found between altered V1 or V2 mRNA levels and DFI. Multivariate regression analysis indicated higher V1 mRNA values, along with lymph node metastases at diagnosis, as independent prognostic factors predicting DFI. In conclusion, the data reported demonstrate that VHL gene expression is deregulated in the majority of PTC tissues. Of particular interest is the apparent protective role exerted by VHL transcripts against PTC recurrences.

SHIP2 Regulates Lumen Generation, Cell Division, and Ciliogenesis through the Control of Basolateral to Apical Lumen Localization of Aurora A and HEF 1.

Lumen formation during epithelial morphogenesis requires the creation of a luminal space at cell interfaces named apical membrane-initiation sites (AMISs). This is dependent upon integrated signaling from mechanical and biochemical cues, vesicle trafficking, cell division, and processes tightly coupled to ciliogenesis. Deciphering relationships between polarity determinants and lumen or cilia generation remains a fundamental issue. Here, we report that Src homology 2 domain-containing inositol 5-phosphatase 2 (SHIP2), a basolateral determinant of polarity, regulates RhoA-dependent actin contractility and cell division to form AMISs. SHIP2 regulates mitotic spindle alignment. SHIP2 is expressed in G1 phase, whereas Aurora A kinase is enriched in mitosis. SHIP2 binds Aurora A kinase and the scaffolding protein HEF1 and promotes their basolateral localization at the expense of their luminal expression connected with cilia resorption. Furthermore, SHIP2 expression increases cilia length. Thus, our findings offer new insight into the relationships among basolateral proteins, lumen generation, and ciliogenesis.

Aggregation dynamics and identification of aggregation-prone mutants of the von Hippel-Lindau tumor suppressor protein.

Quality control mechanisms promote aggregation and degradation of misfolded proteins. In budding yeast, the human von Hippel-Lindau protein (pVHL, officially known as VHL) is misfolded and forms aggregates. Here, we investigated the aggregation of three pVHL isoforms (pVHL213, pVHL160, pVHL172) in fission yeast. The full-length pVHL213 isoform aggregates in highly dynamic small puncta and in large spherical inclusions, either close to the nucleus or to the cell ends. The large inclusions contain the yeast Hsp104 chaperone. Aggregate clearance is regulated by proteasomal degradation. The pVHL160 isoform forms dense foci and large irregularly shaped aggregates. In silico, prediction of pVHL aggregation propensity identified a key aggregation-promoting region within exon 2. Consistently, the pVHL172 isoform, which lacks exon 2, formed rare reduced inclusions. We studied the aggregation propensity of pVHL variants harbouring missense mutations found in kidney carcinomas. We show that the P86L mutation stimulated small aggregate formation, the P146A mutation increased large inclusion formation, whereas the I151S mutant destabilized pVHL. The prefoldin subunit Pac10 (the human homolog VBP-1 binds to pVHL) is required for pVHL stability. Reduction of soluble functional pVHL might be crucial in VHL-related diseases.

Unconventional Functions of Mitotic Kinases in Kidney Tumorigenesis.

Human tumors exhibit a variety of genetic alterations, including point mutations, translocations, gene amplifications and deletions, as well as aneuploid chromosome numbers. For carcinomas, aneuploidy is associated with poor patient outcome for a large variety of tumor types, including breast, colon, and renal cell carcinoma. The Renal cell carcinoma (RCC) is a heterogeneous carcinoma consisting of different histologic types. The clear renal cell carcinoma (ccRCC) is the most common subtype and represents 85% of the RCC. Central to the biology of the ccRCC is the loss of function of the Von Hippel-Lindau gene, but is also associated with genetic instability that could be caused by abrogation of the cell cycle mitotic spindle checkpoint and may involve the Aurora kinases, which regulate centrosome maturation. Aneuploidy can also result from the loss of cell-cell adhesion and apical-basal cell polarity that also may be regulated by the mitotic kinases (polo-like kinase 1, casein kinase 2, doublecortin-like kinase 1, and Aurora kinases). In this review, we describe the "non-mitotic" unconventional functions of these kinases in renal tumorigenesis.

Water-soluble nickel-bis(dithiolene) complexes as photothermal agents.

The remarkable photothermal properties of pegylated nickel-bis(dithiolene) complexes in water can induce cell death under laser irradiation in the near infrared (NIR) region.

Integrative analysis of high-throughput RNAi screen data identifies the FER and CRKL tyrosine kinases as new regulators of the mitogenic ERK-dependent pathways in transformed cells.

BACKGROUND: Cell proliferation is a hallmark of cancer and depends on complex signaling networks that are chiefly supported by protein kinase activities. Therapeutic strategies have been used to target specific kinases but new methods are required to identify combined targets and improve treatment. Here, we propose a small interfering RNA genetic screen and an integrative approach to identify kinase networks involved in the proliferation of cancer cells. RESULTS: The functional siRNA screen of 714 kinases in HeLa cells identified 91 kinases implicated in the regulation of cell growth, most of them never being reported in previous whole-genome siRNA screens. Based on gene ontology annotations, we have further discriminated between two classes of kinases that, when suppressed, result in alterations of the mitotic index and provoke cell-cycle arrest. Extinguished kinases that lead to a low mitotic index mostly include kinases implicated in cytosolic signaling. In contrast, extinguished kinases that result in a high mitotic index mostly include kinases implicated in cell division. By mapping hit kinases in the PhosphPOINT phosphoprotein database, we generated scale-free networks consisting of 449 and 661 protein-protein interactions for kinases from low MI and high MI groups, respectively. Further analyses of the kinase interactomes revealed specific modules such as FER- and CRKL-containing modules that connect three members of the epidermal growth factor receptor (EGFR) family, suggesting a tight control of the mitogenic EGF-dependent pathway. Based on experimental studies, we confirm the involvement of these two kinases in the regulation of tumor cell growth. CONCLUSION: Based on a combined approach of large kinome-wide siRNA screens and ontology annotations, our study identifies for the first time two kinase groups differentially implicated in the control of cell proliferation. We further demonstrate that integrative analysis of the kinase interactome provides key information which can be used to facilitate or optimize target design for new therapeutic strategies. The complete list of protein-protein interactions from the two functional kinase groups will provide a useful database for future investigations.

Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines.

Aurora kinases are serine/threonine kinases that play an essential role in cell division. Their aberrant expression and/or function induce severe mitotic abnormalities, resulting in either cell death or aneuploidy. Overexpression of Aurora kinases is often found in several malignancies, among which is anaplastic thyroid carcinoma (ATC). We have previously demonstrated the in vitro efficacy of Aurora kinase inhibitors in restraining cell growth and survival of different ATC cell lines. In this study, we sought to establish which Aurora might represent the preferential drug target for ATC. To this end, the effects of two selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) on four human ATC cell lines (CAL-62, BHT-101, 8305C, and 8505C) were analysed. Both inhibitors reduced cell proliferation in a time- and dose-dependent manner, with IC50 ranges of 44.3-134.2 nM for MLN8237 and of 9.2-461.3 nM for AZD1152. Immunofluorescence experiments and time-lapse videomicroscopy yielded evidence that each inhibitor induced distinct mitotic phenotypes, but both of them prevented the completion of cytokinesis. As a result, poliploidy increased in all AZD1152-treated cells, and in two out of four cell lines treated with MLN8237. Apoptosis was induced in all the cells by MLN8237, and in BHT-101, 8305C, and 8505C by AZD1152, while CAL-62 exposed to AZD1152 died through necrosis after multiple rounds of endoreplication. Both inhibitors were capable of blocking anchorage-independent cell growth. In conclusion, we demonstrated that either Aurora-A or Aurora-B might represent therapeutic targets for the ATC treatment, but inhibition of Aurora-A appears more effective for suppressing ATC cell proliferation and for inducing the apoptotic pathway.

Sunitinib combined with angiotensin-2 type-1 receptor antagonists induces more necrosis: a murine xenograft model of renal cell carcinoma.

BACKGROUND: Angiotensin-2 type-1 receptor antagonists not are only antihypertensive drugs but also can inhibit VEGF production. We hypothesised that adding telmisartan to sunitinib could potentiate the antiangiogenic effects. MATERIAL AND METHODS: 786-O cell lines were injected in nude mice. After tumor development, mice were divided into 4 groups: the first was the control group (DMSO), the second group was treated with sunitinib alone, the third group was treated with telmisartan alone, and the fourth group was treated with the combination. Drugs were orally administered every day for four weeks. Animals were sacrificed after treatment. Blood and tumor tissues were collected for analysis by immunohistochemistry, Western Blot, and ELISA methods. RESULTS: All animals developed a ccRCC and ten in each group were treated. Using a kinetic model, tumors tended to grow slower in the combination group compared to others (P = 0.06). Compared to sunitinib alone, the addition of telmisartan significantly increased tissue necrosis (P = 0.038). Central microvascular density decreased (P = 0.0038) as well as circulating VEGF (P = 0.003). There was no significant variation in proliferation or apoptosis markers. CONCLUSION: The combination of sunitinib and telmisartan revealed an enhancement of the blockage of the VEGF pathway on renal tumor resulting in a decrease in neoangiogenesis and an increase in necrosis.

Cytoplasmic PAR-3 protein expression is associated with adverse prognostic factors in clear cell renal cell carcinoma and independently impacts survival.

Clear cell renal cell carcinomas (ccRCCs) represent 70% of renal cancers, and several clinical and histolopathological factors are implicated in their prognosis. We recently demonstrated that the overexpression of PAR-3 protein encoded by the PARD3 gene could be implicated in renal oncogenesis. The object of this work was to study the association of intratumoral PAR-3 expression with known prognostic parameters and clinical outcome. In this aim, PAR-3 expression was assessed by immunohistochemistry in ccRCC tumors of 101 patients from 2003 to 2005. The immunostaining of PAR-3 was scored either as membranous (mPAR-3) or as both membranous and cytoplasmic (cPAR-3). Cytoplasmic PAR-3 was significantly associated with worse histopathological and clinical prognostic factors: Fuhrman grades 3 and 4, tumor necrosis, sarcomatoid component, adrenal invasion, renal and hilar fat invasion, eosinophilic component, a noninactivated VHL gene, higher tumor grade, lymph node involvement, metastasis, and worse clinical Eastern Cooperative Oncology Group and S classification scores. After multivariate analysis, 2 parameters were independently associated with cPAR-3: necrosis and eosinophilic components. In addition, cPAR-3 patients had shorter overall and progression-free survivals independently from strong prognostic validated factors like metastases. A cytoplasmic expression of PAR-3 is therefore implicated in worse clinical and pathological cancer features in ccRCC and could be useful to identify patients with high-risk tumors.

All anti-vascular endothelial growth factor drugs can induce 'pre-eclampsia-like syndrome': a RARe study.

BACKGROUND: Specific therapies that target vascular endothelial growth factor (VEGF) and its receptors have improved the survival of patients with metastatic cancers, but can induce side effects. Renal side effects (proteinuria, hypertension and renal failure) are underestimated. METHODS: The French RARe (Reins sous traitement Anti-VEGF Registre) study collects data on patients with cancer who had a renal biopsy because of major renal side effects during treatment with anti-VEGF drugs. RESULTS: We collected 22 renal biopsies performed 16.2±10.6 months after the beginning of treatment; of which 21 had hypertension, mean proteinuria was 2.97±2.00 g/day and mean serum creatinine, 134±117 µmol/L. Thrombotic microangiopathy (TMA) was observed in 21 biopsy specimens, sometimes associated with acute tubular necrosis (ATN; n=4). TMA histological lesions were more important than the biological signs of TMA could suggest. Patients with ATN of >20% had higher serum creatinine levels than those with only TMA (231 versus 95 µmol/L). Nephrin, podocin and synaptopodin were variably down-regulated in all renal biopsies. VEGF was down-regulated in all glomeruli. CONCLUSION: This study underlines the importance of regular clinical and biological cardiovascular and renal checking during all anti-VEGF therapies for cancer for early detection of renal dysfunction. Collaboration between oncologists and nephrologists is essential. In such cases, renal biopsy might help in appreciating the severity of the renal lesions and after multidisciplinary discussion whether or not it is safe to continue the treatment.

Identification of pVHL as a novel substrate for Aurora-A in clear cell renal cell carcinoma (ccRCC).

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer and is often characterized by mutations or deletions of the Von Hippel Lindau (VHL) tumour suppressor gene. Aurora gene family members are implicated in proper mitotic progression and spindle checkpoint function and play a crucial role in cancer progression. In the present study, we assessed the expression of Aurora-A in a cohort of 30 ccRCC with fully characterized VHL status (wt/wt or mut/del) and Fuhrman grade. Aurora-A transcript and protein levels were significantly increased in high Fuhrman grade tumours and in VHLwt/wt tumours. These results suggest that Aurora-A and VHL interact in the ccRCC. We demonstrated that the two proteins interact in vivo and identified the Ser72 on the sequence of VHL as the unique site phosphorylated by Aurora-A.