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Abstract Background Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. Results Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. Conclusion These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.

Background and Aims: The successful plant Fallopia × bohemica presents interesting capacities for control of the soil nitrogen cycle at the adult stage, termed biological inhibition of denitrification (BDI). The BDI strategy allows the plant, via the production of secondary metabolites (procyanidins), to compete with the denitrifying microbial community and to divert nitrate from the soil for its benefit. In this study, we analysed whether seedlings of F. × bohemica can implement BDI at the seedling stage. We also determined whether soil nitrogen availability influences the implementation of BDI and seedling growth., Methods: We sowed achenes of F. × bohemica in soils representing a nitrogen gradient (six treatments) and harvested seedlings after 20 or 40 days of growth. The denitrification and related microbial communities (i.e. functional gene abundances of nirK and nirS), soil parameters (nitrate content and humidity) and plant performance (biomass, growth and root morphology) were determined., Key Results: On soil without addition of nitrogen, BDI was observed after 20 days of growth, whereas a stimulation of denitrification was found after 40 days. The increase of soil N content had few effects on the activity and structure of the soil denitrifying community and on the plant biomasses or the relative growth rates. Correlations between plant and microbial parameters were observed after 20 days of growth, reflecting early and strong chemical interactions between plants and the denitrifying community, which decreased with plant growth after 40 days., Conclusions: This study shows that an early BDI enhances the efficiency of nitrogen acquisition in the first weeks of growth, allowing for a conservative root strategy after 40 days. This switch to a conservative strategy involved resource storage, an altered allocation to above- and below-ground parts and an investment in fine roots. It now seems clear that this storage strategy starts at a very young age with early establishment of BDI, giving this clonal plant exceptional capacities for storage and multiplication., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Despite the significant advances in the management of advanced prostate cancer (PCa), metastatic PCa is currently considered incurable. For further investigations in precision treatment, the development of preclinical models representing the complex prostate tumor heterogeneity are mandatory. Accordingly, we aimed to establish a resource of patient-derived xenograft (PDX) models that exemplify each phase of this multistage disease for accurate and rapid evaluation of candidate therapies., Methods: Fresh tumor samples along with normal corresponding tissues were obtained directly from patients at surgery. To ensure that the established models reproduce the main features of patient's tumor, both PDX tumors at multiple passages and patient's primary tumors, were processed for histological characteristics. STR profile analyses were also performed to confirm patient identity. Finally, the responses of the PDX models to androgen deprivation, PARP inhibitors and chemotherapy were also evaluated., Results: In this study, we described the development and characterization of 5 new PDX models of PCa. Within this collection, hormone-naïve, androgen-sensitive and castration-resistant (CRPC) primary tumors as well as prostate carcinoma with neuroendocrine differentiation (CRPC-NE) were represented. Interestingly, the comprehensive genomic characterization of the models identified recurrent cancer driver alterations in androgen signaling, DNA repair and PI3K, among others. Results were supported by expression patterns highlighting new potential targets among gene drivers and the metabolic pathway. In addition, in vivo results showed heterogeneity of response to androgen deprivation and chemotherapy, like the responses of patients to these treatments. Importantly, the neuroendocrine model has been shown to be responsive to PARP inhibitor., Conclusion: We have developed a biobank of 5 PDX models from hormone-naïve, androgen-sensitive to CRPC primary tumors and CRPC-NE. Increased copy-number alterations and accumulation of mutations within cancer driver genes as well as the metabolism shift are consistent with the increased resistance mechanisms to treatment. The pharmacological characterization suggested that the CRPC-NE could benefit from the PARP inhibitor treatment. Given the difficulties in developing such models, this relevant panel of PDX models of PCa will provide the scientific community with an additional resource for the further development of PDAC research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Béraud, Bidan, Lassalle, Lang, Lindner, Krucker, Masliah-Planchon, Potiron, Lluel, Massfelder, Allory and Misseri.)

Background: Muscle-invasive bladder cancer (MIBC) and upper urinary tract urothelial carcinoma (UTUC) are molecularly heterogeneous. Despite chemotherapies, immunotherapies, or anti-fibroblast growth factor receptor (FGFR) treatments, these tumors are still of a poor outcome. Our objective was to develop a bank of patient-derived xenografts (PDXs) recapitulating the molecular heterogeneity of MIBC and UTUC, to facilitate the preclinical identification of therapies., Methods: Fresh tumors were obtained from patients and subcutaneously engrafted into immune-compromised mice. Patient tumors and matched PDXs were compared regarding histopathology, transcriptomic (microarrays), and genomic profiles [targeted Next-Generation Sequencing (NGS)]. Several PDXs were treated with chemotherapy (cisplatin/gemcitabine) or targeted therapies [FGFR and epidermal growth factor (EGFR) inhibitors]., Results: A total of 31 PDXs were established from 1 non-MIBC, 25 MIBC, and 5 upper urinary tract tumors, including 28 urothelial (UC) and 3 squamous cell carcinomas (SCCs). Integrated genomic and transcriptomic profiling identified the PDXs of three different consensus molecular subtypes [basal/squamous (Ba/Sq), luminal papillary, and luminal unstable] and included FGFR3 -mutated PDXs. High histological and genomic concordance was found between matched patient tumor/PDX. Discordance in molecular subtypes, such as a Ba/Sq patient tumor giving rise to a luminal papillary PDX, was observed (n=5) at molecular and histological levels. Ten models were treated with cisplatin-based chemotherapy, and we did not observe any association between subtypes and the response. Of the three Ba/Sq models treated with anti-EGFR therapy, two models were sensitive, and one model, of the sarcomatoid variant, was resistant. The treatment of three FGFR3-mutant PDXs with combined FGFR/EGFR inhibitors was more efficient than anti-FGFR3 treatment alone., Conclusions: We developed preclinical PDX models that recapitulate the molecular heterogeneity of MIBCs and UTUC, including actionable mutations, which will represent an essential tool in therapy development. The pharmacological characterization of the PDXs suggested that the upper urinary tract and MIBCs, not only UC but also SCC, with similar molecular characteristics could benefit from the same treatments including anti-FGFR for FGFR3-mutated tumors and anti-EGFR for basal ones and showed a benefit for combined FGFR/EGFR inhibition in FGFR3-mutant PDXs, compared to FGFR inhibition alone., Competing Interests: Author’s CB, ML, YM and PL were employed by Urosphere and author FD was employed by Inovarion, Institut Curie, Strasbourg University and Urosphere have a collaboration contract for the transcriptomic, genomic, and pharmacological characterization of the PDX. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lang, Béraud, Cabel, Fontugne, Lassalle, Krucker, Dufour, Groeneveld, Dixon, Meng, Kamoun, Chapeaublanc, De Reynies, Gamé, Rischmann, Bieche, Masliah-Planchon, Beaurepere, Allory, Lindner, Misseri, Radvanyi, Lluel, Bernard-Pierrot and Massfelder.)

The upregulation of PPARγ/RXRα transcriptional activity has emerged as a key event in luminal bladder tumors. It renders tumor cell growth PPARγ-dependent and modulates the tumor microenvironment to favor escape from immuno-surveillance. The activation of the pathway has been linked to PPARG gains/amplifications resulting in PPARγ overexpression and to recurrent activating point mutations of RXRα. Here, we report recurrent mutations of PPARγ that also activate the PPARγ/RXRα pathway, conferring PPARγ-dependency and supporting a crucial role of PPARγ in luminal bladder cancer. These mutations are found throughout the protein-including N-terminal, DNA-binding and ligand-binding domains-and most of them enhance protein activity. Structure-function studies of PPARγ variants with mutations in the ligand-binding domain allow identifying structural elements that underpin their gain-of-function. Our study reveals genomic alterations of PPARG that lead to pro-tumorigenic PPARγ/RXRα pathway activation in luminal bladder tumors and may open the way towards alternative options for treatment.

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.

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., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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 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.

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., (© 2015 UICC.)

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.

Hypoxia, reoxygenation, and the tyrosine phosphatase inhibitor pervanadate activate the transcription factor NF-kappaB, involving phosphorylation of its inhibitor IkappaB-alpha on tyrosine 42. This modification does not lead to degradation of IkappaB by the proteasome/ubiquitin pathway, as is seen on stimulation of cells with proinflammatory cytokines. It is currently unknown how tyrosine-phosphorylated IkappaB is removed from NF-kappaB. Here we show that p85alpha, the regulatory subunit of PI3-kinase, specifically associates through its Src homology 2 domains with tyrosine-phosphorylated IkappaB-alpha in vitro and in vivo after stimulation of T cells with pervanadate. This association could provide a mechanism by which newly tyrosine-phosphorylated IkappaB is sequestered from NF-kappaB. Another mechanism by which PI3-kinase contributed to NF-kappaB activation in response to pervanadate appeared to involve its catalytic p110 subunit. This was evident from the inhibition of pervanadate-induced NF-kappaB activation and reporter gene induction by treatment of cells with nanomolar amounts of the PI3-kinase inhibitor wortmannin. The compound had virtually no effect on tumor necrosis factor- and interleukin-1-induced NF-kappaB activities. Wortmannin did not inhibit tyrosine phosphorylation of IkappaB-alpha or alter the stability of the PI3-kinase complex but inhibited Akt kinase activation in response to pervanadate. Our data suggest that both the regulatory and the catalytic subunit of PI3-kinase play a role in NF-kappaB activation by the tyrosine phosphorylation-dependent pathway.