New players in the landscape of renal cell carcinoma bone metastasis and therapeutic opportunities.

Approximately one-third of advanced renal cell carcinoma (RCC) patients develop osteolytic bone metastases, leading to skeletal complications. In this review, we first provide a comprehensive perspective of seminal studies on bone metastasis of RCC describing the main molecular modulators and growth factor signaling pathways most important for the RCC-stimulated osteoclast-mediated bone destruction. We next focus on newer developments revealing with in-depth details, the bidirectional interplay between renal cancer cells and the immune and stromal microenvironment that can through epigenetic reprogramming, profoundly affect the behaviors of transformed cells. Understanding their mechanistic interactions is of paramount importance for advancing both fundamental and translational research. These new investigations into the landscape of RCC-bone metastasis offer novel insights and identify potential avenues for future therapeutic interventions.

Characterization of RAGE and CK2 Expressions in Human Fetal Membranes.

At the feto-maternal interface, fetal membranes (FM) play a crucial role throughout pregnancy. FM rupture at term implicates different sterile inflammation mechanisms including pathways activated by the transmembrane glycoprotein receptor for advanced glycation end-products (RAGE) belonging to the immunoglobulin superfamily. As the protein kinase CK2 is also implicated in the inflammation process, we aimed to characterize the expressions of RAGE and the protein kinase CK2 as a candidate regulator of RAGE expression. The amnion and choriodecidua were collected from FM explants and/or primary amniotic epithelial cells throughout pregnancy and at term in spontaneous labor (TIL) or term without labor (TNL). The mRNA and protein expressions of RAGE and the CK2α, CK2α', and CK2ß subunits were investigated using reverse transcription quantitative polymerase chain reaction and Western blot assays. Their cellular localizations were determined with microscopic analyses, and the CK2 activity level was measured. RAGE and the CK2α, CK2α', and CK2ß subunits were expressed in both FM layers throughout pregnancy. At term, RAGE was overexpressed in the amnion from the TNL samples, whereas the CK2 subunits were expressed at the same level in the different groups (amnion/choriodecidua/amniocytes, TIL/TNL), without modification of the CK2 activity level and immunolocalization. This work paves the way for future experiments regarding the regulation of RAGE expression by CK2 phosphorylation.

CIGB-300 anticancer peptide regulates the protein kinase CK2-dependent phosphoproteome.

Casein-kinase CK2 is a Ser/Thr protein kinase that fosters cell survival and proliferation of malignant cells. The CK2 holoenzyme, formed by the association of two catalytic alpha/alpha' (CK2α/CK2α') and two regulatory beta subunits (CK2ß), phosphorylates diverse intracellular proteins partaking in key cellular processes. A handful of such CK2 substrates have been identified as targets for the substrate-binding anticancer peptide CIGB-300. However, since CK2ß also contains a CK2 phosphorylation consensus motif, this peptide may also directly impinge on CK2 enzymatic activity, thus globally modifying the CK2-dependent phosphoproteome. To address such a possibility, firstly, we evaluated the potential interaction of CIGB-300 with CK2 subunits, both in cell-free assays and cellular lysates, as well as its effect on CK2 enzymatic activity. Then, we performed a phosphoproteomic survey focusing on early inhibitory events triggered by CIGB-300 and identified those CK2 substrates significantly inhibited along with disturbed cellular processes. Altogether, we provided here the first evidence for a direct impairment of CK2 enzymatic activity by CIGB-300. Of note, both CK2-mediated inhibitory mechanisms of this anticancer peptide (i.e., substrate- and enzyme-binding mechanism) may run in parallel in tumor cells and help to explain the different anti-neoplastic effects exerted by CIGB-300 in preclinical cancer models.

The tyrosine-kinase inhibitor sunitinib targets vascular endothelial (VE)-cadherin: a marker of response to antitumoural treatment in metastatic renal cell carcinoma.

BACKGROUND: Vascular endothelial (VE)-cadherin is an endothelial cell-specific protein responsible for endothelium integrity. Its adhesive properties are regulated by post-translational processing, such as tyrosine phosphorylation at site Y685 in its cytoplasmic domain, and cleavage of its extracellular domain (sVE). In hormone-refractory metastatic breast cancer, we recently demonstrated that sVE levels correlate to poor survival. In the present study, we determine whether kidney cancer therapies had an effect on VE-cadherin structural modifications and their clinical interest to monitor patient outcome. METHODS: The effects of kidney cancer biotherapies were tested on an endothelial monolayer model mimicking the endothelium lining blood vessels and on a homotypic and heterotypic 3D cell model mimicking tumour growth. sVE was quantified by ELISA in renal cell carcinoma patients initiating sunitinib (48 patients) or bevacizumab (83 patients) in the first-line metastatic setting (SUVEGIL and TORAVA trials). RESULTS: Human VE-cadherin is a direct target for sunitinib which inhibits its VEGF-induced phosphorylation and cleavage on endothelial monolayer and endothelial cell migration in the 3D model. The tumour cell environment modulates VE-cadherin functions through MMPs and VEGF. We demonstrate the presence of soluble VE-cadherin in the sera of mRCC patients (n = 131) which level at baseline, is higher than in a healthy donor group (n = 96). Analysis of sVE level after 4 weeks of treatment showed that a decrease in sVE level discriminates the responders vs. non-responders to sunitinib, but not bevacizumab. CONCLUSIONS: These data highlight the interest for the sVE bioassay in future follow-up of cancer patients treated with targeted therapies such as tyrosine-kinase inhibitors.

Modulatory role of the anti-apoptotic protein kinase CK2 in the sub-cellular localization of Fas associated death domain protein (FADD).

The Fas associated death domain protein (FADD) is the key adaptor molecule of the apoptotic signal triggered by death receptors of the TNF-R1 superfamily. Besides its crucial role in the apoptotic machinery, FADD has proved to be important in many biological processes like tumorigenesis, embryonic development or cell cycle progression. In a process to decipher the regulatory mechanisms underlying FADD regulation, we identified the anti-apoptotic kinase, CK2, as a new partner and regulator of FADD sub-cellular localization. The blockade of CK2 activity induced FADD re-localization within the cell. Moreover, cytoplasmic FADD was increased when CK2ß was knocked down. In vitro kinase and pull down assays confirmed that FADD could be phosphorylated by the CK2 holoenzyme. We found that phosphorylation is weak with CK2α alone and optimal in the presence of stoichiometric amounts of CK2α catalytic and CK2ß regulatory subunit, showing that FADD phosphorylation is undertaken by the CK2 holoenzyme in a CK2ß-driven fashion. We found that CK2 can phosphorylate FADD on the serine 200 and that this phosphorylation is important for nuclear localization of FADD. Altogether, our results show for the first time that multifaceted kinase, CK2, phosphorylates FADD and is involved in its sub-cellular localization. This work uncovered an important role of CK2 in stable FADD nuclear localization.

Protein kinase CK2 in breast cancer: the CK2ß regulatory subunit takes center stage in epithelial plasticity.

Structurally, protein kinase CK2 consists of two catalytic subunits (α and α') and two regulatory subunits (ß), which play a critical role in targeting specific CK2 substrates. Compelling evidence shows the complexity of the CK2 cellular signaling network and supports the view that this enzyme is a key component of regulatory protein kinase networks that are involved in several aspects of cancer. CK2 both activates and suppresses the expression of a number of essential oncogenes and tumor suppressors, and its expression and activity are upregulated in blood tumors and virtually all solid tumors. The prognostic significance of CK2α expression in association with various clinicopathological parameters highlighted this kinase as an adverse prognostic marker in breast cancer. In addition, several recent studies reported its implication in the regulation of the epithelial-to-mesenchymal transition (EMT), an early step in cancer invasion and metastasis. In this review, we briefly overview the contribution of CK2 to several aspects of cancer and discuss how in mammary epithelial cells, the expression of its CK2ß regulatory subunit plays a critical role in maintaining an epithelial phenotype through CK2-mediated control of key EMT-related transcription factors. Importantly, decreased CK2ß expression in breast tumors is correlated with inefficient phosphorylation and nuclear translocation of Snail1 and Foxc2, ultimately leading to EMT induction. This review highlights the pivotal role played by CK2ß in the mammary epithelial phenotype and discusses how a modest alteration in its expression may be sufficient to induce dramatic effects facilitating the early steps in tumor cell dissemination through the coordinated regulation of two key transcription factors.

[Strength and specificity of the CMBA screening platform for bioactive molecules discovery]. / Force et spécificité du criblage pour des molécules bioactives au CMBA-Grenoble - Une plate-forme dédiée à la découverte et à l'analyse de molécules bioactives et candidats médicaments.

Used as powerful chemical probes in Life science fundamental research, the application potential of new bioactive molecular entities includes but extends beyond their development as therapeutic drugs in pharmacology. In this review, we wish to point out the methodology of chemical libraries screening on living cells or purified proteins at the CMBA academic platform of Grenoble Alpes University, and strategies employed to further characterize the selected bioactive molecules by phenotypic profiling on human cells. Multiple application fields are concerned by the screening activity developed at CMBA with bioactive molecules previously selected for their potential as tools for fundamental research purpose, therapeutic candidates to treat cancer or infection, or promising compounds for production of bioenergy.

Cancer selective cell death induction by a bivalent CK2 inhibitor targeting the ATP site and the allosteric αD pocket.

Although the involvement of protein kinase CK2 in cancer is well-documented, there is a need for selective CK2 inhibitors suitable for investigating CK2 specific roles in cancer-related biological pathways and further exploring its therapeutic potential. Here, we report the discovery of AB668, an outstanding selective inhibitor that binds CK2 through a bivalent mode, interacting both at the ATP site and an allosteric αD pocket unique to CK2. Using caspase activation assay, live-cell imaging, and transcriptomic analysis, we have compared the effects of this bivalent inhibitor to representative ATP-competitive inhibitors, CX-4945, and SGC-CK2-1. Our results show that in contrast to CX-4945 or SGC-CK2-1, AB668, by targeting the CK2 αD pocket, has a distinct mechanism of action regarding its anti-cancer activity, inducing apoptotic cell death in several cancer cell lines and stimulating distinct biological pathways in renal cell carcinoma.

COL7A1 Expression Improves Prognosis Prediction for Patients with Clear Cell Renal Cell Carcinoma Atop of Stage.

Clear-cell renal cell carcinoma (ccRCC) accounts for 75% of kidney cancers. Due to the high recurrence rate and treatment options that come with high costs and potential side effects, a correct prognosis of patient survival is essential for the successful and effective treatment of patients. Novel biomarkers could play an important role in the assessment of the overall survival of patients. COL7A1 encodes for collagen type VII, a constituent of the basal membrane. COL7A1 is associated with survival in many cancers; however, the prognostic value of COL7A1 expression as a standalone biomarker in ccRCC has not been investigated. With five publicly available independent cohorts, we used Kaplan-Meier curves and the Cox proportional hazards model to investigate the prognostic value of COL7A1, as well as gene set enrichment analysis to investigate genes co-expressed with COL7A1. COL7A1 expression stratifies patients in terms of aggressiveness, where the 5-year survival probability of each of the four groups was 72.4%, 59.1%, 34.15%, and 8.6% in order of increasing expression. Additionally, COL7A1 expression was successfully used to further divide patients of each stage and histological grade into groups of high and low risk. Similar results were obtained in independent cohorts. In vitro knockdown of COL7A1 expression significantly affected ccRCC cells' ability to migrate, leading to the hypothesis that COL7A1 may have a role in cancer aggressiveness. To conclude, we identified COL7A1 as a new prognosis marker that can stratify ccRCC patients.

A new scaffold-free tumoroid model provides a robust preclinical tool to investigate invasion and drug response in Renal Cell Carcinoma.

Clear cell Renal Cell Carcinoma (ccRCC) is one of the most prevalent kidney cancers, which is often asymptomatic and thus discovered at a metastatic state (mRCC). mRCC are highly heterogeneous tumors composed of subclonal populations that lead to poor treatment response rate. Several recent works explored the potential of ccRCC tumoroids culture derived from patients. However, these models were produced following a scaffold-based method using collagen I or Matrigel that exhibit lot variability and whose complexity could induce treatment response modifications and phenotypic alterations. Following the observation that ccRCC tumoroids can create their own niche by secreting extracellular matrix components, we developed the first scaffold-free tumoroid model of ccRCC tumors. Tumoroids from mice as well as from human tumors were generated with high success rate (≥90%) using a magnetic suspension method and standard culture media. Immunofluorescence analysis revealed their self-organization capacities to maintain multiple tumor-resident cell types, including endothelial progenitor cells. Transcriptomic analysis showed the reproducibility of the method highlighting that the majority of gene expression patterns was conserved in tumoroids compared to their matching tumor tissue. Moreover, this model enables to evaluate drug effects and invasiveness of renal cancer cells in a 3D context, providing a robust preclinical tool for drug screening and biomarker assessment in line with alternative ex vivo methods like tumor tissue slice culture or in vivo xenograft models.

CK2ß Is a Gatekeeper of Focal Adhesions Regulating Cell Spreading.

CK2 is a hetero-tetrameric serine/threonine protein kinase made up of two CK2α/α' catalytic subunits and two CK2ß regulatory subunits. The free CK2α subunit and the tetrameric holoenzyme have distinct substrate specificity profiles, suggesting that the spatiotemporal organization of the individual CK2 subunits observed in living cells is crucial in the control of the many cellular processes that are governed by this pleiotropic kinase. Indeed, previous studies reported that the unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition (EMT), a process involved in cancer invasion and metastasis. Moreover, sub-stoichiometric expression of CK2ß compared to CK2α in a subset of breast cancer tumors was correlated with the induction of EMT markers and increased epithelial cell plasticity in breast carcinoma progression. Phenotypic changes of epithelial cells are often associated with the activation of phosphotyrosine signaling. Herein, using phosphotyrosine enrichment coupled with affinity capture and proteomic analysis, we show that decreased expression of CK2ß in MCF10A mammary epithelial cells triggers the phosphorylation of a number of proteins on tyrosine residues and promotes the striking activation of the FAK1-Src-PAX1 signaling pathway. Moreover, morphometric analyses also reveal that CK2ß loss increases the number and the spatial distribution of focal adhesion signaling complexes that coordinate the adhesive and migratory processes. Together, our findings allow positioning CK2ß as a gatekeeper for cell spreading by restraining focal adhesion formation and invasion of mammary epithelial cells.

Structure-based discovery of small molecules targeting different surfaces of protein-kinase CK2.

Protein kinase CK2 is an unfavorable pronostic marker in several cancers and has consequently emerged as a relevant therapeutic target. Several classes of ATP-competitive inhibitors have been identified, showing variable effectiveness. The molecular architecture of this multisubunit enzyme could offer alternative strategies to develop small molecule inhibitors targeting different surfaces of the kinase. Polyoxometalates were identified as original CK2 inhibitors targeting key structural elements located outside the active site. In addition, the CK2 subunit interface represents an exosite distinct from the catalytic cavity that can be targeted by peptides or small molecules to achieve functional effects.

New potent dual inhibitors of CK2 and Pim kinases: discovery and structural insights.

Protein kinase casein kinase 2 (CK2) is a serine/threonine kinase with evidence of implication in growth dysregulation and apoptosis resistance, making it a relevant target for cancer therapy. Several CK2 inhibitors have been developed showing variable efficiency, emphasizing the need to expand the chemical diversity of those inhibitors. We report the identification and characterization of 2,8-difurandicarboxylic acid derivatives as a new class of nanomolar ATP-competitive inhibitors. Selectivity profiling pointed out proviral insertion Moloney virus kinases (Pim kinases) as the only other kinases that are significantly inhibited. By combining structure-activity relationship analysis with structural determination, we were able to determine the binding mode of these inhibitors for both kinases and to explain their strong inhibitory potency. Essential chemical features necessary for activity on both kinases were then identified. The described compounds are not cell permeable: however, they could provide a lead for developing novel inhibitors usable also in vivo. Given the similar but not redundant pathophysiological functions of CK2 and Pim family members, such inhibitors would provide new attractive leads for targeted cancer therapy. This work highlights that 2 functionally related kinases from different kinome branches display exquisite sensitivity to a common inhibitor.

Regulation of epithelial to mesenchymal transition: CK2ß on stage.

Protein kinase CK2 participates in the regulation of fundamental cellular processes. Among these processes, cell polarity and cell morphology are controlled by this enzyme probably through the phosphorylation of key proteins. To further study the involvement of CK2 in these processes, we showed that in epithelial cells, the regulatory CK2ß subunit was required for LKB1-dependent polarization and cell adhesion. Moreover, CK2ß silencing in MCF10A mammary epithelial cells triggered changes in their morphology correlated with the acquisition of mesenchymal phenotype, which were reminiscent to TGFß-induced epithelial-to-mesenchymal-transition (EMT). TGFß has emerged as a major inducer of EMT both in vitro and in vivo. We found that among the TGFß isoforms, TGFß2 expression was strongly induced in CK2ß-knockdown cells. However, the EMT phenotype induced in response to CK2ß silencing was not abolished by blocking the TGFß signaling pathway at TGFß receptor level, suggesting that alternative pathways might be involved. Given the importance of CK2 in tumorigenesis, a dysregulation of CK2ß expression might contribute to EMT induction during cancer progression.

Structure-function analysis of the beta regulatory subunit of protein kinase CK2 by targeting embryonic stem cell.

Programs that govern stem cell maintenance and pluripotency are dependent on extracellular factors and of intrinsic cell modulators. Embryonic stem (ES) cells with a specific depletion of the gene encoding the regulatory subunit of protein kinase CK2 (CK2ß) revealed a viability defect. However, analysis of CK2ß functions along the neural lineage established CK2ß as a positive regulator for neural stem/progenitor cell (NSC) proliferation and multipotency. By using an in vitro genetic conditional approach, we demonstrate in this work that specific domains of CK2ß involved in the regulatory function towards CK2 catalytic subunits are crucial structural determinants for ES cell homeostasis.

Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction.

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL- cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug-gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

Functional analysis of protein kinase CK2 of the human malaria parasite Plasmodium falciparum.

Protein kinase CK2 (casein kinase 2) is a eukaryotic serine/threonine protein kinase with multiple substrates and roles in diverse cellular processes, including differentiation, proliferation, and translation. The mammalian holoenzyme consists of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We report the identification and characterization of a Plasmodium falciparum CK2alpha orthologue, PfCK2alpha, and two PfCK2beta orthologues, PfCK2beta1 and PfCK2beta2. Recombinant PfCK2alpha possesses protein kinase activity, exhibits similar substrate and cosubstrate preferences to those of CK2alpha subunits from other organisms, and interacts with both of the PfCK2beta subunits in vitro. Gene disruption experiments show that the presence of PfCK2alpha is crucial to asexual blood stage parasites and thereby validate the enzyme as a possible drug target. PfCK2alpha is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2alpha enzymes to a small molecule inhibitor. Taken together, our data identify PfCK2alpha as a potential target for antimalarial chemotherapeutic intervention.

Protein kinase CK2 contributes to placental development: physiological and pathological implications.

Preeclampsia (PE) is the most threatening pathology of human pregnancy. Its development is thought to be due to a failure in the invasion of trophoblast cells that establish the feto-maternal circulation. Protein kinase CK2 is a ubiquitous enzyme reported to be involved in the control of cell invasion. CK2 consists of two subunits, a catalytic subunit, CK2α, and a regulatory subunit, CK2ß. To date, no data exist regarding the expression and role of this enzyme in normal and PE pregnancies. We performed studies, at the clinical level using distinctive cohorts from early pregnancy (n = 24) and from PE (n = 23) and age-matched controls (n = 28); in vitro, using trophoblast cell lines; ex vivo, using placental explants; and in vivo, using PE mouse models. We demonstrated that (i) CK2 is more expressed during the late first trimester of pregnancy and is mainly localized in differentiated trophoblast cells, (ii) the inhibition of its enzymatic activity decreased the proliferation, migration, invasion, and syncytialization of trophoblast cells, both in 2D and 3D culture systems, and (iii) CK2 activity and the CK2α/CK2ß protein ratio were increased in PE human placentas. The pattern and profile of CK2 expression were confirmed in gravid mice along with an increase in the PE mouse models. Altogether, our results demonstrate that CK2 plays an essential role in the establishment of the feto-maternal circulation and that its deregulation is associated with PE development. The increase in CK2 activity in PE might constitute a compensatory mechanism to ensure proper pregnancy progress.

Ex-Vivo Treatment of Tumor Tissue Slices as a Predictive Preclinical Method to Evaluate Targeted Therapies for Patients with Renal Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the third type of urologic cancer. At time of diagnosis, 30% of cases are metastatic with no effect of chemotherapy or radiotherapy. Current targeted therapies lead to a high rate of relapse and resistance after a short-term response. Thus, a major hurdle in the development and use of new treatments for ccRCC is the lack of good pre-clinical models that can accurately predict the efficacy of new drugs and allow the stratification of patients into the correct treatment regime. Here, we describe different 3D cultures models of ccRCC, emphasizing the feasibility and the advantage of ex-vivo treatment of fresh, surgically resected human tumor slice cultures of ccRCC as a robust preclinical model for identifying patient response to specific therapeutics. Moreover, this model based on precision-cut tissue slices enables histopathology measurements as tumor architecture is retained, including the spatial relationship between the tumor and tumor-infiltrating lymphocytes and the stromal components. Our data suggest that acute treatment of tumor tissue slices could represent a benchmark of further exploration as a companion diagnostic tool in ccRCC treatment and a model to develop new therapeutic drugs.

Salicylaldehyde derivatives as new protein kinase CK2 inhibitors.

Protein kinase CK2 is a Ser/Thr kinase, with a constitutive activity, that is considered as a promising target for cancer therapy. The currently available CK2 inhibitors lack the potency and the pharmacological properties necessary to be suitable and successful in clinical settings. We report the development of new potent CK2 inhibitors from salicylaldehyde derivatives identified by automated screening of a proprietary small-molecule library. Docking simulations and analysis of the structure-activity relationship for the hits allowed to determine their binding modes on CK2, and to carry out the optimization of their structures. This strategy led to the discovery of potent CK2 inhibitors with novel structures, one of which was able to inhibit CK2 activity in living cells and promote tumor cell death. The essential features required for potent CK2 inhibitory activity of this class of compounds are discussed.