CDK9-55 guides the anaphase-promoting complex/cyclosome (APC/C) in choosing the DNA repair pathway choice.

DNA double-strand breaks (DSBs) contribute to genome instability, a key feature of cancer. DSBs are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). We investigated the role of an isoform of the multifunctional cyclin-dependent kinase 9, CDK9-55, in DNA repair, by generating CDK9-55-knockout HeLa clones (through CRISPR-Cas9), which showed potential HR dysfunction. A phosphoproteomic screening in these clones treated with camptothecin revealed that CDC23 (cell division cycle 23), a component of the E3-ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), is a new substrate of CDK9-55, with S588 being its putative phosphorylation site. Mutated non-phosphorylatable CDC23(S588A) affected the repair pathway choice by impairing HR and favouring error-prone NHEJ. This CDK9 role should be considered when designing CDK-inhibitor-based cancer therapies.

The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment.

Introduction: DNA double-strand breaks are the most toxic lesions repaired through the non-homologous and joining (NHEJ) or the homologous recombination (HR), which is dependent on the generation of single-strand tails, by the DNA end resection mechanism. The resolution of the HR intermediates leads to error-free repair (Gene Conversion) or the mutagenic pathways (Single Strand Annealing and Alternative End-Joining); the regulation of processes leading to the resolution of the HR intermediates is not fully understood. Methods: Here, we used a hydrophilic extract of a new tomato genotype (named DHO) in order to modulate the Camptothecin (CPT) DNA damage response. Results: We demonstrated increased phosphorylation of Replication Protein A 32 Serine 4/8 (RPA32 S4/8) protein in HeLa cells treated with the CPT in combination with DHO extract with respect to CPT alone. Moreover, we pointed out a change in HR intermediates resolution from Gene Conversion to Single Strand Annealing through the modified DNA repair protein RAD52 homolog (RAD52), DNA excision repair protein ERCC-1 (ERCC1) chromatin loading in response to DHO extract, and CPT co-treatment, with respect to the vehicle. Finally, we showed an increased sensitivity of HeLa cell lines to DHO extract and CPT co-treatment suggesting a possible mechanism for increasing the efficiency of cancer therapy. Discussion: We described the potential role of DHO extract in the modulation of DNA repair, in response to Camptothecin treatment (CPT), favoring an increased sensitivity of HeLa cell lines to topoisomerase inhibitor therapy.

HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer.

Acquired resistance to platinum (Pt)-based therapies is an urgent unmet need in the management of epithelial ovarian cancer (EOC) patients. Here, we characterized by an unbiased proteomics method three isogenic EOC models of acquired Pt resistance (TOV-112D, OVSAHO, and MDAH-2774). Using this approach, we identified several differentially expressed proteins in Pt-resistant (Pt-res) compared to parental cells and the chaperone HSP90 as a central hub of these protein networks. Accordingly, up-regulation of HSP90 was observed in all Pt-res cells and heat-shock protein 90 alpha isoform knockout resensitizes Pt-res cells to cisplatin (CDDP) treatment. Moreover, pharmacological HSP90 inhibition using two different inhibitors [17-(allylamino)-17-demethoxygeldanamycin (17AAG) and ganetespib] synergizes with CDDP in killing Pt-res cells in all tested models. Mechanistically, genetic or pharmacological HSP90 inhibition plus CDDP -induced apoptosis and increased DNA damage, particularly in Pt-res cells. Importantly, the antitumor activities of HSP90 inhibitors (HSP90i) were confirmed both ex vivo in primary cultures derived from Pt-res EOC patients ascites and in vivo in a xenograft model. Collectively, our data suggest an innovative antitumor strategy, based on Pt compounds plus HSP90i, to rechallenge Pt-res EOC patients that might warrant further clinical evaluation.

Genomic analysis reveals association of specific SNPs with athletic performance and susceptibility to injuries in professional soccer players.

The development of specific and individualized training programs is a possible way to improve athletic performance and minimize injuries in professional athletes. The information regarding the sport's physical demands and the athletes' physical profile have been, so far, considered as exhaustive for the design of effective training programs. However, it is currently emerging that the genetic profile has to be also taken into consideration. By merging medical and genetic data, it is thus possible to identify the athlete's specific attitude to respond to training, diet, and physical stress. In this context, we performed a study in which 30 professional soccer players, subjected to standard sport medical evaluation and practices, were also screened for genetic polymorphism in five key genes (ACTN3, COL5A1, MCT1, VEGF, and HFE). This genetic analysis represents the central point of a multidisciplinary method that can be adopted by elite soccer teams to obtain an improvement in athletic performance and a concomitant reduction of injuries by tailoring training and nutritional programs. The genetic fingerprinting of single athletes led to the identification of two performance-enhancing polymorphisms (ACTN3 18705C>T, VEGF-634C>G) significantly enriched. Moreover, we derived a genetic model based on the gene set analyzed, which was tentatively used to reduce athletes' predisposition to injuries, by dictating a personalized nutrition and training program. The potential usefulness of this approach is concordant with data showing that this team has been classified as the healthiest and least injured team in Europe while covering the highest distance/match with the highest number of high-intensity actions/match.

SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage.

DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3' single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single Molecule Analysis of Resection Tracks (SMART) has been described as a reliable method to visualize, by immunofluorescence, the long 3' single-strand DNA tails generated upon cell treatment with a S-phase specific DNA damaging agent (such as camptothecin). Then, DNA tract lengths can be measured through an image analysis software (such as Photoshop), to evaluate the processivity of the DNA end-resection machinery. The preparation of DNA fibres is performed in non-denaturing conditions so that the immunofluorescence detects only the specific long 3' single-strand DNA tails, generated from DSB processing.

A new case of radiation-induced breast angiosarcoma.

INTRODUCTION: Radiation-induced breast angiosarcoma is a severe but rare late complication in the breast-preserving management of breast cancer through surgery and radiotherapy. Often the initial diagnosis is complex given its relatively anodyne nature and the fact that it usually presents in the form of typically multifocal reddish-purple papular skin lesions. PRESENTATION OF THE CASE: We describe the clinical and pathologic findings of a 79-year-old woman, who developed a radiation-induced breast angiosarcoma after around 8 years. She initially refused a mastectomy leading to an adaptation in the management of this cancer. DISCUSSION: The average latency of secondary angiosarcoma of the breast following radiation therapy is around six years. Breast angiosarcoma is typically considered to affect the dermis, and is therefore cutaneous in origin. An incisional biopsy of the discoloured skin and underlying mass is necessary. The treatment is surgical resection. The role of chemotherapy has not been clearly defined. Most data originate from retrospective case series studies suggesting that angiosarcomas are relatively sensitive to taxanes and anthracyclines. CONCLUSION: The preferred treatment is always aggressive surgical removal and, as our atypical clinical case suggests, neoadjuvant chemotherapy in very high doses is also needed. A biopsy of any suspicious breast skin lesion after radiotherapy is recommended. Despite the treatment challenges, our case provides enlightening details on the management of such a rare cancer even when faced with unplanned events which do not always allow for a textbook approach.

Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation.

DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.

RBL2/p130 is a direct AKT target and is required to induce apoptosis upon AKT inhibition in lung cancer and mesothelioma cell lines.

The retinoblastoma (RB) protein family includes RB1/p105, RBL1/p107, and RBL2/p130, which are key factors in cell-cycle regulation and stand at the crossroads of multiple pathways dictating cell fate decisions. The role of RB proteins in apoptosis is controversial because they can inhibit or promote apoptosis depending on the context, on the apoptotic stimuli and on their intrinsic status, impacting on the response to antitumoral treatments. Here we identified RBL2/p130 as a direct substrate of the AKT kinase, a key antiapoptotic factor hyperactive in multiple cancer types. We showed that RBL2/p130 and AKT1 physically interact and AKT phosphorylates RBL2/p130 Ser941, located in the pocket domain, but not when this residue is mutated into Ala. We found that pharmacological inhibition of AKT, through the highly selective AKT inhibitor VIII (AKTiVIII), impairs RBL2/p130 Ser941 phosphorylation and increases RBL2/p130 stability, mRNA expression and nuclear levels in both lung cancer and mesothelioma cell lines, mirroring the more extensively studied effects on the p27 cell-cycle inhibitor. Consistently, AKT inhibition reduced cell viability, induced cell accumulation in G0/G1, and triggered apoptosis, which proved to be largely dependent on RBL2/p130 itself, as shown upon RBL2/p130 silencing. AKT inhibition induced RBL2/p130-dependent apoptosis also in HEK-293 cells, in which re-expression of a short hairpin-resistant RBL2/p130 was able to rescue AKTiVIII-induced apoptosis upon RBL2/p130 silencing. Our data also showed that the combination of AKT and cyclin-dependent kinases (CDK) inhibitors, which converge on the re-activation of RBL2/p130 antitumoral potential, could be a promising anticancer strategy.

CDK9 inhibitors in acute myeloid leukemia.

Current treatment for acute myeloid leukemia (AML) is less than optimal, but increased understanding of disease pathobiology and genomics has led to clinical investigation of novel targeted therapies and rational combinations. Targeting the cyclin-dependent kinase 9 (CDK9) pathway, which is dysregulated in AML, is an attractive approach. Inhibition of CDK9 leads to downregulation of cell survival genes regulated by super enhancers such as MCL-1, MYC, and cyclin D1. As CDK9 inhibitors are nonselective, predictive biomarkers that may help identify patients most likely to respond to CDK9 inhibitors are now being utilized, with the goal of improving efficacy and safety.

NONO ubiquitination is mediated by FBW7 and GSK3 ß via a degron lost upon chromosomal rearrangement in cancer.

NONO is an RNA-binding protein involved in transcription, mRNA splicing, DNA repair, and checkpoint activation in response to UV radiation. NONO expression has been found altered in several tumor types, including prostate, colon, breast, melanoma, and in papillary renal carcinoma, in which an X chromosome inversion generates a NONO-TFE3 fusion protein. Upon such rearrangement, NONO loses its C-terminal domain. Through bioinformatics analysis, we identified a putative degron motif, known to be recognized by the Skp1-Cul1-F-box-protein (SCF) complex. Here, we evaluated how this domain could affect NONO protein biology. We showed that NONO interacts with the nuclear FBW7α isoform and its ubiquitination is regulated following modulation of the GSK3ß kinase. Mutation of T428A/T432A within the degron impaired polyubiquitination upon FBW7α and GSK3ß overexpression. Overall, our data suggest that NONO is likely subjected to proteasome-mediated degradation and add NONO to the list of proteins targeted by FBW7, which is itself often deregulated in cancer.

HUR protects NONO from degradation by mir320, which is induced by p53 upon UV irradiation.

UV radiations challenge genomic stability and are a recognized cancer risk factor. We previously found that the RNA-binding protein NONO regulates the intra-S phase checkpoint and its silencing impaired HeLa and melanoma cell response to UV-induced DNA damage. Here we investigated the mechanisms underlying NONO regulation upon UVC treatment. We found that UVC rays induce the expression of mir320a, which can indeed target NONO. However, despite mir320a induction, NONO mRNA and protein expression are not affected by UVC. We found through RNA immunoprecipitation that UVC rays induce the ubiquitous RNA-binding protein HUR to bind NONO 5'UTR in a site overlapping mir320a binding site. Both HUR silencing and its pharmacological inhibition induced NONO downregulation following UVC exposure, whereas concomitant mir320a silencing restored NONO stability. UVC-mediated mir320a upregulation is triggered by p53 binding to its promoter, which lies within a region marked by H3K4me3 and H3K27ac signals upon UVC treatment. Silencing mir320a sensitizes cells to DNA damage. Overall our findings reveal a new mechanism whereby HUR protects NONO from mir320-mediated degradation upon UVC exposure and identify a new component within the complex network of players underlying the DNA damage response adding mir320a to the list of p53-regulated targets upon genotoxic stress.

Abrogating G2/M checkpoint through WEE1 inhibition in combination with chemotherapy as a promising therapeutic approach for mesothelioma.

Malignant mesothelioma (MM) is a very aggressive asbestos-related neoplasm of the serous membranes, whose incidence is increasing worldwide. Although the introduction of new drug combinations, such as cisplatin plus pemetrexed/gemcitabine, has determined an improvement in the patient quality of life, MM remains a universally fatal disease. The observation that key G 1/S checkpoint regulators are often functionally inactivated in MM prompted us to test whether the use of G 2/M checkpoint inhibitors, able to sensitize G 1/S checkpoint-defective cancer cells to DNA-damaging agents, could be successful in MM. We treated six MM cell lines, representative of different histotypes (epithelioid, biphasic, and sarcomatoid), with cisplatin in combination with MK-1775, an inhibitor of the G 2/M checkpoint kinase WEE1. We observed that MK-1775 enhanced the cisplatin cytotoxic effect in all MM cell lines, except the sarcomatoid cell line, which is representative of the most aggressive histotype. As expected, the enhancement in cisplatin toxicity was accompanied by a decrease in the inactive phosphorylated form of cyclin-dependent kinase 1 (CDK1), a key substrate of WEE1, which is indicative of G 2/M checkpoint inactivation. Consistently, we also observed a decrease in G 2/M accumulation and an increase in mitotic entry of DNA-damaged cells and apoptosis, probably due to the loss of the cell ability to arrest cell cycle in response to DNA damage, irrespectively of p53 mutational status. Notably, this treatment did not increase cisplatin cytotoxicity on normal cells, thus suggesting a possible use of MK-1775 in combination with cisplatin for a safe and efficient treatment of epithelioid and biphasic MM.

Molecular mechanism of 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL receptor tyrosine kinase degradation.

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.

Hydrocortisone attenuates cyclosporin A-induced nephrotoxicity in rats.

Cyclosporin A (CsA) is the prototype of immunosuppressant drugs that have revolutionized the management of all transplantation and autoimmune diseases. Side effects of CsA mainly affecting the kidney but also observed in liver and heart, limit the therapeutic use of this drug after organ transplantation. The renal toxicity of CsA is attributed to reduced renal blood flow which leads to hypoxia-reoxygenation injury accompanied by excessive generation of oxygen-derived free radicals. In several therapeutic protocols, CsA is used in association with corticosteroids to obtain better therapeutic results. Recently, our studies showed that hydrocortisone (HY) has a protective effect on CsA-induced cardiotoxicity. In fact our previous results demonstrated that in rat cardiomyocytes, CsA toxicity is due to a calcium overload, which in turn induce lipid peroxidation and determines oxidative stress-induced cell injury. Treatment with HY effectively inhibits CsA-induced toxicity, decreasing lipid peroxidation as well as calcium intracellular concentration. In this study we evaluated in vivo the effects of CsA, used alone or in association with HY, on some parameters of renal dysfunction (blood urea nitrogen; BUN, creatinine, and cholesterol), malondialdheyde (MDA) levels, antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and apoptosis. CsA administration for 24 days resulted in a marked renal oxidative stress, which significantly deranged the renal functions. Treatment with CsA in association with HY significantly improved the renal dysfunction and renal oxidative status. This study clearly suggests the role of oxidative stress in the pathogenesis of CsA-induced nephrotoxicity.

RET is a heat shock protein 90 (HSP90) client protein and is knocked down upon HSP90 pharmacological block.

CONTEXT: Mutations of the RET receptor tyrosine kinase are associated to multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinoma (MTC). The heat shock protein (HSP) 90 chaperone is required for folding and stability of several kinases. HSP90 is specifically inhibited by 17-allyl-amino-17-demethoxygeldanamycin (17-AAG). OBJECTIVE: Our aim was to investigate whether RET protein half-life depends on HSP90 and to dissect the molecular pathway responsible for the degradation of RET upon HSP90 inhibition by 17-AAG. DESIGN: 17-AAG effects were studied in RAT1 fibroblasts exogenously expressing MEN2-associated RET mutants and human MTC-derived cell lines. RESULTS: 17-AAG induced a 26S proteasome-dependent degradation of wild-type RET and MEN2-associated RET mutants. The compound hampered HSP90/RET interaction and stabilized RET binding to HSP70, leading to the recruitment of the HSP70-associated E3 ligase C-terminus of Hsc70-interacting protein. In turn, C-terminus of Hsc70-interacting protein polyubiquitinated RET, promoting its proteasomal degradation. 17-AAG blocked RET downstream effectors and RET-dependent transcriptional activation of gene promoters. In human MTC cells carrying oncogenic RET mutants, HSP90 inhibition induced receptor degradation and signaling hindrance leading to cell cycle arrest. CONCLUSION: RET and MEN2-associated RET mutants rely on HSP90 for protein stability, and HSP90 blockade by 17-AAG promotes RET degradation.