Molecular Determinants of Cellular Response to Anticancer Agents Treatment

The response of cancer cells to treatment with anticancer agents is mediated by several factors, among which the functionality of proteins participating in the control of cell cycle progression and genomic integrity is an important one. The studies reported here were aimed at understanding the role of crucial proteins, participating in key processes in normal cells, in determining cellular sensitivity towards the cytotoxcity of anticancer agents. Four different proteins have been investigated: p53, p73 and the two cell cycle checkpoint proteins CHKl and CHK2. P53 has been selected because of its undisputed role in tumor formation and development, for its high prevalence of mutations in human cancer and for its role in normal cells in response to different stimuli. P73 is a structural homologue of p53 which shares functions with p53 but in addition has other very distinct functions. The two checkpoint proteins CHKl and CHK2 control cell cycle progression subsequent to DNA damage, particularly during the S and G2 phases of the cell cycle, and their importance is currently under intensive elucidation. The studies have been conducted in isogenic cell systems, to minimize as much as possible interference by other alterations invariably present when two different cell types are considered. Using two widely used anticancer agents, cisplatinum (DDP) and taxol, it was shown that for both drugs the presence of functional p53 was associated with resistance to drug-induced cytotoxicity. These results have been obtained in different experimental systems of human cancer cells of epithelial origin such as the colon carcinoma cell line HCT116 and the ovarian cancer cell line A2780. With respect to p73, the work took advantage of the availability in the laboratory of two subclones overexpressing p73 derived from a human ovarian cancer cell line by transfection with the p73 alpha cDNA. These two clones over-express DNA repair genes, particularly those participating in the nucleotide excision repair (NER). These cells are prone to repair lesions recognized by NER. Due to this fact, the p73- overexpressing clones were less susceptible to treatment with DDP and UV irradiation, as both lesions are recognized by NER. Other drugs, such as doxorubicin and topotecan inducing damage which is not repaired by NER, exhibited similar activities in parental and p73-overexpressing clones. For both CHKl and CHK2, experiments which were performed using clones transfected with dominant negative mutants failed to show differences between transfected clones and parental cells in the cytotoxicity of DNA-damaging agents. However the use of inhibitors of these kinases resulted in increased activity of DDP, suggesting that both CHKl and CHK2 may play a role in determining sensitivity of cancer cells to drugs, but that the dominant negative mutants in some way masked these effects. The potential involvement of CHKl in response to stress is underlined by evidence of a link between p53 and CHKl, implying that they mutually regulate each other in a way which controls both the activation and the repression of checkpoint response following DNA damage

Activity of Pan-Class I Isoform PI3K/mTOR Inhibitor PF-05212384 in Combination with Crizotinib in Ovarian Cancer Xenografts and PDX

AbstractThe Phosphatidyl inositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and c-Met signaling pathways are often deregulated in cancer. The two pathways are interconnected and at least c-Met has been implicated in drug resistance. The aim of the study was to assess in ovarian cancer preclinical models, the efficacy and tolerability of a dual PI3K mTOR inhibitor (PF-05212384 or gedatolisib) and a c-Met inhibitor (crizotinib) either as single agents or in combination. In vitro, both PF-05212384 and crizotinib showed a concentration dependent activity in the two ovarian cancer cell lines. The combination of the two did not result in synergistic activity. A subline resistant to gedatolisib was obtained and showed an increased expression of MDR-1 gene. In vivo results show that crizotinib alone did not display any activity in all the tumors investigated, while PF-05212384 alone had some marginal activity. The combination of the two resulted in all the experiments superior to single agents with a good tolerability. Considering that crizotinib did not show activity in the models used, the results indicate that crizotinib is able to potentiate the activity of PF-05212384. Although the activity of the combination was not striking in these three models of ovarian cancer, due to the good tolerability of the combination, the results would suggest the possibility to combine the two drugs in settings in which gedatolisib or crizotinib alone have already some significant activity

DNA Damage Induces p53-dependent Down-regulation of hCHK1

Abstract The levels of the human checkpoint gene hCHK1 were measured in human cancer cells growing in vitro after treatment with the DNA damaging agent cis-dichlorodiammine platinum(II) (DDP). Treatment of human cancer cell lines with DDP induced a decrease in the hCHK1 protein levels starting 6 h after treatment, with a further decline at 24 and 48 h. A similar decrease in the levels of hCHK1 was found at the mRNA level by using Northern blot analysis. By using isogenic cell systems in which p53 was disrupted either by transfection with HPV-E6 or by targeted homologous recombination, we found that the DNA damage-induced down-regulation of hCHK1 was only observable in wild type p53-expressing cells, with only a minor decline in the hCHK1 levels observable 48 h after treatment in cells with disrupted p53. Similarly, treatment of mutant p53-expressing human cancer cell lines with DDP did not result in changes in the levels of hCHK1. The p53-dependent down-regulation of hCHK1 is likely to be at transcriptional levels, as suggested by the lack of down-regulation of the hCHK1 when transfected under the control of a heterologous viral promoter. In addition, p53 is able to down-regulate the luciferase activity under the control of the 5′ flanking region of the hCHK1 gene. The data suggest a strict link between p53 and hCHK1 governing the activation and repression of the G2 checkpoint in which both proteins participate

Questioning the oncogenic role of ΔNp73α in different cell lines expressing p53 or not

The recent finding that the 1p36.3 locus gene encodes an array of different p73 isoforms with apparently distinct and sometimes opposing cellular functions, might explain the difficulty in establishing the protein's role as tumor suppressor. Therefore we need to investigate the roles of each of these splicing variants in cellular functions when expressed alone or in combination with other family members, as well as the genetic background on which the proteins are expressed. We investigated, in two p53 null cell lines, the human SCLC line H1299 and a subline derived from the human colon carcinoma cell line HCT116 (HCT116/379.2), the effects of DeltaNp73alpha overexpression on cell growth and the response to anticancer treatment. We generated three different clones overexpressing DeltaNp73alpha under a tetracycline inducible promoter. Immunofluorescent staining and luciferase reporter assays confirmed that clones HCT116/DeltaNA and H1299/DeltaN7 and H1299/DeltaN11 did express a functional, nuclear localized DeltaNp73alpha protein. The stable overexpression of DeltaNp73alpha protein did not confer any cell growth advantage. Doubling time of clones overexpressing DeltaNp73alpha were comparable to counterparts not expressing it. Clonogenic assays showed that the cytotoxic activity of different DNA damaging agents, such as cDDP, UV light and doxorubicin, were comparable in clones expressing DeltaNp73 or not. The overall data argue against an oncogenic role for this isoform. These findings are independent of the p53 status since they overlap with those previously obtained by our group in HCT116 cell lines, wild type for p53

Tubulo-interstitial lesions mediate renal damage in adriamycin glomerulopathy

Tubulo-interstitial lesions mediate renal damage in adriamycin glomerulopathy. The present study was designed to investigate the relationship between proteinuria, focal sclerosis, and tubulo-interstitial changes in the evolution of renal damage in experimental nephrosis. We utilized an accelerated unilateral model of adriamycin (ADR) nephrosis characterized by morphological changes more severe than in the classical model. The first events in ADR-induced glomerulopathy were epithelial cell damage and proteinuria. Subsequently, tubular casts were formed at the distal level. The cast formation preceded the development of interstitial damage, which was determined by tubular obstruction and breaking of tubular basement membrane (TBM), which in turn promoted an interstitial inflammatory reaction. Despite the severity of tubulo-interstitial damage observed after a long period of heavy proteinuria, the incidence of focal segmental glomerulosclerosis (FSG) was very low. The results of the present study indicate that chronic proteinuria is not necessarily accompanied by the development of focal sclerosis Tubulo-interstitial lesions appear to be the most important determinant for the progression of renal damage in this model

Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2

Allosteric targeting of protein kinases via displacement of the structural αC helix with type III allosteric inhibitors is currently gaining a foothold in drug discovery. Recently, the first crystal structure of CDK2 with an open allosteric pocket adjacent to the αC helix has been described, prospecting new opportunities to design more selective inhibitors, but the structure has not yet been exploited for the structure-based design of type III allosteric inhibitors. In this work we report the results of a virtual screening campaign that resulted in the discovery of the first-in-class type III allosteric ligands of CDK2. Using a combination of docking and post-docking analyses made with our tool BEAR, 7 allosteric ligands (hit rate of 20%) with micromolar affinity for CDK2 were identified, some of them inhibiting the growth of breast cancer cell lines in the micromolar range. Competition experiments performed in the presence of the ATP-competitive inhibitor staurosporine confirmed that the 7 ligands are truly allosteric, in agreement with their design. Of these, compound 2 bound CDK2 with an EC50 value of 3 μM and inhibited the proliferation of MDA-MB231 and ZR-75-1 breast cancer cells with IC50 values of approximately 20 μM, while compound 4 had an EC50 value of 71 μM and IC50 values around 4 μM. Remarkably, the most potent compound 4 was able to selectively inhibit CDK2-mediated Retinoblastoma phosphorylation, confirming that its mechanism of action is fully compatible with a selective inhibition of CDK2 phosphorylation in cells. Finally, hit expansion through analog search of the most potent inhibitor 4 revealed an additional ligand 4g with similar in vitro potency on breast cancer cells

Cancer-derived p53 mutants suppress p53-target gene expression—potential mechanism for gain of function of mutant p53

Tumour-derived p53 mutants are thought to have acquired ‘gain-of-function’ properties that contribute to oncogenicity. We have tested the hypothesis that p53 mutants suppress p53-target gene expression, leading to enhanced cellular growth. Silencing of mutant p53 expression in several human cell lines was found to lead to the upregulation of wild-type p53-target genes such as p21, gadd45, PERP and PTEN. The expression of these genes was also suppressed in H1299-based isogenic cell lines expressing various hot-spot p53 mutants, and silencing of mutant p53, but not TAp73, abrogated the suppression. Consistently, these hot-spot p53 mutants were able to suppress a variety of p53-target gene promoters. Analysis using the proto-type p21 promoter construct indicated that the p53-binding sites are dispensable for mutant p53-mediated suppression. However, treatment with the histone deacetylase inhibitor trichostatin-A resulted in relief of mutant p53-mediated suppression, suggesting that mutant p53 may induce hypo-acetylation of target gene promoters leading to the suppressive effects. Finally, we show that stable down-regulation of mutant p53 expression resulted in reduced cellular colony growth in human cancer cells, which was found to be due to the induction of apoptosis. Together, the results demonstrate another mechanism through which p53 mutants could promote cellular growth

Down-regulation of the Nucleotide Excision Repair gene XPG as a new mechanism of drug resistance in human and murine cancer cells

<p>Abstract</p> <p>Background</p> <p>Drug resistance is one of the major obstacles limiting the activity of anticancer agents. Activation of DNA repair mechanism often accounts for increase resistance to cancer chemotherapy.</p> <p>Results</p> <p>We present evidence that nemorubicin, a doxorubicin derivative currently in clinical evaluation, acts through a mechanism of action different from classical anthracyclines, requiring an intact nucleotide excision repair (NER) system to exert its activity. Cells made resistant to nemorubicin show increased sensitivity to UV damage. We have analysed the mechanism of resistance and discovered a previously unknown mechanism resulting from methylation-dependent silencing of the XPG gene. Restoration of NER activity through XPG gene transfer or treatment with demethylating agents restored sensitivity to nemorubicin. Furthermore, we found that a significant proportion of ovarian tumors present methylation of the XPG promoter.</p> <p>Conclusions</p> <p>Methylation of a NER gene, as described here, is a completely new mechanism of drug resistance and this is the first evidence that XPG gene expression can be influenced by an epigenetic mechanism. The reported methylation of XPG gene could be an important determinant of the response to platinum based therapy. In addition, the mechanism of resistance reported opens up the possibility of reverting the resistant phenotype using combinations with demethylating agents, molecules already employed in the clinical setting.</p

PO-035 LKB1 deficiency renders non-small-cell lung cancer cells sensitive to ERK inhibitor

Introduction Lung Cancer is the first cause of cancer-related death in the world. The alterations in KRAS oncogene are very frequent (25%), but, unfortunately, this protein is at present undruggable. KRAS mutations determine over-activation of important pathways of growth and proliferation (PI3K-AKT-mTOR and MAPK). KRAS-mutated tumours are also frequently co-mutated in LKB1 (50%), an important regulator of metabolic homeostasis and oxidative stress in the cells. LKB1 modulates catabolic processes through AMPK-mediated mTOR inhibition. Thereafter, the inactivation of LKB1 causes in KRAS mutated-tumours further activation of PI3K-AKT-mTOR and MAPK pathways, making them particularly aggressive. The possibility to specifically target tumours with both KRAS and LKB1 alterations represent an important medical need. Material and methods We generated from the NSCLC cell line NCI-H1299 clones over-expressing KRAS WT or KRAS G12C forms. These clones have been subsequently modified through CRISPR-CAS9 system to obtain deletions in LKB1 gene. We successfully generated isogenic cells differing only for the status of KRAS and LKB1 (KRASwt/LKB1wt, KRASmut/LKB1wt, KRASwt/LKB1mut, KRASmut/LKB1mut). These clones were treated with a panel of inhibitors of MAPK and PI3K pathways. Viability was evaluated with MTS assay. Molecular characterizations were performed by western blot analysis. In vivo antitumor activity was determined after subcutaneous injection of NSCLC cells in immunodeficient mice. Results and discussions Using the isogenic system generated we tested the activity of several inhibitors of MAPK and PI3K pathways. The results highlighted a strong response of the clones with deletion in LKB1 to ERK inhibitor, independently from the KRAS status. These results were confirmed 'in vivo', where tumours with LKB1 deletion showed a significant sensitivity to ERK inhibitor, compared to LKB1 WT tumours. At molecular level we tested the activation of proteins related to MAPK and PI3K pathway such as p70, S6, 4-EBP1, ERK. The results showed that the response to ERK inhibitor was mainly due to mTOR signalling inhibition. Conclusion The results obtained highlight a possible strategy to target NSCLC with KRAS-LKB1 co-mutations, that, at moment are those with a worse prognosis. The sensitivity to ERK inhibitor is remarkable, also in presence of KRAS WT, therefore this strategy could be applied to all LKB1-mutated lung tumours, that represent 30% of all NSCLC. These studies are being confirmed in other NSCLC backgrounds and mouse models

Exploiting endocytosis for transfection of mRNA for cytoplasmatic delivery using cationic gold nanoparticles

Gene therapeutics; Gold nanoparticles; SafetyTerapia génica; Nanopartículas de oro; SeguridadTeràpia gènica; Nanopartícules d'or; SeguretatIntroduction: Gene therapy holds promise to cure various diseases at the fundamental level. For that, efficient carriers are needed for successful gene delivery. Synthetic ‘non-viral’ vectors, as cationic polymers, are quickly gaining popularity as efficient vectors for transmitting genes. However, they suffer from high toxicity associated with the permeation and poration of the cell membrane. This toxic aspect can be eliminated by nanoconjugation. Still, results suggest that optimising the oligonucleotide complexation, ultimately determined by the size and charge of the nanovector, is not the only barrier to efficient gene delivery. Methods: We herein develop a comprehensive nanovector catalogue comprising different sizes of Au NPs functionalized with two different cationic molecules and further loaded with mRNA for its delivery inside the cell. Results and Discussion: Tested nanovectors showed safe and sustained transfection efficiencies over 7 days, where 50 nm Au NPs displayed the highest transfection rates. Remarkably, protein expression was increased when nanovector transfection was performed combined with chloroquine. Cytotoxicity and risk assessment demonstrated that nanovectors are safe, ascribed to lesser cellular damage due to their internalization and delivery via endocytosis. Obtained results may pave the way to design advanced and efficient gene therapies for safely transferring oligonucleotides.We acknowledge financial support from the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU) (RTI2018-099965-B-I00, AEI/FEDER,UE) proyectos de I+D+i de programación conjunta internacional MCIN/AEI (CONCORD, PCI2019-103436) cofunded by the European Union and Generalitat de Catalunya (2017-SGR-1431). ICN2 is supported by the Severo Ochoa program from Spanish MINECO (SEV-2017-0706) and is funded by the CERCA Programme/Generalitat de Catalunya