CHK1 expression in Gastric Cancer is modulated by p53 and RB1/E2F1: implications in chemo/radiotherapy response.

Radiation has a limited but relevant role in the adjuvant therapy of gastric cancer (GC) patients. Since Chk1 plays a critical function in cellular response to genotoxic agents, we aimed to analyze the role of Chk1 in GC as a biomarker for radiotherapy resistance. We analyzed Chk1 expression in AGS and MKN45 human GC cell lines by RT-QPCR and WB and in a small cohort of human patient's samples. We demonstrated that Chk1 overexpression specifically increases resistance to radiation in GC cells. Accordingly, abrogation of Chk1 activity with UCN-01 and its expression with shChk1 increased sensitivity to bleomycin and radiation. Furthermore, when we assessed Chk1 expression in human samples, we found a correlation between nuclear Chk1 accumulation and a decrease in progression free survival. Moreover, using a luciferase assay we found that Chk1's expression is controlled by p53 and RB/E2F1 at the transcriptional level. Additionally, we present preliminary data suggesting a posttranscriptional regulation mechanism, involving miR-195 and miR-503, which are inversely correlated with expression of Chk1 in radioresistant cells. In conclusion, Chk1/microRNA axis is involved in resistance to radiation in GC, and suggests Chk1 as a potential tool for optimal stratification of patients susceptible to receive adjuvant radiotherapy after surgery.

Impact of chemotherapy on telomere length in sporadic and familial breast cancer patients.

Recently, we observed that telomeres of BRCA1/2 mutation carriers were shorter than those of controls or sporadic breast cancer patients, suggesting that mutations in these genes might be responsible for this event. Given the contradictory results reported in the literature, we tested whether other parameters, such as chemotherapy, could be modifying telomere length (TL). We performed a cross-sectional study measuring leukocyte TL of 266 sporadic breasts cancer patients treated with first-line chemotherapy, with a median follow-up of 240 days. Additionally, we performed both cross-sectional and longitudinal studies in a series of 236 familial breast cancer patients that included affected and non-affected BRCA1/2 mutation carriers. We have measured in leukocytes from peripheral blood: the TL, percentage of short telomeres (<3 kb), telomerase activity levels and the annual telomere shortening speed. In sporadic cases we found that chemotherapy exerts a transient telomere shortening effect (around 2 years) that varies depending on the drug combination. In familial cases, only patients receiving treatment were associated with telomere shortening but they recovered normal TL after a period of 2 years. Chemotherapy affects TL and should be considered in the studies that correlate TL with disease susceptibility.

Mad2 and BubR1 modulates tumourigenesis and paclitaxel response in MKN45 gastric cancer cells.

Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated ß-galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.

P53 pathway activation by telomerase attrtion inX-DC primary fibroblats occurs in the absence of ribosome biogenesis failure and as consequence of DNA damage

BACKGROUND: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. OBJECTIVES: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. METHODS: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. RESULTS: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. CONCLUSIONS: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence (AU)

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p53 pathway activation by telomere attrition in X-DC primary fibroblasts occurs in the absence of ribosome biogenesis failure and as a consequence of DNA damage.

BACKGROUND: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. OBJECTIVES: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. METHODS: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. RESULTS: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. CONCLUSIONS: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence.

Targeting Chk2 improves gastric cancer chemotherapy by impairing DNA damage repair.

Our results demonstrate that the addition of cisplatin after paclitaxel-induced mitotic arrest was more effective than individual treatment on gastric adenocarcinoma cells (MKN45). However, the treatment did not induce benefits in cells derived from lymph node metastasis (ST2957). Time-lapse microscopy revealed that cell death was caused by mitotic catastrophe and apoptosis induction, as the use of the caspase inhibitor z-VAD-fmk decreased cell death. We propose that the molecular mechanism mediating this cell fate is a slippage suffered by these cells, given that our Western blot (WB) analysis revealed premature cyclin B degradation. This resulted in the cell exiting from mitosis without undergoing DNA damage repair, as demonstrated by the strong phosphorylation of H2AX. A comet assay indicated that DNA repair was impaired, and Western blotting showed that the Chk2 protein was degraded after sequential treatment (paclitaxel-cisplatin). Based on these results, the modulation of cell death during mitosis may be an effective strategy for gastric cancer therapy.

IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

Although many cancers initially respond to cisplatin (CDDP)-based chemotherapy, resistance frequently develops. Insulin-like growth factor-binding protein-3 (IGFBP-3) silencing by promoter methylation is involved in the CDDP-acquired resistance process in non-small cell lung cancer (NSCLC) patients. Our purpose is to design a translational-based profile to predict resistance in NSCLC by studying the role of IGFBP-3 in the phosphatidyl inositol 3-kinase (PI3K) signaling pathway. We have first examined the relationship between IGFBP-3 expression regulated by promoter methylation and activation of the epidermal growth factor receptor (EGFR), insulin-like growth factor-I receptor (IGFIR) and PI3K/AKT pathways in 10 human cancer cell lines and 25 NSCLC patients with known IGFBP-3 methylation status and response to CDDP. Then, to provide a helpful tool that enables clinicians to identify patients with a potential response to CDDP, we have calculated the association between our diagnostic test and the true outcome of analyzed samples in terms of cisplatin IC50; the inhibitory concentration that kills 50% of the cell population. Our results suggest that loss of IGFBP-3 expression by promoter methylation in tumor cells treated with CDDP may activate the PI3K/AKT pathway through the specific derepression of IGFIR signaling, inducing resistance to CDDP. This study also provides a predictive test for clinical practice with an accuracy and precision of 0.84 and 0.9, respectively, (P=0.0062). We present a biomarker test that could provide clinicians with a robust tool with which to decide on the use of CDDP, improving patient clinical outcomes.

Defects in mTR stability and telomerase activity produced by the Dkc1 A353V mutation in dyskeratosis congenita are rescued by a peptide from the dyskerin TruB domain

BACKGROUND: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. MATERIALS AND METHODS: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. RESULTS: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. DISCUSSION: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach (AU)

High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. Cells derived from DC patients present short telomeres at early ages, as a result of mutations in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). However, mutations have been identified only in around 50% of the cases, indicating that other genes could be involved in the development of this disease. Indeed, mutations in TCBA1 or chromosome segment C16orf57 have been described recently. We have used HRM technology to perform genetic analysis in the above mentioned genes, in Spanish patients showing both, some clinical features of DC and short telomeres. The mutations have been identified by PCR amplification of DC genes followed by high resolution melting (HRM) and direct DNA sequencing analysis. We have identified seven new families with DC, three with X-linked DC and four with autosomal dominant DC, in which we have found two novel mutations in DKC1 (p.His68Arg and p.Lys390del) and four novel mutations in TERT gene (p.Pro530Leu, p.Arg698Trp, p.Arg971His and p.Arg698Gln). The results show that the use of HRM analysis enables a rapid and inexpensive identification of mutations in dyskeratosis congenita associated genes.

Acid ceramidase as a chemotherapeutic target to overcome resistance to the antitumoral effect of choline kinase α inhibition.

We have analyzed the response of primary cultures derived from tumor specimens of non small cell lung cancer (NSCLC) patients to choline kinase α (ChoKα) inhibitors. ChoKα inhibitors have been demonstrated to increase ceramides levels specifically in tumor cells, and this increase has been suggested as the mechanism that explain its proapoptotic effect in cancer cells. Here, we have investigated the molecular mechanism associated to the intrinsic resistance, and found that other enzyme involved in lipid metabolism, acid ceramidase (ASAH1), is specifically upregulated in resistant tumors. NSCLC cells with acquired resistance to ChoKα inhibitors also display increased levels of ASAH1. Accordingly, ASAH1 inhibition synergistically sensitizes lung cancer cells to the antiproliferative effect of ChoKα inhibitors. Thus, the determination of the levels of ASAH1 predicts sensitivity to targeted therapy based on ChoKα specific inhibition and represents a model for combinatorial treatments of ChoKα inhibitors and ASAH1 inhibitors. Considering that ChoKα inhibitors have been recently approved to enter Phase I clinical trials by the Food and Drug Administration (FDA), these findings are anticipating critical information to improve the clinical outcome of this family of novel anticancer drugs under development.

Treatment for ALK-mutated non-small-cell lung cancer: a new miracle in the research race.

The discovery of anaplastic lymphoma kinase (ALK) rearrangements in a subset of patients with nonsmall- cell lung cancer (NSCLC) and its potential blockage by specific inhibitors such as crizotinib has been one of the latest advances in the treatment of this disease. In this article, we will review the most important clinical aspects of ALK alterations in NSCLC patients and the pending questions to answer: the most effective means of diagnosing ALK-rearranged NSCLC, and efficacy, toxicity profile and potential mechanisms of resistance to crizotinib.

Treatment for ALK-mutated non-small-cell lung cancer: a new miracle in the research race

The discovery of anaplastic lymphoma kinase (ALK) rearrangements in a subset of patients with nonsmall- cell lung cancer (NSCLC) and its potential blockage by specific inhibitors such as crizotinib has been one of the latest advances in the treatment of this disease. In this article, we will review the most important clinical aspects of ALK alterations in NSCLC patients and the pending questions to answer: the most effective means of diagnosing ALK-rearranged NSCLC, and efficacy, toxicity profile and potential mechanisms of resistance to crizotinib (AU)

DUSP1/MKP1 promotes angiogenesis, invasion and metastasis in non-small-cell lung cancer.

DUSP1/MKP1 is a dual-specific phosphatase that regulates MAPKs activity, with an increasingly recognized role in tumor biology. To understand more about the involvement of DUSP1 in lung cancer, we performed gene expression analyses of parental and DUSP1-interfered H460 non-small-cell lung cancer (NSCLC) cells. Downregulation of DUSP1 induced changes in the expression levels of genes involved in specific biological pathways, including angiogenesis, MAP kinase phosphatase activity, cell-cell signaling, growth factor and tyrosine-kinase receptor activity. Changes in the expression of some of these genes were due to modulation of c-Jun-N-terminal kinase and/or p38 activity by DUSP1. Complementary functional assays were performed to focus on the implication of DUSP1 in angiogenesis and metastasis. In H460 cells, interference of DUSP1 resulted in a diminished capacity to invade through Matrigel, to grow tumors in nude mice and also to induce metastasis through tail-vein injection. Furthermore, the angiogenic potential of H460 cells was also impaired, correlating with a decrease in VEGFC production and indicating that DUSP1 could be required to induce angiogenesis. Finally, we studied whether a similar relationship occurred in patients. In human NSCLC specimens, DUSP1 was mainly expressed in those tumor cells close to CD31 vascular structures and a statistically significant correlation was found between VEGFC and DUSP1 expression. Overall, these results provide evidence for a role of DUSP1 in angiogenesis, invasion and metastasis in NSCLC.

Checkpoint kinase 1 modulates sensitivity to cisplatin after spindle checkpoint activation in SW620 cells.

Aneuploidy is a common feature of tumours that arise by errors in chromosome segregation during mitosis. The aim of this study was to evaluate possible signaling pathways involved in sensitization to chemotherapy in cells with chromosomal instability. We designed a screen using the fission yeast Squizossaccharomyces pombe, to isolate strains showing a phenotype of chromosome mis-segregation and higher sensitivity to the antitumoral drug Bleomycin. We examined differences in gene expression using a comparative analysis of genome-wide expression of the wild type strain and one of the mutants. The results revealed a set of genes involved in cell cycle control, including Mad3/BubR1 and Chk1. We then studied the levels of these two proteins in colorectal cancer human cell lines with different genomic content. Among these, SW620 cells showed higher BubR1 and Chk1 mRNA levels than control cells under normal conditions. Since Chk1 is required for both S and G2/M checkpoints, and the microtubule-destabilizing agent, nocodazole induces mitotic arrest, we attempted to investigate the potential anticancer effects of nocodazole in combination with cisplatin. These studies showed that SW620 cells undergo synergistic cell death after spindle checkpoint activation followed by cisplatin treatment, suggesting a role of Chk1 in this checkpoint, very likely dependent on BubR1 protein. Importantly, Chk1-depleted SW620 cells lost this synergistic effect. In summary, we propose that Chk1 could be a biomarker predictive of the efficacy of chemotherapy across different types of tumors with aneuploidy. These findings may be potentially very useful for the stratification of patients for treatment.

IGFBP-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small-cell lung cancer.

Cisplatin-based chemotherapy is the paradigm of non-small-cell lung cancer (NSCLC) treatment; however, it also induces de novo DNA-hypermethylation, a process that may be involved in the development of drug-resistant phenotypes by inactivating genes required for drug-cytotoxicity. By using an expression microarray analysis, we aimed to identify those genes reactivated in a set of two cisplatin (CDDP) resistant and sensitive NSCLC cell lines after epigenetic treatment. Gene expression, promoter methylation and CDDP-chemoresponse were further analyzed in three matched sets of sensitive/resistant cell lines, 23 human cancer cell lines and 36 NSCLC specimens. Results revealed specific silencing by promoter hypermethylation of IGFBP-3 in CDDP resistant cells, whereas IGFBP-3 siRNA interference, induced resistance to CDDP in sensitive cells (P<0.001). In addition, we found a strong correlation between methylation status and CDDP response in tumor specimens (P<0.001). Thus, stage I patients, whose tumors harbor an unmethylated promoter, had a trend towards increased disease-free survival (DFS). We report that a loss of IGFBP-3 expression, mediated by promoter-hypermethylation, results in a reduction of tumor cell sensitivity to cisplatin in NSCLC. Basal methylation status of IGFBP-3 before treatment may be a clinical biomarker and a predictor of the chemotherapy outcome, helping to identify patients who are most likely to benefit from CDDP therapy alone or in combination with epigenetic treatment.

Telomerase deficiency and cancer susceptibility syndromes

Telomeres from most eukaryotes are composed of repeats of guanine-rich sequences whose main function is to preserve the end of the chromosomes. Telomeres are synthesised by a reverse transcriptase enzyme, telomerase (TERT), which forms part of a ribonucleoprotein complex containing also a RNA template molecule (TERC) and dyskerin. Exhaustion of telomeres during cell divisions triggers a DNA damage response that induces a senescence phenotype. The DNA damage machinery plays an essential role in maintaining the integrity of the genome and also detecting telomere shortening. However in some syndromes that involved mutations either in the telomerase complex genes or those involved in maintaining DNA secondary structure, such as the recQ helicase WRN, a higher frequency in the development of different types of malignancies is observed. We here describe the biology of some of these diseases, together with the molecular modifications in the telomerase complex genes and the impact of these alterations on the development of particular types of cancer (AU)

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hCCR4/cNOT6 targets DNA-damage response proteins.

Radio and chemotherapy are the election options besides surgical resection, in cancer treatment. However, resistance to chemotherapy limits the effectiveness of therapy in the clinic. An improved knowledge of the mechanisms underlying the resistance to treatment would generate new therapeutic strategies. Genetic suppressor elements (GSEs) are short, biologically active, cDNA fragments that interfere with the function of their cognate gene. By selection of genetic suppressor elements (GSEs) conferring resistance to cisplatin, we identified the GSE11, that corresponds to the hCCR4/CNOT6 gene that mediates cellular sensitivity to the drug. Expression of GSE11-hCCR4 reduces hCCR4 protein levels in cells. Targeting hCCR4 with GSE11 or with siRNA, decreases sensitivity of mammalian cells to DNA-damaging agents. Overexpression of hCCR4 targets Chk2 following exposure to cisplatin, without interfering with the upstream ATM/ATR pathway, however histone gammaH2AX is strongly phosphorylated in these cells compared to control cells. Our results uncover a new function for a human protein involved in chemotherapy response. This finding introduces a new pharmacological target in the treatment of solid tumours.