Rosario Machado-Pinilla

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.

MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the most frequent and therapy-refractive sub-class of lung cancer. Improving apoptosis induction in NSCLC represents a logical way forward in treating this tumor. Cisplatin, a commonly used therapeutic agent in NSCLC, induces activation of N-terminal-c-Jun kinase (JNK) that, in turn, mediates induction of apoptosis. In analysing surgical tissue samples of NSCLC, we found that expression of MKP1/CL100, a negative regulator of JNK, showed a strong nuclear staining for tumor cells, whereas, in normal bronchial epithelia, MKP1 was localized in the cytoplasm as well as in nuclei. In the NSCLC-derived cell lines H-460 and H-23, we found that MKP1 was constitutively expressed. Expressing a small-interfering RNA (siRNA) vector for MKP1 in H-460 cells resulted in a more efficient activation by cisplatin of JNK and p38 than in the parental cells, and this correlated with a 10-fold increase in sensitivity to cisplatin. A similar response was also observed in H-460 and H-23 cells when treated with the MKP1 expression inhibitor RO-31-8220. Moreover, expression of a siRNA-MKP2, an MKP1-related phosphatase, had no effect on H-460 cell viability response to cisplatin. Tumors induced by H-460 cells expressing MKP1 siRNA grew slower in nu−/nu− mice and showed more susceptibility to cisplatin than parental cells, and resulted in an impaired growth of the tumor in mice. On the other hand, overexpression of MKP1 in the H-1299 NSCLC-derived cell line resulted in further resistance to cisplatin. Overall, the results showed that inhibition of MKP1 expression contributes to a slow down in cell growth in mice and an increase of cisplatin-induced cell death in NSCLC. As such, MKP1 can be an attractive target in sensitizing cells to cisplatin to increase the effectiveness of the drug in treating NSCLC.

MKP1 repression is required for the chemosensitizing effects of NF-κB and PI3K inhibitors to cisplatin in non-small cell lung cancer

Treatment of non-small cell lung cancer (NSCLC) with cisplatin has a level of antitumor activity still modest. We have shown previously that MKP1/DUSP1 inhibits cisplatin-induced apoptosis in NSCLC cells and is overexpressed in tumors from most patients with stage I-II NSCLC. Here, using different NSCLC cell lines we found that MKP1 and NF-kappaB are differentially expressed. We studied whether targeting MKP1, NF-kappaB or both affects cisplatin-induced cell death. MKP1 is expressed in H460 and H727 cells. H727 and H1299 cells showed constitutive phosphorylation of Akt and increased NF-kappaB activity than did H460 cells. H460-MKP1-siRNA-expressing cells (but not H727-MKP1-siRNA or H1299-MKP1-siRNA cells) exhibit a marked increase in cisplatin response compared with parental cells. Treatment with the PI3K inhibitor LY294002 or the NF-kappaB inhibitor BAY11-7082 enhanced cisplatin antitumor activity in parental H1299 cells but only weakly affected responses of H727 and H460 cells. MKP1-siRNA expression enhanced the chemosensitization effect of LY294002 and BAY11-7082 on H727 and H460 cells. Additionally, NSCLC cell lines with higher NF-kappaB-constitutive activation were the most sensitive to PS-341 (Bortezomib), a non-specific NF-kappaB inhibitor. This finding suggests the proteasome as a suitable strategy in treating NSCLC tumors with high constitutive NF-kappaB activity. Altogether, these results showed that either an activated PI3K/Akt/NF-kappaB pathway and/or high MKP1 was linked to reduced sensitivity to cisplatin in NSCLC cells. Inhibition of NF-kappaB or PI3K potently enhanced cisplatin cytotoxicity in cells with endogenous or genetically induced low MKP1 levels. These findings support the potential improvement in cisplatin responses by co-targeting NF-kappaB or Akt and MKP1.

Expression of the Genetic Suppressor Element 24.2 (GSE24.2) Decreases DNA Damage and Oxidative Stress in X-Linked Dyskeratosis Congenita Cells

The predominant X-linked form of Dyskeratosis congenita results from mutations in DKC1, which encodes 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. Here we have found that an increased basal and induced DNA damage response occurred in X-DC cells in comparison with normal cells. DNA damage that is also localized in telomeres results in increased heterochromatin formation and senescence. Expression of a cDNA coding for GSE24.2 rescues both global and telomeric DNA damage. Furthermore, transfection of bacterial purified or a chemically synthesized GSE24.2 peptide is able to rescue basal DNA damage in X-DC cells. We have also observed an increase in oxidative stress in X-DC cells and expression of GSE24.2 was able to diminish it. Altogether our data indicated that supplying GSE24.2, either from a cDNA vector or as a peptide reduces the pathogenic effects of Dkc1 mutations and suggests a novel therapeutic approach.

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

BackgroundThe 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 MethodsHere, 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.ResultsExpression 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.