Jose Antonio Rodriguez

Global Histone Modifications Predict Prognosis of Resected Non–Small-Cell Lung Cancer

PURPOSEnEpigenetic modifications may contribute to the development and progression of cancer. We investigated whether epigenetic changes involving multiple histones influence prognosis of non-small-cell lung cancer (NSCLC) patients.nnnPATIENTS AND METHODSnWe used immunohistochemistry to assess histone 3 lysine 4 dimethylation (H3K4diMe), and acetylation of histone 2A lysine 5 (H2AK5Ac), histone 2B lysine 12, histone 3 lysine 9 (H3K9Ac), and histone 4 lysine 8 in resected tumor samples of 138 NSCLC patients. Data were analyzed using a recursive partitioning analysis (RPA).nnnRESULTSnThe RPA classified the patients into seven distinct prognostic groups based on TNM stage (first node), histology, and histone modifications: H3K4diMe (< or 85% tumor cells), H3K9Ac (< or 68% tumor cells), and H2AK5Ac (< or 5% tumor cells). The seven groups were associated with significantly different disease-free (P < .0001) and overall survival (P < .0001). Interestingly, the four groups determined by stage I patients (below the first node) displayed dramatic differences in survival (median, 10 months in adenocarcinoma patients with H3K9Ac 68% v 147 months in nonadenocarcinoma patients with H3K4diMe 85%). A Cox model retained age and RPA groups as the sole independent factors significantly influencing overall survival.nnnCONCLUSIONnThe prognostic influence of epigenetic changes involving multiple histones, in particular H2A and H3, is greater in early NSCLC, and evaluation of these changes may help in selecting early-stage NSCLC patients for adjuvant treatment. Our observations provide a rationale for the use of a combination of standard chemotherapy with drugs interacting with histone modifications, such as histone deacetylase inhibitors.

Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells.

We have previously reported that the microtubule stabilizing agents (MSAs) paclitaxel, epothilone B and discodermolide induce caspase-independent cell death in non-small cell lung cancer (NSCLC) cells. Here we present two lines of evidence indicating a central role for the lysosomal protease cathepsin B in mediating cell death. First, inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against drug-induced cell death in several NSCLC cells. Second, MSAs trigger disruption of lysosomes and release and activation of cathepsin B. Interestingly, inhibition of cathepsin B prevents the appearance of multinucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in this novel cell death pathway.

Predictive Factors for Outcome in a Phase II Study of Gefitinib in Second-Line Treatment of Advanced Esophageal Cancer Patients

PURPOSEnThe efficacy of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib was assessed in a phase II study in patients with advanced esophageal cancer. Several biologic features were investigated as potential markers of gefitinib activity.nnnPATIENTS AND METHODSnPatients with advanced esophageal cancer, who had failed one line of prior chemotherapy, were administered gefitinib 500 mg/d. Response was evaluated every 8 weeks. Tumor material obtained before gefitinib treatment was investigated for gene mutations in EGFR, k-ras, and PIK3CA; protein expression levels of EGFR, p-Akt, and p-Erk; and EGFR gene amplification.nnnRESULTSnOf the 36 enrolled patients, one (2.8%) achieved a partial response, 10 (27.8%) had stable disease, 17 (47.2%) experienced progression on treatment, and eight (22.2%) were not assessable for response. The progression-free survival time was 59 days, and the median overall survival time was 164 days. Although EGFR or PIK3CA mutations were absent, k-ras mutations were found in two patients with progressive disease. High EGFR gene copy number was identified in two patients experiencing partial response or progressive disease. A higher disease control rate (response plus stable disease) was observed in females (P = .038) and in patients with squamous cell carcinoma (SCC; P = .013) or high EGFR expression (P = .002).nnnCONCLUSIONnGefitinib has a modest activity in second-line treatment of advanced esophageal cancer. However, the patient outcome was significantly better in female patients and in patients demonstrating high EGFR expression or SCC histology. The selection of esophageal cancer patients for future studies with EGFR-TKIs based on the level of EGFR expression in their tumors or SCC histology should be considered.

Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells

In this study, we have characterized a panel of NSCLC cell lines with differential sensitivity to gefitinib for activating mutations in egfr, pik3ca, and k‐ras, and basal protein expression levels of PTEN. The egfr mutant NSCLC cell line H1650 as well as the egfr wild type cell lines H292 and A431 were highly sensitive to gefitinib treatment, indicating that other factors determine gefitinib‐sensitivity in egfr wild type cells. Activating k‐ras mutations were specifically detected in gefitinib‐resistant cells, suggesting that the occurrence of k‐ras mutations is correlated with resistance to EGFR antagonists. No pik3ca mutations were detected within the panel of cell lines, and PTEN protein expression levels did not correlate with gefitinib sensitivity. Gefitinib effectively blocked Akt and Erk phosphorylation in two gefitinib‐sensitive NSCLC cell lines, further supporting our previous findings that persistent activity of the PI3K/Akt and/or Ras/Erk pathways is associated with gefitinib‐resistance of NSCLC cell lines. Gefitinib‐resistant NSCLC cell lines, showing EGFR‐independent activity of the PI3K/Akt or Ras/Erk pathways, were treated with gefitinib in combination with specific inhibitors of mTOR, P13K, Ras, and MEK. Additive cytotoxicity was observed in A549 cells co‐treated with gefitinib and the MEK inhibitor U0126 or the farnesyl transferase inhibitor SCH66336 and in H460 cells treated with gefitinib and the PI3K inhibitor LY294002, but not in H460 cells treated with gefitinib and rapamycin. These data suggest that combination treatment of NSCLC cells with gefitinib and specific inhibitors of the PI3K/Akt and Ras/Erk pathways may provide a successful strategy.

Molecular Mechanisms Underlying the Synergistic Interaction of Erlotinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, with the Multitargeted Antifolate Pemetrexed in Non-Small-Cell Lung Cancer Cells

Because the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and the multitargeted antifolate pemetrexed are registered in the treatment of second-line non-small-cell lung cancer (NSCLC), empirical combinations of these drugs are being tested. This study investigated molecular mechanisms underlying their combination in six NSCLC cell lines. Cells were characterized by heterogeneous expression of pemetrexed determinants, including thymidylate synthase (TS) and dihydrofolate reductase (DHFR), and mutations potentially affecting chemosensitivity. Pharmacological interaction was studied using the combination index (CI) method, whereas cell cycle, apoptosis induction, and EGFR, extracellular signal-regulated kinases 1 and 2, and Akt phosphorylation were studied by flow cytometry, fluorescence microscopy, and enzyme-linked immunosorbent assays. Reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and activity assays were performed to assess whether erlotinib influenced TS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays demonstrated that EGFR and k-Ras mutations were related to erlotinib sensitivity, whereas TS and DHFR expression were related to pemetrexed sensitivity. Synergistic cytotoxicity was found in all cells, most pronounced with pemetrexed + erlotinib (24 h) → erlotinib (48 h) sequence (CI, 0.09-0.40), which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination (-70.6% in H1650). Erlotinib significantly reduced TS expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and Western blot, and the combination decreased TS in situ activity in all cells. Erlotinib and pemetrexed showed a strong synergism in NSCLC cells, regardless of their genetic characteristics. Induction of apoptosis, modulation of EGFR and Akt phosphorylation, and changes in the expression of critical genes involved in pemetrexed activity contribute to this synergistic interaction and support the clinical investigation of these markers.

Frequent overexpression of aurora B kinase, a novel drug target, in non–small cell lung carcinoma patients

The serine/threonine protein kinase aurora B, a key regulator of mitosis, is emerging as a novel drug target for cancer treatment. Aurora B overexpression has been previously documented by immunohistochemistry in several types of human tumors. We assessed aurora B expression in a series of 160 non–small cell lung cancer (NSCLC) samples (60% stage I, 21% stage II, 11% stage III, and 8% stage IV). In addition, we determined the expression of survivin and p16, two molecules also involved in cell cycle control. Aurora B was expressed selectively in tumor cells compared with normal epithelium. Aurora B expression was significantly correlated with expression of survivin in the nucleus (P < 0.0001), but not with expression of p16 (P = 0.134). High aurora B expression levels were significantly associated with older age (P = 0.012), male sex (P = 0.013), squamous cell carcinoma histology (P = 0.001), poor tumor differentiation grade (P = 0.007), and lymph node invasion (P = 0.037), in the subset of radically resected patients in our series. In addition, aurora B expression predicted shorter survival for the patients with adenocarcinoma histology, at both univariate (P = 0.020) and multivariate (P = 0.012) analysis. Survivin expression levels were neither associated with patient clinicopathologic characteristics nor with survival. However, expression of survivin in the nucleus was preferentially detected in stage I and II than in stage III and IV (P = 0.007) in the overall series of NSCLC samples. Taken together, our results suggest that aurora B may represent a valid target in NSCLC. [Mol Cancer Ther 2006;5(11):2905–13]

EGFR inhibitors: what have we learned from the treatment of lung cancer?

Tyrosine kinase inhibitors directed against the epidermal growth factor receptor (EGFR) are the first molecular-targeted agents to be approved in the US and other countries for the treatment of advanced non-small-cell lung cancer after failure of chemotherapy. Some patient characteristics, such as never-smoking, female gender, East Asian origin, adenocarcinoma histology, and bronchioloalveolar subtype, are associated with a greater benefit from treatment with EGFR inhibitors. Recently, studies have identified gene mutations targeting the kinase domain of the EGFR that are related to the response to inhibitors. Most EGFR mutations predict a higher benefit from treatment compared with wild-type receptors and are correlated with clinical features related to better outcome; some EGFR mutations, however, confer drug resistance. The analysis of material usually available from lung cancer patients, using techniques such as direct sequencing to determine EGFR mutational status, can be technically challenging. In this regard, high EGFR copy number and EGFR protein detected by immunohistochemistry can also be used to select those patients who would benefit from treatment. Prospective validation of biological and clinical markers of sensitivity needs to be performed.

Yeast associated with spontaneous fermentations of white wines from the "Txakoli de Bizkaia" region (Basque Country, North Spain).

The microbiota of eight spontaneous fermentation of white wine from different grape varieties and different wineries from the Txakoli de Bizkaia region (Basque country, North Spain), in 1996 and 1997 campaigns was studied. The yeast population was higher in grapes harvested in 1997, in which late summer and early autumn was warmer and drier. Eight species belonging to five genera were identified in total. The most frequent genera in grapes were Rhodotorula in 1996 and Kloeckera in 1997. Saccharomyces bayanus was the most frequent species during vigorous and final fermentation, and it was occasionally isolated from grapes and must. Only another Saccharomyces spp., i.e., S. kluyvery, was identified in some samples from 1997.

EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens.

Background: Mutational analysis of the Epidermal Growth Factor Receptor (EGFR) and K-ras genes to select non-small cell lung cancer (NSCLC) patients for treatment with novel EGFR tyrosine kinase inhibitors is an appealing possibility currently under investigation. Although frozen tumor tissue would probably be the optimal source for analysis, the most common source of tumor material is fixed and paraffin embedded (FPE) archival specimens. Here, we evaluate how different procedures of tissue sample processing and preservation may affect the outcome of EGFR and K-ras mutation analysis. Furthermore, we compare the sensitivity of the analysis using genomic DNA (gDNA) versus RNA. Methods: We used PCR amplification and direct sequencing to analyze EGFR and K-ras genes in paired FPE and frozen tumor samples corresponding to 47 NSCLC patients. In frozen samples, the analysis was carried out using both gDNA and RNA extracted in parallel. Results: Whereas 100% of frozen samples were successfully amplified, the rate of successful PCR amplification in FPE samples was approximately 50%. We detected three previously described EGFR point mutations in 2 samples. In ten other samples, a K-ras mutation was observed. These mutations were detected in DNA extracted from frozen samples as well as in DNA obtained from FPE tissue. In addition, 10 nucleotide changes, were detected in FPE samples that were not detected in the frozen specimens. Upon re-analysis, these nucleotide changes could not be confirmed and were most likely the result of paraffin embedding and fixation procedures. All mutations found in gDNA were also detected in the corresponding RNA and, in two cases, the presence of the mutant allele was easier to identify by using RNA. Conclusions: Our results indicate that RNA extracted from frozen tissue is the preferred source for EGFR and K-ras mutation testing. When analyzing FPE samples, reducing the size of the amplified fragments would increase PCR success rate, and care should be taken to control for false-positive results.

USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy

Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.