Purpose: Epigenetic 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. Patients and Methods: We 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). Results: The 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. Conclusion: The 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.

BRCA1 is a tumor suppressor with several important nuclear functions. BRCA1 has no known cytoplasmic functions. We show here that the two previously identified nuclear localization signals (NLSs) are insufficient for nuclear localization of BRCA1 due to the opposing action of an NH₂-terminal nuclear export signal. In transfected breast cancer cells, BRCA1 nuclear localization requires both the NLSs and NH₂-terminal RING domain region; mutating either of these sequences shifts BRCA1 to the cytoplasm. The BRCA1 RING element mediates nuclear import via association with BARD1, and this is not affected by cancer-associated RING mutations. Moreover, BARD1 directly masks the BRCA1 nuclear export signal, and the resulting block to nuclear export is requisite for efficient import and nuclear localization of ectopic and endogenous BRCA1. Our results explain why BRCA1 exon 11 splice variants, which lack the NLSs but retain the RING domain, are frequently detected in the nucleus and in nuclear foci in vivo. In fact, co-expression of BARD1 promoted formation of DNA damage-induced nuclear foci comprising ectopic wild-type or NLS-deficient BRCA1, implicating BARD1 in nuclear targeting of BRCA1 for DNA repair. Our identification of BARD1 as a BRCA1 nuclear chaperone has regulatory implications for its reported effects on BRCA1 protein stability, ubiquitin ligase activity, and DNA repair.

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.

Purpose: The 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. Patients and Methods: Patients 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. Results: Of 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). Conclusion: Gefitinib 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.

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 anin vitromodel 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.

Purpose: Erlotinib has proven activity in pretreated patients with advanced non–small cell lung cancer (NSCLC). We evaluated erlotinib in the frontline treatment of advanced NSCLC and assessed biological predictors of outcome. Experimental Design: In this phase II study, chemotherapy-naive patients with stage IIIB/IV NSCLC received oral erlotinib (150 mg/d) until disease progression or unacceptable toxicity occurred. Tumor response was assessed every 6 weeks, and samples were analyzed for potential molecular markers of treatment response and survival. The primary end point was the proportion of patients without disease progression after 6 weeks of treatment. Results: Fifty-three patients were eligible. The overall rate of nonprogression at 6 weeks was 52.8% (28 of 53 patients). Tumor response rate was 22.7%, with 1 complete response, 11 partial responses, and 16 cases of stable disease. Responses were seen across most patient clinical characteristics. The median duration of tumor response was 333 days; median overall survival was 391 days; and median time to disease progression was 84 days. Erlotinib was well tolerated, the main treatment-related adverse events being mild-to-moderate rash and diarrhea. Histologic material for biological studies was available in 29 cases. Four of five responders and one patient with stable disease had a classic epidermal growth factor receptor tyrosine kinase mutation. Two progressing patients exhibited epidermal growth factor receptor point mutations (one with T790M mutation), and K-ras mutations were detected in 10 nonresponders. Conclusions: Erlotinib shows significant antitumor activity in the first-line treatment of advanced NSCLC and may be a viable alternative to chemotherapy. Patient selection cannot easily be based on clinical or biological variables.