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 NH 2 -terminal nuclear export signal. In transfected breast cancer cells, BRCA1 nuclear localization requires both the NLSs and NH 2 -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.The tumor suppressor, BRCA1, was the first susceptibility gene linked to breast and ovarian cancer (1). Germ-line mutations of BRCA1 are found in ϳ50% of patients with inherited breast cancer and up to 90% of families with breast and ovarian cancer susceptibility (1, 2). The role of BRCA1 as a tumor suppressor is not fully defined, although accumulated evidence suggests that BRCA1 plays a role in transcriptional regulation (3), cell cycle control (4, 5), and cell survival responses to DNA damage (6 -8).BRCA1 is a large gene of 24 exons that encodes a 1,863-amino acid protein (1). The BRCA1 protein contains several protein-interaction domains: an NH 2 -terminal RING domain common to many regulatory proteins (1), two tandem copies of the BRCT (BRCA1 carboxyl terminus) motif at the COOH terminus (9), and both nuclear import (10, 11) and export signals (12). The BRCT domain is found in a variety of proteins, including 53BP1, RAD9, RAD4, Crb2, and RAP1, all of which are associated with cell cycle regulation and DNA repair (13). The BRCT motifs of BRCA1 appear to be critical for its transcription activation function (3, 14), and cancer mutations in this COOH-terminal region impair transcriptional activity (3,15). This is likely due to altered association with specific proteins, such as the RNA polymerase II holoenzyme, which normally interacts with the COOH terminus of BRCA1 (16). The NH 2 -terminal RING domain of BRCA1 mediates association with proteins including BARD1 (17) and BAP1 (18). BARD1 is similar in primary structure to BRCA1, in that it also contains an NH 2 -terminal RING finger and two COOHterminal BRCT domains (17). BRCA1 and BARD1 i...
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.
We have previously reported that the microtubule stabilizing agents (
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.
Because the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and the multitargeted antifolate pemetrexed are registered in the treatment of second-line nonsmall-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 signalregulated 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 ϩ erlotinib (24 h) 3 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 (Ϫ70.6% 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.Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the Western world. Chemotherapy represents the backbone of treatment of advanced NSCLC, which represents more than 50% of cases diagnosed. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.042382.ABBREVIATIONS: NSCLC, non-small-cell lung cancer; 5-FU, 5-fluorouracil; AI, apoptotic index; BCRP, breast cancer resistance protein; CI, combination index; DHFR, dihydrofolate reductase; EGFR, epidermal growth factor receptor; FA, fraction affected; FPGS, folyl-polyglutamate synthetase; GARFT, glycinamide ribonucleotide formyltransferase; MRPs, multidrug-related protein; PI3K, phosphatidylinositide 3-kinase; RFC, reduced folate carrier; TKI, tyrosine-kinase inhibitor; TS, thymidylate synthase; LY294002, (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one ...
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.
Altered expression of XPO1, the main nuclear export receptor in eukaryotic cells, has been observed in cancer, and XPO1 has been a focus of anticancer drug development. However, mechanistic evidence for cancer-specific alterations in XPO1 function is lacking. Here, genomic analysis of 42,793 cancers identified recurrent and previously unrecognized mutational hotspots in XPO1. XPO1 mutations exhibited striking lineage specificity, with enrichment in a variety of B-cell malignancies, and introduction of single amino acid substitutions in XPO1 initiated clonal, B-cell malignancy in vivo. Proteomic characterization identified that mutant XPO1 altered the nucleocytoplasmic distribution of hundreds of proteins in a sequence-specific manner that promoted oncogenesis. XPO1 mutations preferentially sensitized cells to inhibitors of nuclear export, providing a biomarker of response to this family of drugs. These data reveal a new class of oncogenic alteration based on change-of-function mutations in nuclear export signal recognition and identify therapeutic targets based on altered nucleocytoplasmic trafficking. SIGNIFICANCE: Here, we identify that heterozygous mutations in the main nuclear exporter in eukaryotic cells, XPO1, are positively selected in cancer and promote the initiation of clonal B-cell malignancies. XPO1 mutations alter nuclear export signal recognition in a sequence-specific manner and sensitize cells to compounds in clinical development inhibiting XPO1 function.
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 gefitinibsensitivity 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. ' 2005 Wiley-Liss, Inc.Key words: EGFR; Erk; Akt/PKB; mTOR; SCH66336; rapamycin The EGFR II (ErbB1, HER1) is the prototypic member of the ErbB family of RTKs, which further consists of ErbB2-4 (HER2-4). The ErbB receptors share a similar protein structure, consisting of an extracellular ligand binding domain, a single transmembrane domain and an intracellular C-terminal domain with tyrosine kinase activity. Upon specific binding of EGF-like ligands to the extracellular domain, ErbB receptors dimerize, either as homodimers or as heterodimers, and undergo autophosphorylation at specific tyrosine residues within the intracellular domain. These phosphorylated tyrosines serve as docking sites for adapter molecules such as Grb2 and the p85 subunit of PI3K, which activate downstream signalling pathways. These pathways, including the Ras/MAPK and Akt/mTOR kinase cascades, in turn, regulate transcription factors and other proteins involved in cell proliferation, survival, motility and differentiation 1 (Fig. 1). Aberrantly high EGFR activity is common in several tumor types, including NSCLC, and correlates with a more aggressive disease, resistance to chemotherapy and poor patient prognosis.Monoclonal antibodies to the extracellular domain of EGFR, such as cetuximab, and EGFR tyrosine kinase inhibitors, including gefitinib and erlotinib, specifically block E...
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