Jose A. Rodriguez

Erlotinib for Frontline Treatment of Advanced Non–Small Cell Lung Cancer: a Phase II Study

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.

Integration of Gene Dosage and Gene Expression in Non-Small Cell Lung Cancer, Identification of HSP90 as Potential Target

Background Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease. Methodology and Principal Findings In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. Conclusions We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

Bortezomib, but not cisplatin, induces mitochondria-dependent apoptosis accompanied by up-regulation of noxa in the non–small cell lung cancer cell line NCI-H460

Defects in the apoptotic machinery may contribute to chemoresistance of non–small cell lung cancer (NSCLC) cells. We have previously showed a deficiency in mitochondria-dependent caspase-9 activation in NSCLC H460 cells after exposure to cisplatin, a drug widely used to treat NSCLC. Here we show that, unlike cisplatin, the novel anticancer agent bortezomib efficiently induces caspase-9 activation and apoptosis in H460 cells. A comparative analysis of molecular events underlying cell death in bortezomib-treated versus cisplatin-treated H460 cells revealed that bortezomib, but not cisplatin, caused a rapid and abundant release of cytochrome c and Smac/DIABLO from mitochondria. This was associated with a marked increase in levels of the BH3-only proapoptotic protein Noxa and the antiapoptotic protein Mcl-1. Taken together, our data show that bortezomib, by promoting a proapoptotic shift in the levels of proteins involved in mitochondrial outer-membrane permeabilization, is a potent activator of the mitochondrial pathway of apoptosis in NSCLC cells. Our preclinical results support further investigation of bortezomib-based therapies as a possible new treatment modality for NSCLC. [Mol Cancer Ther 2007;6(3):1046–53]

Epidermal growth factor receptor (EGFR) gene copy number detection in non-small-cell lung cancer; a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization.

Aims:  The epidermal growth factor receptor (EGFR) is an important target for anticancer therapy. In non‐small‐cell lung cancer (NSCLC), mutations in the tyrosine kinase domain of EGFR and EGFR gene copy number have been demonstrated to identify patients most likely to benefit from EGFR tyrosine kinase inhibitors. EGFR gene copy number has been assessed mainly by fluorescence in situ hybridization (FISH), a method requiring the use of a fluorescence microscope and often hampered by the rapid fading of the fluorescent signal. These limitations of FISH can be overcome by using chromogenic in situ hybridization (CISH). To test the applicability of CISH for EGFR gene copy number testing in NSCLC, a comparison of CISH and FISH was performed.

Genetic heterogeneity in patients with multiple neoplastic lung lesions: a report of three cases.

Introduction: It is important to determine the relation among the various lesions in patients presenting with multiple malignant lung tumors to define the best treatment approach. A better understanding of the molecular alterations present in the different lesions may help in defining this relation. Methods: We performed a detailed molecular analysis of several tumor specimens obtained from three patients presenting with multiple lung lesions. Tumor specimens were analyzed for epidermal growth factor receptor (EGFR) and k-ras mutations by direct DNA sequencing. In addition, a genome-wide chromosomal copy number analysis was performed on DNA extracted from the various lesions using array-based comparative genomic hybridization. Results: In one case, a deletion of 15 base pairs in exon 19 of EGFR was present in all tumor sites analyzed. Furthermore, a similar pattern of chromosomal aberrations was observed among the various lesions, suggesting that they share the same clonal origin. In the other two cases, in contrast, we identified distinct k-ras genotypes among the various lesions from the same patient. These lesions, moreover, showed different chromosomal aberration patterns, indicating that they may have different underlying pathways of tumorigenesis. Conclusion: Our results show that EGFR and k-ras mutation analysis, combined with chromosomal copy number profiling, can help in defining the relationship among different tumors in one patient.

Comparative proteomics analysis of caspase-9-protein complexes in untreated and cytochrome c/dATP stimulated lysates of NSCLC cells

Apoptosis is a process of cellular suicide executed by caspases. Impaired activation of caspase-9 may contribute to chemoresistance in cancer. Activation of caspase-9 occurs after binding to Apaf-1 and formation of the apoptosome in the presence of cytochrome c/(d)ATP. We used a proteomics approach to identify proteins in caspase-9-protein complexes in extracts derived from NSCLC cells with(out) cytochrome c/dATP. Using co-immunoprecipitation, one-dimensional gel electrophoresis and tandem mass spectrometry, 38 proteins were identified of which 24 differential interactors. The differential interactors can be functionally assigned to cytoskeletal (re)organization and cell motility, catalytic activity, and transcriptional processes and apoptosis. The interaction of caspase-9 with Apaf-1 was confirmed and acetylserotonin-O-methyltransferase-like protein was identified as a candidate substrate of caspase-9. Novel interactors were found including galectin-3, swiprosin-1 and the membrane-cytoskeleton linkers Ezrin/Radixin/Moesin. Co-immunoprecipitation and Western blot experiments confirmed the interaction of caspase-9 with several identified binding partners. A large number of cytoskeletal proteins associated with unprocessed caspase-9 may indicate a scaffold function of this structure and/or may act as caspase substrates during apoptosis. Together, our results indicate that proteomic analysis of the caspase-9-associated protein complexes is a powerful exploratory approach to identify novel caspase substrates and/or regulators of caspase-9-dependent apoptosis.

TUCAN/CARDINAL/CARD8 and apoptosis resistance in non-small cell lung cancer cells

BackgroundActivation of caspase-9 in response to treatment with cytotoxic drugs is inhibited in NSCLC cells, which may contribute to the clinical resistance to chemotherapy shown in this type of tumor. The aim of the present study was to investigate the mechanism of caspase-9 inhibition, with a focus on a possible role of TUCAN as caspase-9 inhibitor and a determinant of chemosensitivity in NSCLC cells.MethodsCaspase-9 processing and activation were investigated by Western blot and by measuring the cleavage of the fluorogenic substrate LEHD-AFC. Proteins interaction assays, and RNA interference in combination with cell viability and apoptosis assays were used to investigate the involvement of TUCAN in inhibition of caspase-9 and chemosensitivity NSCLC.ResultsAnalysis of the components of the caspase-9 activation pathway in a panel of NSCLC and SCLC cells revealed no intrinsic defects. In fact, exogenously added cytochrome c and dATP triggered procaspase-9 cleavage and activation in lung cancer cell lysates, suggesting the presence of an inhibitor. The reported inhibitor of caspase-9, TUCAN, was exclusively expressed in NSCLC cells. However, interactions between TUCAN and procaspase-9 could not be demonstrated by any of the assays used. Furthermore, RNA interference-mediated down-regulation of TUCAN did not restore cisplatin-induced caspase-9 activation or affect cisplatin sensitivity in NSCLC cells.ConclusionThese results indicate that procaspase-9 is functional and can undergo activation and full processing in lung cancer cell extracts in the presence of additional cytochrome c/dATP. However, the inhibitory protein TUCAN does not play a role in inhibition of procaspase-9 and in determining the sensitivity to cisplatin in NSCLC.

Subcellular localization of CrmA: identification of a novel leucine-rich nuclear export signal conserved in anti-apoptotic serpins

The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments.

Immunohistochemical detection of nuclear survivin in NSCLC: a comparison of commercial antibodies

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Apoptosis Pathways and New Anticancer Agents

Apoptosis or programmed cell death is a physiologic process that determines tissue homeostasis and provides an effective way to remove unwanted cells, such as those that have accumulated oncogenic mutations. Inhibition of apoptosis disrupts the balance between cell proliferation and cell death and has been recognized as one of six key mechanisms that are essential for the generation of fully transformed malignant cells [1]. Cells that undergo apoptosis are characterized by morphologic changes that include cytoplasmic shrinkage, plasma membrane blebbing, and chromatin condensation in the nucleus, which facilitate the efficient inflammation-free removal of apoptotic cells by macrophages [2]. At the molecular level, proteolytic enzymes such as caspases play an important role as the executors of apoptosis leading to cell death. Apoptosis is distinct from passive nonregulated cell death (necrosis), and, in general, is referred to as caspase-dependent cell death. The term “classical apoptosis” has been coined to distinguish it from other forms of programmed cell death that display a mixture of morphology or molecular features or both representing caspaseor noncaspase-dependent cell death. We focus on the therapeutic exploitation of the core apoptotic machinery that regulates caspase-dependent cell death. Two main caspase activation pathways have been identified (see also Fig. 12-1). One route, known as the intrinsic or mitochondrial pathway, is triggered upon disruption of mitochondria, e.g., because of DNA damage induced by cytotoxic agents, and causes the release of cytochrome c into the cytoplasm [3, 4]. Together with dATP, cytochrome c is a cofactor for the assembly of the apoptosome, which contains Apaf-1 and procaspase-9, and leads to the processing and activation of caspase-9. The second route, the so-called extrinsic or death receptor pathway, is initiated through specific cell membrane receptors, such as Fas/CD95 and tumor necrosis factor (TNF) family receptors, that upon ligand binding recruit the cytosolic death-domain-containing protein FADD (Fas-associated protein with death domain), which is able to bind and activate procaspase-8 in a complex named the death-inducing signaling complex (DISC). Both caspase-8 and -9 can activate the effector caspases-3, -6, and -7 provided that the caspase inhibitory effect of the inhibitor of apoptosis proteins (IAP) is relieved by Smac/Diablo, a proapoptotic protein that is also released from the mitochondria. The IAP family comprises proteins that contain one or more baculovirus IAP repeat (BIR) domains, which mediate in some IAP the interaction with caspases [5, 6]. The most potent caspase-inhibitory IAP is X-linked IAP (XIAP). When released in the cytosol, Smac binds to XIAP facilitating caspase activation. The activation of the effector caspases leads to the cleavage of various substrates, which results in the characteristic morphologic features of apoptotic cell death. An important class of regulators of apoptosis are the BCL-2 family proteins [7–9], comprising both antiapoptotic members, such as BCL-2, BCL-X L , and MCL-1, as well as proapoptotic members such as BAX and BAK, that share homology throughout four or three BCL-2 homology domains, respectively. Their primary mode of action has been assumed to be the regulation of mitochondrial integrity; however, they also appear to be involved in maintaining the integrity of other intracellular membrane structures, such as the endoplasmatic reticulum. Upon apoptosis activation, BAX and BAK translocate from the cytoplasm to the mitochondrial membrane where they oligomerize to form porelike structures, thereby causing mitochondrial outer membrane permealization (MOMP) and the release of apoptogenic factors, such as cytochrome c and Smac. The BH3-only proteins constitute a third class of proapoptotic BCL-2 proteins, which includes BID, BAD, BIK, PUMA, NOXA, BMF, and HRK. These proteins share homology in only one region, the BH3 domain. The BH3-only proteins appear to function as sentinels for the detection of cellular damage or aberrations; for example, BIM is activated by microtubule disarray, whereas