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

Signaling Networks Associated with AKT Activation in Non-Small Cell Lung Cancer (NSCLC): New Insights on the Role of Phosphatydil-Inositol-3 kinase

Aberrant activation of PI3K/AKT signalling represents one of the most common molecular alterations in lung cancer, though the relative contribution of the single components of the cascade to the NSCLC development is still poorly defined. In this manuscript we have investigated the relationship between expression and genetic alterations of the components of the PI3K/AKT pathway [KRAS, the catalytic subunit of PI3K (p110α), PTEN, AKT1 and AKT2] and the activation of AKT in 107 surgically resected NSCLCs and have analyzed the existing relationships with clinico-pathologic features. Expression analysis was performed by immunohistochemistry on Tissue Micro Arrays (TMA); mutation analysis was performed by DNA sequencing; copy number variation was determined by FISH. We report that activation of PI3K/AKT pathway in Italian NSCLC patients is associated with high grade (G3–G4 compared with G1–G2; n = 83; p<0.05) and more advanced disease (TNM stage III vs. stages I and II; n = 26; p<0.05). In addition, we found that PTEN loss (41/104, 39%) and the overexpression of p110α (27/92, 29%) represent the most frequent aberration observed in NSCLCs. Less frequent molecular lesions comprised the overexpression of AKT2 (18/83, 22%) or AKT1 (17/96, 18%), and KRAS mutation (7/63, 11%). Our results indicate that, among all genes, only p110α overexpression was significantly associated to AKT activation in NSCLCs (p = 0.02). Manipulation of p110α expression in lung cancer cells carrying an active PI3K allele (NCI-H460) efficiently reduced proliferation of NSCLC cells in vitro and tumour growth in vivo. Finally, RNA profiling of lung epithelial cells (BEAS-2B) expressing a mutant allele of PIK3 (E545K) identified a network of transcription factors such as MYC, FOS and HMGA1, not previously recognised to be associated with aberrant PI3K signalling in lung cancer

Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion

The Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser435 by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser897 by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinoma—whereas conditional knockout of another RCP cargo, α5 integrin, does not suppress pancreatic cancer metastasis—indicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo

Relation between outcomes and localisation of p-mTOR expression in gastric cancer

The mammalian target of rapamycin (mTOR), a Ser/Thr protein kinase that mediates intracellular signalling related to cell growth, proliferation, and differentiation, has received considerable interest as a possible target for cancer treatment. We evaluated the correlation of mTOR expression with clinicopathological features, outcomes, and the expression of Akt, an upstream regulator of mTOR, in gastric cancer. Tumour samples were obtained from 109 patients with gastric adenocarcinomas who underwent a radical gastrectomy. The expressions of phosphorylated mTOR (p-mTOR) and phosphorylated Akt (p-Akt) in the cytoplasm and in the nucleus were analysed by immunohistochemical staining. Cytoplasmic p-mTOR expression positively correlated with the depth of tumour invasion (T1 vs T2–4, P=0.003), involved lymph nodes (P=0.010), and tumour stage (I vs II–IV, P=0.002). In contrast, nuclear p-mTOR expression negatively correlated with these variables (P<0.001,=0.035, and <0.001). Cytoplasmic p-mTOR expression was associated with significantly poorer relapse-free survival (RFS, P=0.037) and overall survival (OS, P=0.024), whereas nuclear p-mTOR expression was associated with better RFS and OS (P=0.029, 0.059). Neither cytoplasmic nor nuclear p-Akt expression was associated with any clinicopathological factor or with survival. Localisation of p-mTOR may play an important role in tumour progression and outcomes in patients with gastric cancer

Deletions at 14q in malignant mesothelioma detected by microsatellite marker analysis

Previous molecular cytogenetic studies by comparative genomic hybridization (CGH) on primary tumours of human malignant mesothelioma have revealed that loss of genetic material at chromosome 14q is one of the most frequently occurring aberrations. Here we further verify the frequency and pattern of deletions at 14q in mesothelioma. A high-resolution deletion mapping analysis of 23 microsatellite markers was performed on 18 primary mesothelioma tumours. Eight of these had previously been analysed by CGH. Loss of heterozygosity or allelic imbalance with at least one marker was detected in ten of 18 tumours (56%). Partial deletions of varying lengths were more common than loss of all informative markers, which occurred in only one tumour. The highest number of tumours with deletions at a specific marker was detected at 14q11.1–q12 with markers D14S283 (five tumours), D14S972 (seven tumours) and D14S64 (five tumours) and at 14q23–q24 with markers D14S258 (five tumours), D14S77 (five tumours) and D14S284 (six tumours). We conclude from these data that genomic deletions at 14q are more common than previously reported in mesothelioma. Furthermore, confirmation of previous CGH results was obtained in all tumours but one. This tumour showed deletions by allelotyping, but did not show any DNA copy number change at 14q by CGH. Although the number of tumours allelotyped was small and the deletion pattern was complex, 14q11.1–q12 and 14q23–q24 were found to be the most involved regions in deletions. These regions provide a good basis for further molecular analyses and may highlight chromosomal locations of tumour suppressor genes that could be important in the tumorigenesis of malignant mesothelioma. © 1999 Cancer Research Campaig

Integrated Analyses of Copy Number Variations and Gene Expression in Lung Adenocarcinoma

Numerous efforts have been made to elucidate the etiology and improve the treatment of lung cancer, but the overall five-year survival rate is still only 15%. Identification of prognostic biomarkers for lung cancer using gene expression microarrays poses a major challenge in that very few overlapping genes have been reported among different studies. To address this issue, we have performed concurrent genome-wide analyses of copy number variation and gene expression to identify genes reproducibly associated with tumorigenesis and survival in non-smoking female lung adenocarcinoma. The genomic landscape of frequent copy number variable regions (CNVRs) in at least 30% of samples was revealed, and their aberration patterns were highly similar to several studies reported previously. Further statistical analysis for genes located in the CNVRs identified 475 genes differentially expressed between tumor and normal tissues (p<10−5). We demonstrated the reproducibility of these genes in another lung cancer study (p = 0.0034, Fisher's exact test), and showed the concordance between copy number variations and gene expression changes by elevated Pearson correlation coefficients. Pathway analysis revealed two major dysregulated functions in lung tumorigenesis: survival regulation via AKT signaling and cytoskeleton reorganization. Further validation of these enriched pathways using three independent cohorts demonstrated effective prediction of survival. In conclusion, by integrating gene expression profiles and copy number variations, we identified genes/pathways that may serve as prognostic biomarkers for lung tumorigenesis

Gene expression profiling for molecular distinction and characterization of laser captured primary lung cancers

<p>Abstract</p> <p>Methods</p> <p>We examined gene expression profiles of tumor cells from 29 untreated patients with lung cancer (10 adenocarcinomas (AC), 10 squamous cell carcinomas (SCC), and 9 small cell lung cancer (SCLC)) in comparison to 5 samples of normal lung tissue (NT). The European and American methodological quality guidelines for microarray experiments were followed, including the stipulated use of laser capture microdissection for separation and purification of the lung cancer tumor cells from surrounding tissue.</p> <p>Results</p> <p>Based on differentially expressed genes, different lung cancer samples could be distinguished from each other and from normal lung tissue using hierarchical clustering. Comparing AC, SCC and SCLC with NT, we found 205, 335 and 404 genes, respectively, that were at least 2-fold differentially expressed (estimated false discovery rate: < 2.6%). Different lung cancer subtypes had distinct molecular phenotypes, which also reflected their biological characteristics. Differentially expressed genes in human lung tumors which may be of relevance in the respective lung cancer subtypes were corroborated by quantitative real-time PCR.</p> <p>Genetic programming (GP) was performed to construct a classifier for distinguishing between AC, SCC, SCLC, and NT. Forty genes, that could be used to correctly classify the tumor or NT samples, have been identified. In addition, all samples from an independent test set of 13 further tumors (AC or SCC) were also correctly classified.</p> <p>Conclusion</p> <p>The data from this research identified potential candidate genes which could be used as the basis for the development of diagnostic tools and lung tumor type-specific targeted therapies.</p