As with other epithelial cancers, lung cancer develops over a period of several years or decades via a series of progressive morphological changes accompanied by molecular alterations that commence in histologically normal epithelium. However the development of lung cancer presents certain unique features that complicates this evaluation. Anatomically the respiratory tree may be divided into central and peripheral compartments having different gross and histological anatomies as well as different functions. In addition, there are three major forms of lung cancer and many minor forms. Many of these forms arise predominantly in either the central or peripheral compartments. Squamous cell and small cell carcinomas predominantly arise in the central compartment, while adenocarcinomas predominantly arise peripherally. Large cell carcinomas are not a single entity but consist of poorly differentiated forms of the other types and, possibly, some truly undifferentiated “stem cell like” tumors. The multistage origin of squamous cell carcinomas, because of their central location, can be followed more closely than the peripherally arising adenocarcinomas. Squamous cell carcinomas arise after a series of reactive, metaplastic, premalignant and preinvasive changes. However, long term observations indicate that not all tumors follow a defined histologic course, and the clinical course, especially of early lesions, is difficult to predict. Peripheral adenocarcinomas are believed to arise from precursor lesions known as atypical adenomatous hyperplasias and may have extensive in situ growth before becoming invasive. Small cell carcinomas are believed to arise from severely molecularly damaged epithelium without going through recognizable preneoplastic changes. The molecular changes that occur prior to the onset on invasive cancers are extensive. As documented in this chapter, they encompass all of the six classic Hallmarks of Cancer other than invasion and metastasis, which by definition occur beyond preneoplasia. A study of preinvasive lung cancer has yielded much valuable biologic information that impacts on clinical management.
Lung cancer; squamous cell carcinoma; adenocarcinomas; small cell lung carcinoma; preneoplasia; carcinoma in situ; atypical adenomatous hyperplasia; tumor suppressor genes; oncogenes; apoptosis; telomerase; angiogenesis
Lung cancer cell lines have made a substantial contribution to lung cancer translational research and biomedical discovery. A systematic approach to initiating and characterizing cell lines from small cell and non–small cell lung carcinomas has led to the current collection of more than 200 lung cancer cell lines, a number that exceeds those for other common epithelial cancers combined. The ready availability and widespread dissemination of the lines to investigators worldwide have resulted in more than 9000 citations, including multiple examples of important biomedical discoveries. The high (but not perfect) genomic similarities between lung cancer cell lines and the lung tumor type from which they were derived provide evidence of the relevance of their use. However, major problems including misidentification or cell line contamination remain. Ongoing studies and new approaches are expected to reveal the full potential of the lung cancer cell line panel.
Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT-5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT-5A but not in ACC-MESO-1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in two MPM cell lines (ACC-MESO-1 and H290) but not in MeT-5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21WAF1/CIP1 and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC-MESO-1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM.
apoptosis; BMAL1; mesothelioma; targeted therapy; mitotic catastrophe
Gazdar and Minna discuss the context and implications of a research article that examines the transformation potential and response to inhibitors of specific
EGFR mutations found in lung cancer.
The last decade has seen significant advances in our understanding of lung cancer biology and management. Identification of key driver events in lung carcinogenesis has contributed to the development of targeted lung cancer therapies, heralding the era of personalised medicine for lung cancer. As a result, histological subtyping and molecular testing has become of paramount importance, placing increasing demands on often small diagnostic specimens. This has triggered the review and development of the first structured classification of lung cancer in small biopsy/cytology specimens and a new classification of lung adenocarcinoma from the IASLC/ATS/ERS. These have enhanced the clinical relevance of pathological diagnosis, and emphasise the role of the modern surgical pathologist as an integral member of the multidisciplinary team, playing a crucial role in clinical trials and determining appropriate and timely management for patients with lung cancer.
Lung Neoplasms; pathology; non-small-cell lung carcinoma (NSCLC); small cell lung carcinoma (SCLC)
Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never-smoker lung cancer patients including patients with EGFR and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never-smoker and 30 current-smoker lung cancer patients, and determined the mtDNA content. All the patients’ samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex-I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P=0.006) mtDNA mutation in the never-smokers compared to the current-smoker lung cancer patients. MtDNA mutation was significantly higher (P=0.026) in the never-smoker Asian compared to the current-smoker Caucasian patients’ population. MtDNA mutation was significantly (P=0.007) associated with EGFR gene mutation in the never-smoker patients. We also observed a significant increase (P=0.037) in mtDNA content among the never-smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex-I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never-smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never-smoker lung cancer patients.
Lung cancer; never-smokers; MtDNA mutation; Respiratory Complex-I; EGFR mutation
Malignant mesothelioma (MM) is a neoplasm arising from mesothelial cells lining the pleural, peritoneal, and pericardial cavities. Over 20 million people in the US are at risk of developing MM due to asbestos exposure. MM mortality rates are estimated to increase by 5-10% per year in most industrialized countries until about 2020. The incidence of MM in men has continued to rise during the past 50 years, while the incidence in women appears largely unchanged. It is estimated that about 50-80% of pleural MM in men and 20-30% in women developed in individuals whose history indicates asbestos exposure(s) above that expected from most background settings. While rare for women, about 30% of peritoneal mesothelioma in men has been associated with exposure to asbestos. Erionite is a potent carcinogenic mineral fiber capable of causing both pleural and peritoneal MM. Since erionite is considerably less widespread than asbestos, the number of MM cases associated with erionite exposure is smaller. Asbestos induces DNA alterations mostly by inducing mesothelial cells and reactive macrophages to secrete mutagenic oxygen and nitrogen species. In addition, asbestos carcinogenesis is linked to the chronic inflammatory process caused by the deposition of a sufficient number of asbestos fibers and the consequent release of pro-inflammatory molecules, especially HMGB-1, the master switch that starts the inflammatory process, and TNF-alpha by macrophages and mesothelial cells. Genetic predisposition, radiation exposure and viral infection are co-factors that can alone or together with asbestos and erionite cause MM.
Mesothelioma; Genetics; Asbestos; Erionite; SV40
IKBKB (IKK-β/IKK-2), which activates NF-κB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in regulation of NF-κB signaling. We investigated complex component gene disruption as a novel genetic mechanism of NF-κB activation in non-small cell lung cancer (NSCLC).
644 tumor- and 90 cell line-genomes were analyzed for gene-dosage status of the individual complex components and IKBKB. Gene expression of these genes, and NF-κB target genes were analyzed in 48 tumors. IKBKB protein levels were assessed in tumors with and without complex or IKBKB genetic disruption. Complex component knockdown was performed to assess effects of the E3-ligase complex on IKBKB and NF-κB levels, and phenotypic importance of IKBKB expression was measured by pharmacological inhibition.
We observed strikingly frequent genetic disruption (42%) and aberrant expression (63%) of the E3-ligase complex and IKBKB in the samples examined. While both adenocarcinomas and squamous cell carcinomas showed complex disruption, the patterns of gene disruption differed. IKBKB levels were elevated with complex disruption, knockdown of complex components increased activated forms of IKBKB and NF-κB proteins, and IKBKB inhibition detriments cell viability, highlighting the biological significance of complex disruption. NF-κB target genes were overexpressed in samples with complex disruption, further demonstrating the effect of complex disruption on NF-κB activity.
Gene dosage alteration is a prominent mechanism that disrupts each component of the KEAP1-CUL3-RBX1 complex and its NF-κB stimulating substrate, IKBKB. Here we show that, multiple component disruption of this complex represents a novel mechanism of NF-κB activation in NSCLC.
KEAP1; CUL3; RBX1; IKBKB; NF-κB signaling; genetic disruption
Non-small cell lung cancer (NSCLC) is the major cause of cancer-related deaths in the USA and worldwide. Most patients present with advanced disease, and treatment options for these patients are generally limited to platinum-based chemotherapy and a few targeted therapies. Targeted agents currently in use for NSCLC inhibit oncogenic receptor tyrosine kinase pathways, such as the epidermal growth factor receptor (EGFR) pathway. While current EGFR-targeted agents, including erlotinib and gefitinib, may result in dramatic responses, they demonstrate efficacy in only a fraction of patients, and resistance to these agents frequently develops. In order to select patients most likely to benefit from blockade of EGFR pathways, investigators have focused on identifying molecular correlates of response to anti-EGFR therapy. New strategies to minimize the risk of resistance to EGFR inhibition have been employed in the development of next-generation EGFR tyrosine kinase inhibitors, such as PF00299804 and BIBW 2992; these include irreversibility of target binding, inhibition of multiple EGFR family receptors, and/or simultaneous inhibition of EGFR and other oncogenic pathways.
Epidermal growth factor receptor; NSCLC; Targeted therapy; Resistance
Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers including lung cancer, and its contribution to increased proliferation through upregulation of cell cycle accelerators such as cyclins A and E has been well established in breast and gastric cancers. Nevertheless, very little is known about its role in supporting the survival of cancer cells. In addition, the functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. In this study, we show that RNAi-mediated knockdown of EpCAM suppresses proliferation and clonogenic growth of three EpCAM-expressing lung cancer cell lines (H3255, H358, and HCC827), but does not induce cell cycle arrest in any of these. In addition, EpCAM knockdown inhibits invasion in the highly invasive H358 but not in less invasive H3255 cells in a Transwell assay. Of note, the EpCAM knockdown induces massive apoptosis in the three cell lines as well as in another EpCAM-expressing lung cancer cell line, HCC2279, but to a much lesser extent in a cdk4/hTERT immortalized normal human bronchial epithelial cell line, HBEC4, suggesting that EpCAM could be a therapeutic target for lung cancer. Finally, EpCAM knockdown partially restores contact inhibition in HCC827, in association with p27Kip1 upregulation. These results indicate that EpCAM could contribute substantially to the pathogenesis of lung cancer, especially cancer cell survival, and suggest that EpCAM targeted therapy for lung cancer may have potential.
Detection of lung cancer at early stages could potentially increase survival rates. One promising approach is the application of suitable lung cancer-specific biomarkers to specimens obtained by non-invasive methods. Thus far, clinically useful biomarkers that have high sensitivity have proven elusive. Certain genes, which are involved in cellular pathways such as signal transduction, apoptosis, cell to cell communication, cell cycles and cytokine signaling are down-regulated in cancers and may be considered as potential tumor suppressor genes. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. Using quantitative real time PCR, we tested 11 genes (3-OST-2, RASSF1A, DcR1, DcR2, P16, DAPK, APC, ECAD, HCAD, SOCS1, SOCS3) for levels of methylation within their promoter sequences in non-small cell lung cancers (NSCLC), adjacent non-malignant lung tissues, in peripheral blood mononuclear cells (PBMC) from cancer free patients, in sputum of cancer patients and controls. Of all the 11 genes tested 3-OST-2 showed the highest levels of promoter methylation in tumors combined with lowest levels of promoter methylation in control tissues. 3-OST-2 followed by, RASSF1A showed increased levels of methylation with advanced tumor stage (P<0.05). Thus, quantitative analysis of 3-OST-2 and RASSF1A methylation appears to be a promising biomarker assay for NSCLC and should be further explored in a clinical study. Our preliminary data on the analysis of sputum DNA specimens from cancer patients further support these observations.
Real time PCR; Tumor suppressor gene; Non-small cell lung cancer
The CXC chemokine interleukin-8 (IL-8) is an angiogenic growth factor that is overexpressed in various cancers, including non-small cell lung cancer (NSCLC). Previously, IL-8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS-driven NSCLC. Using microarray analysis, we identified IL-8 as the most downregulated gene by shRNA-mediated KRAS knockdown in NCI-H1792 NSCLC cells where IL-8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL-8, while IL-8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL-8 expression was downregulated by shRNA-mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL-8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL-8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL-8 expression, while the p38 inhibitor increased IL-8 expression. Attenuation of IL-8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL-8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL-8 expression in NSCLC; IL-8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS-mutated adenocarcinomas; and IL-8 plays a role in cell growth and migration in oncogenic KRAS-driven NSCLC.
non-small cell lung cancer; KRAS; interleukin-8; molecular target
Emerging evidence suggests that aberrant expression of oncogenes contributes to development of lung malignancy. The thyroid transcription factor 1 (TITF-1) gene functions as a lineage survival gene abnormally expressed in a significant fraction of NSCLCs, in particular lung adenocarcinomas.
To better characterize TITF-1 abnormality: patterns in NSCLC, we studied TITF-1’s gene copy number using fluorescent in situ hybridization (FISH) and quantitative PCR, as well as its protein expression by immunohistochemistry analysis in a tissue microarray comprised of surgically resected NSCLC (N=321) including 204 adenocarcinomas and 117 squamous cell carcinomas (SCCs). TITF-1 copy number and protein expression were correlated with patients’ clinicopathologic characteristics, and in a subset of adenocarcinomas with EGFR and KRAS mutation status.
We found that increased TITF-1 protein expression was prevalent in lung adenocarcinomas only and was significantly associated with female gender (p<0.001), never smokers (p=0.004), presence of EGFR mutations (p=0.05) and better overall survival (all stages, p=0.0478. stages I and II, p=0.002). TITF-1 copy number gain (CBG) was detected by FISH analysis in both adenocarcinomas (18.9%; high CNG, 8.3%) and SCCs (20.1%; high CNG, 3.0%), and correlated significantly with the protein product (p=0.004) and presence of KRAS mutations (p=0.008) in lung adenocarcinomas. Moreover, multivariate analysis revealed that TITF-1 copy number gain was an independent predictor of poor survival of NSCLC (p=0.039).
Our integrative study demonstrates that the protein versus genomic expression patterns of TITF-1 have opposing roles in lung cancer prognosis and may occur preferentially in different subsets of NSCLC patients with distinct oncogene mutations.
NSCLC; TITF-1; gene copy gain; lineage-specific oncogenes
DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover novel such alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adaptor protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to EGF-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contributes to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlighting a role of adapter proteins as primary genetic drivers of tumorigenesis.
CRKL; lung cancer; DNA amplification; genomic profiling; adapter protein
Oncogenic KRAS is found in >25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we performed microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines (HBECs) with and without oncogenic KRAS. We found that while the MAPK pathway is significantly down-regulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-EGFR, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment but may offer possibilities of combining anti-KRAS strategies with other targeted drugs.
Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for the ALDH1A1, ALDH3A1 and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH+ lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared to their ALDH− counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH+ cells. Suppression of the Notch pathway by treatment with either a gamma-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH+ lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH+ component, implicating Notch signaling in lung cancer stem cell maintenance.
Lung cancer; cancer stem cells; ALDH; Notch; self renewal
To investigate the frequency of xenotropic murine leukemia virus (MLV) presence in human cell lines established from mouse xenografts and to search for the evidence of horizontal viral spread to other cell lines.
Six of 23 (26%) mouse DNA free xenograft cultures were strongly positive for MLV and their sequences had greater than 99% homology to known MLV strains. Four of five available supernatant fluids from these viral positive cultures were strongly positive for RT activity. Three of these supernatant fluids were studied to confirm the infectivity of the released virions for other human culture cells. Of the 78 non-xenograft derived cell lines maintained in the xenograft culture-containing facilities, 13 (17%) were positive for MLV, including XMRV, a virus strain first identified in human tissues. By contrast, all 50 cultures maintained in a xenograft culture-free facility were negative for viral sequences.
We examined xenograft tumor cell lines from seven independent laboratories and 128 non-xenografted tumor cell lines. Cell line DNA was examined for mouse DNA contamination, and by 3 Taqman qPCR assays targeting the gag, env or pol regions of MLV. Sequencing was used for viral strain identification. Supernatant fluids were tested for reverse transcriptase (RT) activity.
Human cultures derived after mouse xenografting frequently contain and release highly infectious xenotropic MLV viruses. Laboratories working with xenograft-derived human cultures should be aware of the risk of contamination with potentially biohazardous human-tropic mouse viruses and their horizontal spread to other cultures.
xenograft cultures; xenotropic murine leukemia virus; retrovirus; XMRV virus; cell cultures; lung cancer; prostate cell line
Somatic mutations and copy number alterations (as a result of deletion or amplification of large portions of a chromosome) are major drivers of human lung cancers. Detailed analysis of lung cancer–associated chromosomal amplifications could identify novel oncogenes. By performing an integrative cytogenetic and gene expression analysis of non–small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines and tumors, we report here the identification of a frequently recurring amplification at chromosome 11 band p13. Within this region, only TNF receptor–associated factor 6 (TRAF6) exhibited concomitant mRNA overexpression and gene amplification in lung cancers. Inhibition of TRAF6 in human lung cancer cell lines suppressed NF-κB activation, anchorage-independent growth, and tumor formation. In these lung cancer cell lines, RAS required TRAF6 for its oncogenic capabilities. Furthermore, TRAF6 overexpression in NIH3T3 cells resulted in NF-κB activation, anchorage-independent growth, and tumor formation. Our findings show that TRAF6 is an oncogene that is important for RAS-mediated oncogenesis and provide a mechanistic explanation for the previously apparent importance of constitutive NF-κB activation in RAS-driven lung cancers.
Steroid receptor coactivator-3 (SRC-3) is a histone acetyltransferase and nuclear hormone receptor (NHR) co activator, located on 20q12, which is amplified in several epithelial cancers and well studied in breast cancer. However, its possible role in lung cancer pathogenesis is unknown. We found SRC-3 over-expressed in 27% of NSCLC patients (N=311) by immunohistochemistry, which correlated with poor disease-free (p=0.0015) and overall (p=0.0008) survival. Twenty-seven percent of NSCLCs exhibited SRC-3 gene amplification, and we found lung cancer cell lines expressed higher levels of SRC-3 than immortalized human bronchial epithelial cells (HBECs), which in turn expressed higher level of SRC-3 than cultured primary human HBECs. siRNA-mediated down-regulation of SRC-3 in high-expressing (but not low expressing) lung cancer cells significantly inhibited tumor cell growth and induced apoptosis. Finally, we found that SRC-3 expression is inversely correlated with gefitinib sensitivity and that SRC-3 knockdown results in EGFR-TKI-resistant lung cancers becoming more sensitive to gefitinib. Together these data suggest that SRC-3 may be an important oncogene and therapeutic target for lung cancer.
Multiple cell lines (estimated at 300–400) have been established from human small cell (SCLC) and non-small cell lung cancers (NSCLC). These cell lines have been widely dispersed to and used by the scientific community worldwide, with over 8000 citations resulting from their study. However, there remains considerable skepticism on the part of the scientific community as to the validity of research resulting from their use. These questions center around the genomic instability of cultured cells, lack of differentiation of cultured cells and absence of stromal–vascular–inflammatory cell compartments. In this report we discuss the advantages and disadvantages of the use of cell lines, address the issues of instability and lack of differentiation. Perhaps the most important finding is that every important, recurrent genetic and epigenetic change including gene mutations, deletions, amplifications, translocations and methylation-induced gene silencing found in tumors has been identified in cell lines and vice versa. These “driver mutations” represented in cell lines offer opportunities for biological characterization and application to translational research. Another potential shortcoming of cell lines is the difficulty of studying multistage pathogenesis in vitro.To overcome this problem, we have developed cultures from central and peripheral airways that serve as models for the multistage pathogenesis of tumors arising in these two very different compartments. Finally the issue of cell line contamination must be addressed and safeguarded against. A full understanding of the advantages and shortcomings of cell lines is required for the investigator to derive the maximum benefit from their use.
Lung cancer; Cell lines; Preneoplasia; Oncogenes; Tumor suppressor genes; Genetic instability
We found that among four master epithelial-to-mesenchymal transition (EMT)-inducing genes (ZEB1, SIP1, Snail, and Slug) ZEB1expression was most significantly correlated with the mesenchymal phenotype (high Vimentin and low E-cadherin expression) in non-small cell lung cancer (NSCLC) cell lines and tumors. Furthermore, ZEB1 knockdown with RNA interference in three NSCLC cell lines with high ZEB1 expression suppressed to varying degrees mass culture growth and liquid colony formation but in all cases dramatically suppressed soft agar colony formation. In addition, ZEB1 knockdown induced apoptosis in one of the three lines, indicating that the growth inhibitory effects of ZEB1 knockdown occurs in part through the activation of the apoptosis pathway. These results suggest that inhibiting ZEB1 function may be an attractive target for NSCLC therapeutic development.
Lung cancer; Epidermal growth factor receptor; Anchorage-independent growth; EMT; MicroRNA; RNA interference
Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC often harbors oncogenic K-RAS mutations that lead to the aberrant activation of several intracellular networks including the phosphoinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Oncogenic K-RAS predicts poor prognosis and resistance to treatment with ionizing radiation (IR). Oncogenic K-Ras expression in the respiratory epithelium is sufficient to initiate NSCLC tumorigenesis, which requires the catalytic subunit of PI3K. Thus, effective inhibition of the PI3K signaling should lead to significant antitumor effects. However, therapy with rapamycin analogs has yielded disappointing results, due in part to compensatory upregulation of AKT. We hypothesized that dual PI3K/mTOR blockade would overcome these limitations. We tested this hypothesis with BEZ235 a novel dual PI3K/mTOR inhibitor that has recently entered clinical development. We found that BEZ235 induces a striking anti-proliferative effect both in transgenic mice with oncogenic K-RAS induced NSCLC and in NSCLC cell lines expressing oncogenic K-RAS. We determined that treatment with BEZ235 was not sufficient to induce apoptosis. However, we found that dual PI3K/mTOR blockade effectively sensitizes NSCLC expressing oncogenic K-RAS to the pro-apoptotic effects of IR both in vitro and in vivo. We conclude that dual PI3K/mTOR blockade in combination with IR may benefit patients with NSCLC expressing oncogenic K-RAS. These findings may have general applicability in cancer therapy, since aberrant activation of PI3K occurs frequently in human cancer.