Search

Small-cell lung cancer (SCLC) is initially sensitive to platinum doublet chemotherapy, providing dramatic clinical benefit. Unfortunately, most SCLCs relapse and become resistant to further therapy. In this issue of Cancer Cell , Thomas et al. show that some platinum-resistant SCLCs benefit from combination therapy with topotecan plus the ATR (ataxia telangiectasia–mutated and rad3-related) inhibitor berzosertib. [ABSTRACT FROM AUTHOR]

Grainyhead-like 2 (GRHL2) is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT). It has been previously shown that GRHL2 can confer both oncogenic and tumor-suppressive roles in human cancers, including breast, pancreatic and colorectal cancers. However, its role in lung cancer remains elusive. In the present study, a meta-analysis of multiple gene expression datasets with clinical data revealed that GRHL2 expression was increased in lung cancer compared with that in the normal tissues. Copy number analysis of GRHL2, performed using datasets of whole exome sequencing involving 151 lung cancer cell lines, revealed frequent amplifications, suggesting that the increased GRHL2 expression may have resulted from gene amplification. A survival meta-analysis of GRHL2 using The Cancer Genome Atlas (TCGA) dataset showed no association of GRHL2 expression with overall survival. GRHL2 expression was found to be associated with EMT status in lung cancer in TCGA dataset and lung cancer cell lines. GRHL2 knockdown induced partial EMT in the hTERT/Cdk4-immortalized normal lung epithelial cell line HBEC4KT without affecting proliferation measured by CCK-8 assays. In addition, GRHL2 silencing caused three lung cancer cell lines, H1975, H2009 and H441, to undergo partial EMT. However, the proliferative effects differed significantly. GRHL2 silencing promoted proliferation but not colony formation in H1975 cells whilst suppressing colony formation without affecting proliferation in H2009 cells, but it did not affect proliferation in H441 cells. These results suggest cell type-dependent effects of GRHL2 knockdown. Downstream, GRHL2 silencing enhanced the phosphorylation of AKT and ERK, assessed by western blotting with phospho-specific antibodies, in HBEC4KT, H1975 and H2009 cell lines but not in the H441 cell line. By contrast, transient GRHL2 overexpression did not affect A549 cell proliferation, which lack detectable endogenous expression of the GRHL2 protein. However, GRHL2 overexpression did suppress E-cadherin expression in A549 cells. These results suggested that GRHL2 does not only function as a tumor suppressor of EMT but can also behave as an oncogene depending on the lung cancer cell-type context. [ABSTRACT FROM AUTHOR]

It is well established that lung tumors induce the formation of lymphatic vessels. However, the molecular mechanisms controlling tumor lymphangiogenesis in lung cancer have not been fully delineated. In the present study, we identify a panel of non-small cell lung cancer (NSCLC) cell lines that induce lymphangiogenesis and use genome-wide mRNA expression to characterize the molecular mechanisms regulating tumor lymphangiogenesis. We show that Calu-1, H1993, HCC461, HCC827, and H2122 NSCLC cell lines form tumors that induce lymphangiogenesis whereas Calu-3, H1155, H1975, and H2073 NSCLC cell lines form tumors that do not induce lymphangiogenesis. By analyzing genome-wide mRNA expression data, we identify a 17-gene expression signature that distinguishes lymphangiogenic from non-lymphangiogenic NSCLC cell lines. Importantly, VEGF-C is the only lymphatic growth factor in this expression signature and is approximately 50-fold higher in the lymphangiogenic group than in the non-lymphangiogenic group. We show that forced expression of VEGF-C by H1975 cells induces lymphangiogenesis and that knockdown of VEGF-C in H1993 cells inhibits lymphangiogenesis. Additionally, we demonstrate that the triple angiokinase inhibitor, nintedanib (small molecule that blocks all FGFRs, PDGFRs, and VEGFRs), suppresses tumor lymphangiogenesis in H1993 tumors. Together, these data suggest that VEGF-C is the dominant driver of tumor lymphangiogenesis in NSCLC and reveal a specific therapy that could potentially block tumor lymphangiogenesis in NSCLC patients. [ABSTRACT FROM AUTHOR]

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity. [ABSTRACT FROM AUTHOR]

NSCLC (non-small cell lung cancer) often exhibits resistance to paclitaxel treatment. Identifying the elements regulating paclitaxel response will advance efforts to overcome such resistance in NSCLC therapy. Using in vitro approaches, we demonstrated that over-expression of the microRNA miR-337-3p sensitizes NCI-H1155 cells to paclitaxel, and that miR-337- 3p mimic has a general effect on paclitaxel response in NSCLC cell lines, which may provide a novel adjuvant strategy to paclitaxel in the treatment of lung cancer. By combining in vitro and in silico approaches, we identified STAT3 and RAP1A as direct targets that mediate the effect of miR-337-3p on paclitaxel sensitivity. Further investigation showed that miR-337-3p mimic also sensitizes cells to docetaxel, another member of the taxane family, and that STAT3 levels are significantly correlated with taxane resistance in lung cancer cell lines, suggesting that endogenous STAT3 expression is a determinant of intrinsic taxane resistance in lung cancer. The identification of a miR-337-3p as a modulator of cellular response to taxanes, and STAT3 and RAP1A as regulatory targets which mediate that response, defines a novel regulatory pathway modulating paclitaxel sensitivity in lung cancer cells, which may provide novel adjuvant strategies along with paclitaxel in the treatment of lung cancer and may also provide biomarkers for predicting paclitaxel response in NSCLC. [ABSTRACT FROM AUTHOR]

It was only 3 years ago that an acquired translocation of EML4 with ALK leading to the expression of an EML4-ALK oncoprotein in non-small cell lung cancer (NSCLC) was reported. Tumor cells expressing EML4-ALK are “addicted” to its continued function. Now, crizotinib, an oral ALK inhibitor, is demonstrated to provide dramatic clinical benefit with little toxicity in patients having such advanced NSCLC, and a mechanism of clinical resistance to crizotinib is identified. Such therapy “targeted” at oncogenic proteins provides “personalized” medicine and prompts genome-wide mutation analysis of human tumors to find other therapeutic targets. [ABSTRACT FROM AUTHOR]

AZD6244 and MK2206 are targeted small-molecule drugs that inhibit MEK and AKT respectively. The efficacy of this combination in lung cancer is unknown. Our previous work showed the importance of activated AKT in mediating resistance of non-small cell lung cancer (NSCLC) to AZD6244. Thus we hypothesized that dual inhibition of both downstream MEK and AKT pathways would induce synergistic antitumor activity. In this study, we evaluated the efficacy of AZD6244 and MK2206 individually on a large panel of lung cancer cell lines. Then, we treated 28 human lung cancer cell lines with a combination of AZD6244 and MK2206 at clinically applicable drug molar ratios. The AZD6244-MK2206 combination therapy resulted in a synergistic effect on inhibition of lung cancer cell growth compared to the results of single drug treatment alone. MK2206 enhanced AZD6244-induced Bim overexpression and apoptosis in A549 and H157 cells. When we tested the combination of AZD6244 and MK2206 at ratios of 8:1, 4:1, 2:1, and 1:8, we found that the synergistic effect of the combination therapy was ratio-dependent. At ratios of 8:1, 4:1, and 2:1, the drug combination consistently demonstrated synergy, whereas decreasing the ratio to 1:8 resulted in a loss of synergy and produced an additive or antagonistic effect in most cell lines. Furthermore, the AZD6244-MK2206 combination therapy showed synergy in the suppression of A549 and H157 xenograft tumor growth and increased mean animal survival time. The AZD6244-MK2206 combination therapy resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. In addition, a significant increase of apoptosis was detected in tumor tissue from mice treated with AZD6244-MK2206 compared with that from the single agent treated mice. Our study suggests that the combination of AZD6244 and MK2206 has a significant synergistic effect on tumor growth in vitro and in vivo and leads to increased survival rates in mice bearing highly aggressive human lung tumors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Smac mimetics target cancer cells in a TNFα-dependent manner, partly via proteasome degradation of cellular inhibitor of apoptosis 1 (clAP1) and cIAP2. Degradation of clAPs triggers the release of receptor interacting protein kinase (RIPK1) from TNF receptor I (TNFR1) to form a caspase-8 activating complex together with the adaptor protein Fas-associated death domain (FADD). We report here a means through which cancer cells mediate resistance to Smac mimetic/TNFa-induced apoptosis and corresponding strategies to overcome such resistance. These human cancer cell lines evades Smacmimetic-induced apoptosis by up-regulation of cIAP2, which although initially degraded, rebounds and is refractory to subsequent degradation. cIAP2 is induced by TNFα via NF-κB and modulation of the NF-κB signal renders otherwise resistant cells sensitive to Smac mimetics. In addition, other signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), have the potential to concurrently regulate cIAP2. Using the PI3K inhibitor, LY294002, cIAP2 up-regulation was suppressed and resistance to Smac mimeticsinduced apoptosis was also overcome. [ABSTRACT FROM AUTHOR]

Background: There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer. Results: Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers. Conclusion: The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy. [ABSTRACT FROM AUTHOR]

Background: Recent studies have shown that microRNAs (miRNAs) play roles in tumorigenesis and are reliable classifiers of certain cancer types and subtypes. However, the role of miRNAs in the pathogenesis and diagnosis of small cell carcinoma (SCLC), the majority of which represent the most aggressive lung tumors, has not been investigated. Methods: In order to explore miRNA involvement in the pathogenesis of small cell lung carcinoma (SCLC) and the potential role of miRNAs in SCLC diagnosis, we compared the miRNA expression profile of a set of SCLC cell lines to that of a set of non-small cell lung cancer (NSCLC) cell lines and normal immortalized human bronchial epithelial cells (HBECs) using microarray analysis. Results: Our results show that miRNA profiles reliably distinguish SCLC cell lines from NSCLC and HBEC cell lines. Further analysis of the miRNA expression profile of the two subtypes of lung cancer cell lines indicates that the expression levels of the majority of the miRNAs that are differentially expressed in SCLC cells relative to NSCLC cells and HBECs show a progressive trend from HBECs to NSCLC cells to SCLC cells. Conclusions: The distinctive miRNA expression signature of SCLCs relative to NSCLCs and HBECs suggests that miRNA profiles have the potential to serve as a diagnostic marker of SCLC lung tumors. The progressive trend of miRNA profile changes from HBECs to NSCLCs to SCLCs suggests a possible pathological relationship between SCLCs and NSCLCs, and suggests that the increasing dysregulation of miRNA expression may play a role in lung tumor progression. The specific role of these miRNAs in lung tumor pathogenesis and differentiation need to be investigated further in future studies. [ABSTRACT FROM AUTHOR]

Background: Breast cancer cell lines have been used widely to investigate breast cancer pathobiology and new therapies. Breast cancer is a molecularly heterogeneous disease, and it is important to understand how well and which cell lines best model that diversity. In particular, microarray studies have identified molecular subtypes-luminal A, luminal B, ERBB2- associated, basal-like and normal-like-with characteristic gene-expression patterns and underlying DNA copy number alterations (CNAs). Here, we studied a collection of breast cancer cell lines to catalog molecular profiles and to assess their relation to breast cancer subtypes. Methods: Whole-genome DNA microarrays were used to profile gene expression and CNAs in a collection of 52 widely-used breast cancer cell lines, and comparisons were made to existing profiles of primary breast tumors. Hierarchical clustering was used to identify gene-expression subtypes, and Gene Set Enrichment Analysis (GSEA) to discover biological features of those subtypes. Genomic and transcriptional profiles were integrated to discover within high-amplitude CNAs candidate cancer genes with coordinately altered gene copy number and expression. Findings: Transcriptional profiling of breast cancer cell lines identified one luminal and two basal-like (A and B) subtypes. Luminal lines displayed an estrogen receptor (ER) signature and resembled luminal-A/B tumors, basal-A lines were associated with ETS-pathway and BRCA1 signatures and resembled basal-like tumors, and basal-B lines displayed mesenchymal and stem/progenitor-cell characteristics. Compared to tumors, cell lines exhibited similar patterns of CNA, but an overall higher complexity of CNA (genetically simple luminal-A tumors were not represented), and only partial conservation of subtype-specific CNAs. We identified 80 high-level DNA amplifications and 13 multi-copy deletions, and the resident genes with concomitantly altered gene-expression, highlighting known and novel candidate breast cancer genes. Conclusions: Overall, breast cancer cell lines were genetically more complex than tumors, but retained expression patterns with relevance to the luminal-basal subtype distinction. The compendium of molecular profiles defines cell lines suitable for investigations of subtype-specific pathobiology, cancer stem cell biology, biomarkers and therapies, and provides a resource for discovery of new breast cancer genes. [ABSTRACT FROM AUTHOR]

Abundant evidence suggests that a unifying principle governing the molecular pathology of cancer is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Anomalous proteins engaged in support of this tumorigenic regulatory environment most probably represent optimal intervention targets in a heterogeneous population of cancer cells. The advent of RNA-mediated interference (RNAi)-based functional genomics provides the opportunity to derive unbiased comprehensive collections of validated gene targets supporting critical biological systems outside the framework of preconceived notions of mechanistic relationships. We have combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide synthetic library of chemically synthesized small interfering RNAs for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. NCI-H1155, a human non-small-cell lung cancer line, was employed in a paclitaxel-dependent synthetic lethal screen designed to identify gene targets that specifically reduce cell viability in the presence of otherwise sublethal concentrations of paclitaxel. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, we isolated a panel of 87 genes that represent major focal points of the autonomous response of cancer cells to the abrogation of microtubule dynamics. Here we show that several of these targets sensitize lung cancer cells to paclitaxel concentrations 1,000-fold lower than otherwise required for a significant response, and we identify mechanistic relationships between cancer-associated aberrant gene expression programmes and the basic cellular machinery required for robust mitotic progression. [ABSTRACT FROM AUTHOR]

Silencing the expression of human papillomavirus (HPV) oncoproteins should have therapeutic benefits for cervical cancer. The authors' objective was to study RNA interference of the HPV 18 E6/E7 bicistronic mRNA with E6 small interfering RNA (siRNA) and E7 siRNA and determine the effect of each siRNA on oncoprotein expression, resultant cell growth, and downstream molecular effects. RNA interference was used to knockdown HPV 18 E6 and E7 oncoproteins on the HPV 18 positive cervical cancer cell lines HeLa and C4I. Western blotting was used to assay for each oncoprotein expression and select downstream molecular targets. Cell cycle analyses, cell viability assays, and colony formation assays were performed to determine the effect of treatment by both HPV 18 E6 siRNA and E7 siRNA. The transfection reagent oligofectamine and Tax siRNA were used as negative controls. Transfection with E6 siRNA caused complete loss of E6 but not E7 oncoprotein. However, E7 siRNA induced complete loss of both E6 and E7 oncoproteins. E6 siRNA mediated the reexpression of p53 protein and a moderate decrease in phosphorylated retinoblastoma protein expression (pRb), resulting in decreased colony formation. Transfection with E7 siRNA mediated a robust increase in p53 expression and complete loss of pRb, resulting in a marked decrease in colony formation compared to the E6 siRNA (P = .001). Flow cytometry revealed significantly increased apoptotic cells with E7 siRNA compared to E6 siRNA and control. RNA interference targeting the E7 portion of the bicistronic HPV 18 mRNA can silence both E6 and E7 oncoproteins and is most effective in cervical cancer growth inhibition. [ABSTRACT FROM AUTHOR]

TSLC1 (tumor suppressor in lung cancer-1, IGSF4) encodes a member of the immunoglobulin superfamily molecules, which is involved in cell-cell adhesion. TSLC1 is connected to the actin cytoskeleton by DAL-1 (differentially expressed in adenocarcinoma of the lung-1, EPB41L3) and it directly associates with MPP3, one of the human homologues of a Drosophila tumor suppressor gene, Discs large. Recent data suggest that aberrant promoter methylation is important for TSLC1 inactivation in lung carcinomas. However, little is known about the other two genes in this cascade, DAL-1 and MPP3. Thus, we investigated the expression and methylation patterns of these genes in lung cancer cell lines, primary lung carcinomas and nonmalignant lung tissue samples. By reverse transcription-polymerase chain reaction, loss of TSLC1 expression was observed in seven of 16 (44%) non-small-cell lung cancer (NSCLC)cell lines and in one of 11 (9%) small-cell lung cancer (SCLC)cell lines, while loss of DAL-1 expression was seen in 14 of 16 (87%) NSCLC cell lines and in four of 11 (36%) SCLC cell lines. By contrast, MPP3 expression was found in all tumor cell lines analysed. Similar results were obtained by microarray analysis. TSLC1 methylation was seen in 13 of 39 (33%)NSC LC cell lines, in one of 11 (9%)SCLC cell lines and in 100 of 268 (37%) primary NSCLCs. DAL-1 methylation was observed in 17 of 39 (44%) NSCLC cell lines, in three of 11 (27%) SCLC cell lines and in 147 of 268 (55%)primary NSCLCs. In tumors of NSCLC patients with stage II-III disease, DAL-1 methylation was seen at a statistically significant higher frequency compared to tumors of patients with stage I disease. A significant correlation between loss of expression and methylation of the genes in lung cancer cell lines was found. Overall, 65% of primary NSCLCs had either TSLC1 or DAL-1 methylated. Methylation of one of these genes was detected in 59% of NSCLC cell lines; however, in SCLC cell lines, methylation was much less frequently observed. The majority of nonmalignant lung tissue samples was not TSLC1 or DAL-1 methylated. Re-expression of TSLC1 and DAL-1 was seen after treatment of lung cancer cell lines with 5-aza-2′-deoxycytidine. Our results suggest that methylation of TSLC1 and/or DAL-1, leading to loss of their expression, is an important event in the pathogenesis of NSCLC. [ABSTRACT FROM AUTHOR]

Recently, the human SRBC (hSRBC) gene, a candidate tumor suppressor gene (TSG), has been mapped to the chromosomal region 11p15.5–p15.4 where frequent allele loss has been described in lung cancer. Aberrant methylation (referred to as methylation) of the promoter region of TSGs has been identified as an important mechanism for gene silencing. Loss of hSRBC protein expression occurs frequently in lung cancer cell lines and sodium bisulfite sequencing of the promoter region of hSRBC in several lung cancer cell lines suggested that methylation plays an important role in inactivating hSRBC. To determine the methylation status of hSRBC in a large collection of primary lung cancer samples, corresponding nonmalignant lung tissues and lung cancer cell lines (N=52), we designed primers for a methylation-specific PCR assay. Methylation was detected in 41% of primary non-small-cell lung cancers (NSCLC) (N=107) and in 80% of primary small-cell lung cancers (SCLC) (N=5), but was seen only in 4% of corresponding nonmalignant lung tissues (N=103). In all, 79% of lung cancer cell lines were methylated and the frequency of hSRBC methylation was significantly higher in SCLC (100%) than in NSCLC (58%) cell lines. Normal hSRBC protein expression was detected in only 18% of primary NSCLCs (N=93) by immunostaining and a significant association between loss of protein expression and methylation was found. hSRBC re-expression was observed after treatment of lung cancer cells with the demethylating agent 5-aza-2′-deoxycytidine. In addition, 45% of the 76 hSRBC immunostaining-negative NSCLCs did not have hSRBC promoter methylation, indicating that other mechanisms of hSRBC expression silencing also exist. Both hSRBC immunostaining and methylation results did not correlate with clinicopathological characteristics of these patients. Our findings suggest that hSRBC is a candidate TSG involved in lung cancer pathogenesis, where expression is frequently inactivated by methylation and other mechanisms.Oncogene (2005) 24, 6249–6255. doi:10.1038/sj.onc.1208775; published online 6 June 2005 [ABSTRACT FROM AUTHOR]

RASSF1A:, a major member of the RASSF1 gene family, is silenced by promoter methylation at a high frequency in a large number of human solid tumors. Controlled expression of RASSF1A reverts the tumorigenic phenotype of several human cancer cell lines. Here we investigated another main isoform, RASSF1C, and compared it with RASSF1A in the gene inactivation test (GIT), based on a tetracycline regulation system. In the small-cell lung cancer (SCLC) line U2020, only RASSF1A has shown growth inhibitory activity in vitro, while in the prostate cell line LNCaP and renal cell carcinoma (RCC) line KRC/Y both RASSF1A and RASSF1C showed similar (approximately 90%) suppressing activity in vitro. Both RASSF1C and RASSF1A suppressed the tumorigenicity of the KRC/Y RCC cell line in SCID mice. Mutations, deletions and loss of expression of RASSF1A and RASSF1C transgenes were identified in all 15 grown SCID tumors. In contrast, the mutant RASSF1A containing Cys65Arg and Val211Ala had reduced growth suppression activity both in vitro and in vivo and did not show any further changes in four grown SCID tumors. In addition, RASSF1C was shown to induce cell cycle arrest in KRC/Y cells. These results strongly imply that like RASSF1A the RASSF1C gene could serve a tumor suppressor function.Oncogene (2004) 23, 5941-5949. doi:10.1038/sj.onc.1207789 Published online 21 June 2004 [ABSTRACT FROM AUTHOR]

We recently demonstrated that RASSF1A, a new tumour-suppressor gene located at 3p21.3 is frequently inactivated by promoter region hypermethylation in a variety of human cancers including lung, breast, kidney and neuroblastoma. We have identified another member of the RASSF1 gene family by in silico sequence analysis using BLAST searches. NORE1 located at 1q32.1 exists in three isoforms (NORE1Aα, NORE1Aβ and NORE1B). Both NORE1A and NORE1B isoforms have separate CpG islands spanning their first exons. NORE1Aα Produces a 418 aa protein containing a Ras-association (RA) domain and a diacylglycerol (DAG) binding domain. NORE1Aβ produces a C-terminal truncation of the RA domain. NORE1B also contains the RA domain but not the DAG domain. NORE1 is the human homologue of the mouse Ras effector Nore1. No inactivating somatic mutations were found in lung tumour lines; however, NORE1A promoter region CpG island was hypermethylated in primary tumours and tumour cell lines. NORE1A promoter was methylated in 10/25 breast, 4/40 SCLC, 3/17 NSCLC, 1/6 colorectal and 3/9 kidney tumour cell lines, while NORE1B promoter was unmethylated in the same tumour cell lines. While 24% (6/25) of primary NSCLC underwent NORE1A methylation, methylation in SCLC was a rare event (0/22); (P=0.0234). NORE1A expression in tumour cell lines was reactivated after treatment with a demethylating agent. There was no correlation between NORE1A and RASSF1A methylation status in NSCLC. Our results demonstrate that NORE1A is inactivated in a subset of human cancers by CpG island promoter hypermethylation, and in lung cancer this hypermethylation may be histological type specific.Oncogene (2003) 22, 947–954. doi:10.1038/sj.onc.1206191 [ABSTRACT FROM AUTHOR]

The CACNA2D2 gene, a new subunit of the Ca2+-channel complex, was identified in the homozygous deletion region of chromosome 3p21.3 in human lung and breast cancers. Expression deficiency of the CACNA2D2 in cancer cells suggests a possible link of it to Ca2+ signaling in the pathogenesis of lung cancer and other cancers. We investigated the effects of overexpression of CACNA2D2 on intracellular Ca2+ contents, mitochondria homeostasis, cell proliferation, and apoptosis by adenoviral vector-mediated wild-type CACNA2D2 gene transfer in 3p21.3-deficient nonsmall cell lung cancer cell lines. Exogenous expression of CACNA2D2 significantly inhibited tumor cell growth compared with the controls. Overexpression of CACNA2D2 induced apoptosis in H1299 (12.5%), H358 (13.7%), H460 (22.3%), and A549 (50.1%) cell lines. Levels of intracellular free Ca2+ were elevated in AdCACNA2D2-transduced cells compared with the controls. Mitochondria membrane depolarization was observed prior to apoptosis in Ad-CACNA2D2 and Adp53-transduced H460 and A549 cells. Release of cyt c into the cytosol, caspase 3 activation, and PARP cleavage were also detected in these cells. Together, these results suggest that one of the pathways in CACNA2D2-induced apoptosis is mediated through disruption of mitochondria membrane integrity, the release of cyt c, and the activation of caspases, a process that is associated with regulation of cytosolic free Ca2+ contents.Oncogene (2003) 22, 615–626. doi:10.1038/sj.onc.1206134 [ABSTRACT FROM AUTHOR]

We have previously reported that the key apoptosis related gene caspase 8 (CASP8) is frequently silenced in small cell lung cancer (SCLC) tumors and cell lines usually, but not always, by aberrant promoter methylation. Because CASP8 is a key component of the death-inducing signaling complex (DISC) when specific death receptors (including DR4, DR5, FAS) are activated by their specific ligands (TRAIL/FASL), we examined expression of the components of the DISC complex in lung cancer cell lines. MYC family members are frequently amplified (MYC+ve) in SCLC, and MYC is a potent inducer of apoptosis. We examined 34 SCLC lines (12 of which were MYC+ve) and 22 NSCLC lines. CASP8 gene expression was frequently lost (79%) at message and protein levels in SCLC but not in non-SCLC (NSCLC). MYC amplification was present in 45% of SCLC cell lines, which had lost CASP8 expression, but not in any of the CASP8 positive lines. The frequency of CASP8 loss was significantly higher in MYC+ve SCLC compared to MYC-ve SCLC or in NSCLC. Analyses of other DISC components showed significantly higher rates of loss of expression of CASP10, DR5, FAS and FASL in SCLC compared to NSCLC. The loss of expression of proapoptotic DISC components was significantly higher in MYC+ve SCLC cell lines and these lines were completely resistant to TRAIL. Expression of CASP10 (a caspase closely related to CASP8) was frequently absent at the protein level in both SCLC and NSCLC lines. Expression of c-FLIP (proteolytically inactive homolog of CASP8) was inversely related to expression of CASP8. Our major conclusions are: (a) The death receptor pathway is differently inactivated at multiple levels in lung cancer cell lines; and (b) MYC amplification in SCLC is associated with inactivation of most components of the DISC complex, with resistance to TRAIL and with expression of c-FLIP. These findings may have considerable clinical and therapeutic implications.Oncogene (2002) 21, 8510–8514. doi:10.1038/sj... [ABSTRACT FROM AUTHOR]

Recently, constitutively active mutants of MEK (MAP/ERK kinase) were shown to be capable of transforming cells to tumorigenicity suggesting that MEK can function as a dominant oncogene and potentially play a role in human carcinogenesis. Human lung cancer cells exhibit mutations in other components of the MAP kinase signaling pathway such as the Her-2/neu and ras oncogenes. Thus, the coding sequences of both MEK-1 and MEK-2 cDNAs from human lung cancer cell lines were screened by single strand conformation polymorphism analysis and DNA sequencing for alterations in these two genes. In 37 lung cancer cell lines we found: an allelic variant in MEK-1 cDNA, nt 783 G→A, (no amino acid change); a MEK-2 cDNA change (nt 977 C→T mutation leading to 298 Pro→Leu change); a MEK-2 cDNA change nt 537 C→T (no amino acid change); and a frequent MEK-2 cDNA germline polymorphism nt 744, A→C (no amino acid change) with an allele frequency of 0.5 for each form. These results suggest that mutations in the MEK-1 and MEK-2 gene occur at a very low frequency in human lung cancer. [ABSTRACT FROM AUTHOR]

We studied PTEN/MMAC1, a newly discovered candidate tumor suppressor gene at 10q23.3, for mutations in lung cancer. One hundred and thirty-six lung cancer cell line DNAs (66 small cell lung cancers, SCLC, 61 non-small cell lung cancers, NSCLC, four mesotheliomas, five extrapulmonary small cell cancers) were analysed for PTEN/MMAC1 homozygous deletions and five (8%) SCLC lines showed homozygous deletions interrupting the PTEN/MMAC1 gene. Using single stranded conformation polymorphism (SSCP) analysis, we screened the PTEN/MMAC1 open reading frame of 53 lung cancer cell line cDNAs for point mutations and found that 3/35 SCLCs and 3/18 NSCLCs contained homozygous amino acid sequence altering mutations. Northern blot analysis revealed that expression of the PTEN/MMAC1 gene was considerably lower in all the tumor cell lines with point mutations while no expression was detected for cell lines with PTEN/MMAC1 homozygous deletions. Mutation analysis of 22 uncultured, microdissected, primary SCLC tumors and metastases showed two silent mutations, and two apparent homozygous deletions. We also discovered a processed pseudogene (PTEN2) which has 98.5% nt identity to PTEN/MMAC1, that needs to be accounted for in cDNA mutation analysis. Our findings suggest that genetic abnormalities of the PTEN/MMAC1 gene are only involved in a relatively small subset of lung cancers. [ABSTRACT FROM AUTHOR]

The authors discuss the report made by Johannes Kratz and colleagues regarding the molecular prognostic signature for lung cancer. The authors say that the signature will use a 14-gene mRNA expression assay to provide an early-stage prognosis of non-squamous non-small-cell lung cancer (NSCLC) which will run by technical validation guidelines. According to the author, the assay used improved prognostic accuracy and add that the signature will be of great interest.

Comments on the targeted therapies for killing cancer cells. Expression of the mda-7 gene in human pancreatic cancer cells; Suppression of tumor cell growth; Induction of the apoptosis process.

Ibrutinib, which irreversibly inhibits Bruton tyrosine kinase, was evaluated for antitumor activity in a panel of non–small cell lung cancer (NSCLC) cell lines and found to selectively inhibit growth of NSCLC cells carrying mutations in the epidermal growth factor receptor (EGFR) gene, including T790M mutant and erlotinib-resistant H1975 cells. Ibrutinib induced dose-dependent inhibition of phosphor-EGFR at both Y1068 and Y1173 sites, suggesting ibrutinib functions as an EGFR inhibitor. Survival was analyzed by Kaplan–Meier estimation and log-rank test. All statistical tests were two-sided. In vivo study showed that ibrutinib statistically significantly suppressed H1975 tumor growth and prolonged survival of the tumor bearing mice (n = 5 per group). The mean survival times for solvent- and erlotinib-treated mice were both 17.8 days (95% confidence interval [CI] = 14.3 to 21.3 days), while the mean survival time for ibrutinib-treated mice was 29.8 days (95% CI = 26.0 to 33.6 days, P = .008). Our results indicate that ibrutinib could be a candidate drug for treatment of EGFR-mutant NSCLC, including erlotinib-resistant tumors. [ABSTRACT FROM PUBLISHER]

Mitochondria (MT) participate in most metabolic activities of mammalian cells. A near-unidirectional mitochondrial transfer from T cells to cancer cells was recently observed to "metabolically empower" cancer cells while "depleting immune cells," providing new insights into tumor-T cell interaction and immune evasion. Here, we leverage single-cell RNA-seq technology and introduce MERCI, a statistical deconvolution method for tracing and quantifying mitochondrial trafficking between cancer and T cells. Through rigorous benchmarking and validation, MERCI accurately predicts the recipient cells and their relative mitochondrial compositions. Application of MERCI to human cancer samples identifies a reproducible MT transfer phenotype, with its signature genes involved in cytoskeleton remodeling, energy production, and TNF-α signaling pathways. Moreover, MT transfer is associated with increased cell cycle activity and poor clinical outcome across different cancer types. In summary, MERCI enables systematic investigation of an understudied aspect of tumor-T cell interactions that may lead to the development of therapeutic opportunities. [Display omitted] • Cancer cells "hijack" mitochondria from nearby T cells • Development of MERCI for tracing intercellular MT transfer • Genomic inference of receiver cancer cells at single-cell resolution using MERCI • Receiver cancer cells exhibit a distinct phenotype from the rest Recent work observes mitochondrial transfer from T cells to cancer cells. Zhang et al. systematically investigate this process using single-cell sequencing data from human cancers and identify a distinct phenotype related to mitochondrial transfer. Genes and pathways associated with this phenotype may serve as future therapeutic targets. [ABSTRACT FROM AUTHOR]

Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo, LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. [ABSTRACT FROM AUTHOR]

[Display omitted] To identify potential new reagents and biomarkers for early lung cancer detection we combined the use of a novel preclinical isogenic model of human lung epithelial cells comparing non-malignant cells with those transformed to full malignancy using defined oncogenic changes and our on-bead two color (red and green stained cells) (OBTC) peptoid combinatorial screening methodology. The preclinical model used normal parent lung epithelial cells (HBEC3-KT, labeled with green dye) and isogenic fully malignant transformed derivatives (labeled with a red dye) via the sequential introduction of key genetic alterations of p53 knockdown, oncogenic KRAS and overexpression of cMYC (HBEC3p53, KRAS, cMYC). Using the unbiased OBTC screening approach, we tested 100,000 different peptoids and identified only one (named JM3A) that bound to the surface of the HBEC3p53, KRAS, cMYC cells (red cells) but not HBEC3-KT cells (green cells). Using the JM3A peptoid and proteomics, we identified the protein bound as vimentin using multiple validation approaches. These all confirmed the cell surface expression of vimentin (CSV) on transformed (HBEC3p53, KRAS, cMYC) but not on untransformed (HBEC3-KT) cells. JM3A coupled with fluorophores was able to detect and stain cell surface vimentin on very early stage lung cancers but not normal lung epithelial cells in a fashion comparable to that using anti-vimentin antibodies. We conclude: using a combined isogenic preclinical model of lung cancer and two color screening of a large peptoid library, we have identified differential expression of cell surface vimentin (CSV) after malignant transformation of lung epithelial cells, and developed a new peptoid reagent (JM3A) for detection of CSV which works well in staining of early stage NSCLCs. This new, highly specific, easy to prepare, CSV detecting JM3A peptoid provides an important new reagent for identifying cancer cells in early stage tumors as well as a resource for detection and isolating of CSV expressing circulating tumor cells. [ABSTRACT FROM AUTHOR]

VEGF receptor-2 (VEGFR-2 or kinase insert domain receptor; KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected non-small-cell lung carcinoma (NSCLC; n 248). n KDR copy number gains (CNG), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR = 5.16; P = 0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations, we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear hypoxia inducible factor-1α (HIF-1α) in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11, and 21 by PCR-based sequencing; however, two variant single nucleotide polymorphism genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade. Cancer Res; 71(16); 5512-21. ©2011 AACR. [ABSTRACT FROM AUTHOR]

The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin-resistant MCF-7 cells and determined the functional status of epidermal growth factor receptor (EGFR), MAPK, and AKT signaling pathways by phosphoreceptor tyrosine kinase and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signaling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL-2 expression. We investigated the expression of genes encoding the ligands for the ERBB signaling cascade and found a selective up-regulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGFR (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time, and knockdown of amphiregulin expression by specific short interfering RNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate the generality and importance of our findings, we examined amphiregulin expression and cisplatin resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have thus identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells. [ABSTRACT FROM AUTHOR]

Summary: A small-molecule mimetic of Smac/Diablo that specifically counters the apoptosis-inhibiting activity of IAP proteins has been shown to enhance apoptosis induced by cell surface death receptors as well as chemotherapeutic drugs. Survey of a panel of 50 human non-small-cell lung cancer cell lines has revealed, surprisingly, that roughly one-quarter of these lines are sensitive to the treatment of Smac mimetic alone, suggesting that an apoptotic signal has been turned on in these cells and is held in check by IAP proteins. This signal has now been identified as the autocrine-secreted cytokine tumor necrosis factor alpha (TNFα). In response to autocrine TNFα signaling, the Smac mimetic promotes formation of a RIPK1-dependent caspase-8-activating complex, leading to apoptosis. [Copyright &y& Elsevier]

The epidermal growth factor receptor (EGFR) gene product is a receptor tyrosine kinase (TK) that affects many important downstream pathways. The recent finding that mutations in EGFR predict the response of lung cancers to therapies that target the TK domain of the gene product has generated considerable interest. The mutations are associated with adenocarcinoma histology, oriental origin, female gender and never-smoker status. Most mutations target structures in the TK domain that appear to be essential for the phosphorylation function of the gene. Cancer cells with mutant EGFR genes might become physiologically dependent on the continued activity of the gene for the maintenance of their malignant phenotype; however, this might also be a target for therapy. [Copyright &y& Elsevier]

Discovery of ligands specific to receptor(s) on a surface of a cancer cell could impact clinical issues including functional diagnosis and cell-specific drug delivery. Using a phage display approach, we have isolated 20-mer peptide ligands that bind to 3 different human lung tumor cell lines, NCI-H1299, NCI-H2009, and A549. The panning protocol is unbiased with no selection pressure towards binding a particular cellular receptor. The isolated phage bind to their target cells 24–300 times better than a control phage. Furthermore, the isolated peptides display remarkable cell-specificities and are able to discriminate between normal and cancerous cells as well as different lung tumor cells. The cell-specificities are not coincident with tumor classes indicating that the peptides are able to recognize cell-surface features that are not represented within the classification of tumor type. The isolated peptides are functional outside of the context of the phage and multimerization of the peptide increases its affinity for its given cell type, thus expanding their utility in clinical situations. [Copyright &y& Elsevier]