David S. Shames

Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer

Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein–coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ∼27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.

The mutation spectrum revealed by paired genome sequences from a lung cancer patient

Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung carcinomas in smokers being the predominant form of the disease. Although previous studies have identified important common somatic mutations in lung cancers, they have primarily focused on a limited set of genes and have thus provided a constrained view of the mutational spectrum. Recent cancer sequencing efforts have used next-generation sequencing technologies to provide a genome-wide view of mutations in leukaemia, breast cancer and cancer cell lines. Here we present the complete sequences of a primary lung tumour (60× coverage) and adjacent normal tissue (46×). Comparing the two genomes, we identify a wide variety of somatic variations, including >50,000 high-confidence single nucleotide variants. We validated 530 somatic single nucleotide variants in this tumour, including one in the KRAS proto-oncogene and 391 others in coding regions, as well as 43 large-scale structural variations. These constitute a large set of new somatic mutations and yield an estimated 17.7 per megabase genome-wide somatic mutation rate. Notably, we observe a distinct pattern of selection against mutations within expressed genes compared to non-expressed genes and in promoter regions up to 5 kilobases upstream of all protein-coding genes. Furthermore, we observe a higher rate of amino acid-changing mutations in kinase genes. We present a comprehensive view of somatic alterations in a single lung tumour, and provide the first evidence, to our knowledge, of distinct selective pressures present within the tumour environment.

Involvement of AGO1 and AGO2 in mammalian transcriptional silencing

Duplex RNAs complementary to messenger RNA inhibit translation in mammalian cells by RNA interference (RNAi). Studies have reported that RNAs complementary to promoter DNA also inhibit gene expression. Here we show that the human homologs of Argonaute-1 (AGO1) and Argonaute-2 (AGO2) link the silencing pathways that target mRNA with pathways mediating recognition of DNA. We find that synthetic antigene RNAs (agRNAs) complementary to transcription start sites or more upstream regions of gene promoters inhibit gene transcription. This silencing occurs in the nucleus, requires high promoter activity and does not necessarily require histone modification. AGO1 and AGO2 associate with promoter DNA in cells treated with agRNAs, and inhibiting expression of AGO1 or AGO2 reverses transcriptional and post-transcriptional silencing. Our data indicate key linkages and important mechanistic distinctions between transcriptional and post-transcriptional silencing pathways in mammalian cells.

Multiple Oncogenic Changes (K-RASV12, p53 Knockdown, Mutant EGFRs, p16 Bypass, Telomerase) Are Not Sufficient to Confer a Full Malignant Phenotype on Human Bronchial Epithelial Cells

We evaluated the contribution of three genetic alterations (p53 knockdown, K-RAS(V12), and mutant EGFR) to lung tumorigenesis using human bronchial epithelial cells (HBEC) immortalized with telomerase and Cdk4-mediated p16 bypass. RNA interference p53 knockdown or oncogenic K-RAS(V12) resulted in enhanced anchorage-independent growth and increased saturation density of HBECs. The combination of p53 knockdown and K-RAS(V12) further enhanced the tumorigenic phenotype with increased growth in soft agar and an invasive phenotype in three-dimensional organotypic cultures but failed to cause HBECs to form tumors in nude mice. Growth of HBECs was highly dependent on epidermal growth factor (EGF) and completely inhibited by EGF receptor (EGFR) tyrosine kinase inhibitors, which induced G1 arrest. Introduction of EGFR mutations E746-A750 del and L858R progressed HBECs toward malignancy as measured by soft agar growth, including EGF-independent growth, but failed to induce tumor formation. Mutant EGFRs were associated with higher levels of phospho-Akt, phospho-signal transducers and activators of transcription 3 [but not phospho-extracellular signal-regulated kinase (ERK) 1/2], and increased expression of DUSP6/MKP-3 phosphatase (an inhibitor of phospho-ERK1/2). These results indicate that (a) the HBEC model system is a powerful new approach to assess the contribution of individual and combinations of genetic alterations to lung cancer pathogenesis; (b) a combination of four genetic alterations, including human telomerase reverse transcriptase overexpression, bypass of p16/RB and p53 pathways, and mutant K-RAS(V12) or mutant EGFR, is still not sufficient for HBECs to completely transform to cancer; and (c) EGFR tyrosine kinase inhibitors inhibit the growth of preneoplastic HBEC cells, suggesting their potential for chemoprevention.

A Translational View of the Molecular Pathogenesis of Lung Cancer

Molecular genetic studies of lung cancer have revealed that clinically evident lung cancers have multiple genetic and epigenetic abnormalities, including DNA sequence alterations, copy number changes, and aberrant promoter hypermethylation. Together, these abnormalities result in the activation of oncogenes and inactivation of tumor-suppressor genes. In many cases these abnormalities can be found in premalignant lesions and in histologically normal lung bronchial epithelial cells. Findings suggest that lung cancer develops through a stepwise process from normal lung epithelial cells towards frank malignancy, which usually occurs as a result of cigarette smoking. Lung cancer has a high morbidity because it is difficult to detect early and is frequently resistant to available chemotherapy and radiotherapy. New, rationally designed early detection, chemoprevention, and therapeutic strategies based on the growing understanding of the molecular changes important to lung cancer are under investigation. For example, methylated tumor DNA sequences in sputum or blood are being investigated for early detection screening, and new treatments that specifically target molecules such as vascular endothelial growth factor and the epidermal growth factor receptor are becoming available. Meanwhile, global gene expression signatures from individual tumors are showing potential as prognostic and therapeutic indicators, such that molecular typing of individual tumors for therapy selection is not far away. Finally, the recent development of a model system of immortalized human bronchial epithelial cells, along with a paradigm shift in the conception of cancer stem cells, promises to improve the situation for patients with lung cancer. These advances highlight the translation of molecular discoveries on lung cancer pathogenesis from the laboratory to the clinic.

Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells

Background Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes. Methodology/Principal Findings We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. Conclusions MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

RNA Interference-Mediated Knockdown of DNA Methyltransferase 1 Leads to Promoter Demethylation and Gene Re-Expression in Human Lung and Breast Cancer Cells

DNA methyltransferase 1 (DNMT1) is required to maintain DNA methylation patterns in mammalian cells, and is thought to be the predominant maintenance methyltransferase gene. Recent studies indicate that inhibiting DNMT1 protein expression may be a useful approach for understanding the role of DNA methylation in tumorigenesis. To this end, we used RNA interference to specifically down-regulate DNMT1 protein expression in NCI-H1299 lung cancer and HCC1954 breast cancer cells. RNA interference-mediated knockdown of DNMT1 protein expression resulted in >80% reduction of promoter methylation in RASSF1A, p16(ink4A), and CDH1 in NCI-H1299; and RASSF1A, p16(ink4A), and HPP1 in HCC1954; and re-expression of p16(ink4A), CDH1, RASSF1A, and SEMA3B in NCI-H1299; and p16(ink4A), RASSF1A, and HPP1 in HCC1954. By contrast, promoter methylation and lack of gene expression was maintained when these cell lines were treated with control small interfering RNAs. The small interfering RNA treatment was stopped and 17 days later, all of the sequences showed promoter methylation and gene expression was again dramatically down-regulated, indicating the tumor cells still were programmed for these epigenetic changes. We saw no effects on soft agar colony formation of H1299 cells 14 days after DNMT1 knockdown indicating that either these genes are not functioning as tumor suppressors under these conditions, or that more prolonged knockdown or other factors are also required to inhibit the malignant phenotype. These results provide direct evidence that loss of DNMT1 expression abrogates tumor-associated promoter methylation and the resultant silencing of multiple genes implicated in the pathogenesis of human lung and breast cancer.

DNA methylation in health, disease, and cancer

The spatial arrangement and three-dimensional structure of DNA in the nucleus is controlled through the interdigitation of DNA binding proteins such as histones and their modifiers, the Polycomb-Trithorax proteins, and the DNA methyltransferase enzymes. DNA methylation forms the foundation of chromatin and is crucial to epigenetic gene regulation in mammals. Disease pathogenesis mediated through infectious agents, inflammation, aging, or genetic damage often involves changes in gene expression. In particular, cellular transformation coincides with multiple changes in chromatin architecture, many of which appear to affect genome integrity and gene expression. Infectious agents, such as viruses directly affect genome structure and induce methylation of particular sequences to suppress host immune responses. Hyperproliferative tissues such as those in the gastrointestinal tract and colon have been shown to gradually acquire aberrant promoter hypermethylation. Here we review recent findings on altered DNA methylation in human disease, with particular focus on cancer and the increasingly large number of genes subject to tumor-specific promoter hypermethylation and the possible role of aberrant methylation in tumor development.

Detection and Dynamic Changes of EGFR Mutations from Circulating Tumor DNA as a Predictor of Survival Outcomes in NSCLC Patients Treated with First-line Intercalated Erlotinib and Chemotherapy

Purpose: Blood-based circulating-free (cf) tumor DNA may be an alternative to tissue-based EGFR mutation testing in NSCLC. This exploratory analysis compares matched tumor and blood samples from the FASTACT-2 study. Experimental Design: Patients were randomized to receive six cycles of gemcitabine/platinum plus sequential erlotinib or placebo. EGFR mutation testing was performed using the cobas tissue test and the cobas blood test (in development). Blood samples at baseline, cycle 3, and progression were assessed for blood test detection rate, sensitivity, and specificity; concordance with matched tumor analysis (n = 238), and correlation with progression-free survival (PFS) and overall survival (OS). Results: Concordance between tissue and blood tests was 88%, with blood test sensitivity of 75% and a specificity of 96%. Median PFS was 13.1 versus 6.0 months for erlotinib and placebo, respectively, for those with baseline EGFR mut+ cfDNA [HR, 0.22; 95% confidence intervals (CI), 0.14–0.33, P < 0.0001] and 6.2 versus 6.1 months, respectively, for the EGFR mut− cfDNA subgroup (HR, 0.83; 95% CI, 0.65–1.04, P = 0.1076). For patients with EGFR mut+ cfDNA at baseline, median PFS was 7.2 versus 12.0 months for cycle 3 EGFR mut+ cfDNA versus cycle 3 EGFR mut− patients, respectively (HR, 0.32; 95% CI, 0.21–0.48, P < 0.0001); median OS by cycle 3 status was 18.2 and 31.9 months, respectively (HR, 0.51; 95% CI, 0.31–0.84, P = 0.0066). Conclusions: Blood-based EGFR mutation analysis is relatively sensitive and highly specific. Dynamic changes in cfDNA EGFR mutation status relative to baseline may predict clinical outcomes. Clin Cancer Res; 21(14); 3196–203. ©2015 AACR.

Vascular Endothelial Growth Factor Receptor 2 Mediates Macrophage Infiltration into Orthotopic Pancreatic Tumors in Mice

Macrophages are an abundant inflammatory cell type in the tumor microenvironment that can contribute to tumor growth and metastasis. Macrophage recruitment into tumors is mediated by multiple cytokines, including vascular endothelial growth factor (VEGF), which is thought to function primarily through VEGF receptor (VEGFR) 1 expressed on macrophages. Macrophage infiltration is affected by VEGF inhibition. We show that selective inhibition of VEGFR2 reduced macrophage infiltration into orthotopic pancreatic tumors. Our studies show that tumor-associated macrophages express VEGFR2. Furthermore, peritoneal macrophages from tumor-bearing animals express VEGFR2, whereas peritoneal macrophages from non-tumor-bearing animals do not. To our knowledge, this is the first time that tumor-associated macrophages have been shown to express VEGFR2. Additionally, we found that the cytokine pleiotrophin is sufficient to induce VEGFR2 expression on macrophages. Pleiotrophin has previously been shown to induce expression of endothelial cell markers on macrophages and was present in the microenvironment of orthotopic pancreatic tumors. Finally, we show that VEGFR2, when expressed by macrophages, is essential for VEGF-stimulated migration of tumor-associated macrophages. In summary, tumor-associated macrophages express VEGFR2, and selective inhibition of VEGFR2 reduces recruitment of macrophages into orthotopic pancreatic tumors. Our results show an underappreciated mechanism of action that may directly contribute to the antitumor activity of angiogenesis inhibitors that block the VEGFR2 pathway.