Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers

Background: Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported. Results: We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker. Conclusions: This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research

Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer

Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies

Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types

<div><p>Gene fusions, like <i>BCR/ABL1</i> in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a “breakpoint analysis” pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving <i>ROS1</i> tyrosine kinase in angiosarcoma (<i>CEP85L/ROS1</i>), <i>SLC1A2</i> glutamate transporter in colon cancer (<i>APIP/SLC1A2</i>), <i>RAF1</i> kinase in pancreatic cancer (<i>ATG7/RAF1</i>) and anaplastic astrocytoma (<i>BCL6/RAF1</i>), <i>EWSR1</i> in melanoma (<i>EWSR1/CREM</i>), <i>CDK6</i> kinase in T-cell acute lymphoblastic leukemia (<i>FAM133B/CDK6</i>), and <i>CLTC</i> in breast cancer (<i>CLTC/VMP1</i>). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including <i>EGFRvIII</i> and <i>FIP1L1/PDGFRA</i>. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis.</p></div

Discovery and characterization of <i>EWSR1/CREM</i> in melanoma.

<p>(<i>A</i>) Array CGH heatmap displaying intragenic <i>EWSR1</i> breakpoints identified in the SH-4 and CHL-1 melanoma cell lines. (<i>B</i>) Paired-end RNA-seq identification of <i>EWSR1/CREM</i> in CHL-1. Paired-end reads supporting the rearrangement are depicted along with the predicted gene fusion structure. CREM contributes a basic leucine zipper motif (ZIP), while EWSR1 contributes the EWS Activation Domain (EAD). (<i>C</i>) RT-PCR verification of <i>EWSR1/CREM</i> in CHL-1. (<i>D</i>) Quantitative RT-PCR using primers flanking the gene fusion junction verifies <i>EWSR1/CREM</i> knockdown following transfection of an siRNA pool targeting the 3′ end of <i>CREM</i>. (<i>E</i>, <i>F</i>, <i>G</i>) Transfection of CHL-1 with <i>CREM</i>-targeting siRNA pool results in (<i>E</i>) decreased cell proliferation, (<i>F</i>) decreased invasion, and (<i>G</i>) a higher fraction of senescent cells, compared to non-targeting control (NTC). **<i>P</i><0.01 (two-sided Student's t-test).</p

Discovery of new cell line models for the known rearrangements, <i>EGFRvIII</i> and <i>FIP1L1/PDGFRA</i>.

<p>(<i>A</i>) Heatmap depicting genomic breakpoints within <i>EGFR</i> in the glioblastoma cell lines, CAS-1 and DKMG. (<i>B</i>) Identification of <i>EGFRvIII</i> in DKMG cells by paired-end RNA-seq. Paired-end reads supporting the rearrangement are depicted. (<i>C</i>) Verification of <i>EGFRvIII</i> expression by RT-PCR (top panel) and Western blotting (bottom panel) in DKMG. RT-PCR was done using primers flanking the exon 1/exon 8 junction of <i>EGFRvIII</i>, and Western blotting was done using an antibody specific to the EGFRvIII isoform. Control samples include U87 glioblastoma cells without <i>EGFR</i> rearrangement, U87-vIII cells engineered to express exogenous <i>EGFRvIII</i>, and A431 epidermoid carcinoma cells with <i>EGFR</i> amplification. (<i>D</i>) RBA identification of expression-level breakpoint within <i>PDGFRA</i> in SUPT13 T-ALL cells. ***<i>P<10⁻¹¹</i> (Student's t-test). (<i>E</i>) RNA-seq identification of <i>FIP1L1/PDGFRA</i>. (<i>F</i>) RT-PCR validation of <i>FIP1L1/PDGFRA</i> expression in SUPT13. (<i>G</i>) SUPT13 cells are sensitive to imatinib (IC₅₀ = 0.036 µM). K562 is a positive control CML cell line harboring BCR/ABL1 with known sensitivity to imatinib (IC₅₀ = 0.18 µM).</p

DBA discovery of recurrent rearrangements of <i>CLTC</i> and <i>VMP1</i> across diverse cancer types.

<p>(<i>A</i>) Heatmap depicting focal deletions between <i>CLTC</i> and <i>VMP1</i> in the breast cancer cell lines BT-549 and HCC1954. (<i>B</i>) Discovery of the recurrent <i>CLTC/VMP1</i> rearrangement in BT-549 (<i>left</i> panel) and HCC1954 (<i>right</i> panel) by paired-end RNA-seq. (<i>C</i>) RT-PCR verification of <i>CLTC/VMP1</i> fusion in BT-549 and HCC1954. (<i>D</i>) Heatmap depicting focal deletions disrupting <i>CLTC</i>, <i>PTRH2</i> and/or <i>VMP1</i> in various cancer types (see legend). (<i>E</i>) A renal cell carcinoma line, RXF393, was also profiled by exon microarray where an expression breakpoint was evident within <i>CLTC</i>. ***<i>P<10⁻⁹</i> (Student's t-test).</p

Identification and characterization of novel <i>RAF1</i> gene fusions in pancreatic cancer and anaplastic astrocytoma.

<p>(<i>A</i>) Array CGH heatmaps displaying intragenic <i>RAF1</i> genomic breakpoints identified in the PL5 pancreatic cancer cell line (<i>left panel</i>) and the D-538MG anaplastic astrocytoma cell line (<i>right panel</i>). Unsmoothed log₂ ratios are displayed. (<i>B</i>) Identification of <i>ATG7/RAF1</i> (left) and <i>BCL6/RAF1</i> (right) in PL5 and D-538MG cells, respectively, by paired-end RNA-seq. A subset of the paired-end reads supporting each gene fusion is displayed. Both gene fusions are in-frame and the <i>RAF1</i> serine threonine kinase domain (STK) is retained in both fusions. (<i>C</i>) Experimental validation of gene fusions by RT-PCR, using primers flanking the respective gene fusion junction. (<i>D</i>) Western blotting verifies knockdown of ATG7/RAF1 in PL5 following transfection of a <i>RAF1</i>-targeting siRNA pool. ATG7/RAF1 protein levels were monitored using an anti-<i>RAF1</i> antibody, with anti-<i>GAPDH</i> providing a loading control. (<i>E</i>) Decreased cell proliferation and (<i>F</i>) invasion rates of PL5 following transfection of a <i>RAF1</i>-targeting siRNA pool, compared to transfection of a non-targeting control (NTC) siRNA pool. ** <i>P</i><0.01 (two-sided Student's t-test). (<i>G</i>) Break-apart FISH demonstrates rearrangement of <i>BRAF</i> in a pancreatic cancer case from the TMA, as evidenced by physical separation of the red and green probes (arrows) flanking <i>BRAF</i> (single interphase nucleus shown).</p

Breakpoint analysis for discovering novel cancer gene rearrangements.

<p>Schematic depiction of the approach and workflow, demonstrated by example of the rediscovery of a known gene fusion, <i>SET/NUP214</i>, in the T-ALL cell line LOUCY. Various publicly-available and in-house exon microarray and high-density CGH/SNP array experiments were analyzed. RNA breakpoint analysis (RBA) identifies significant transitions in exon expression level, which may reflect elevated expression of exons distal (3′ partner) or proximal (5′ partner) to a gene fusion junction. To identify such transitions a “walking” Student's t-test was applied, comparing expression levels of proximal and distal exons. Candidate rearrangements were subsequently filtered for those disrupting genes of the Cancer Gene Census, with directional orientation (i.e. being the 5′ or 3′ partner) consistent with known rearrangements of that gene. RBA candidates were further filtered using a Bonferroni correction to adjust for multiple t-tests. DNA breakpoint analysis (DBA) screens for intragenic DNA copy number transitions, which may reflect unbalanced chromosomal rearrangements leading to the formation of gene fusions. The fused lasso method (FDR 1%) followed by a copy number smoothing algorithm was applied to identify CNAs. Copy number transitions were filtered for those disrupting any annotated gene and then further filtered for those disrupting genes of the Cancer Gene Census. We included only candidate breakpoints where the directional orientation of the copy number transition was consistent with known rearrangements of that gene. Several candidates were then validated using molecular and cytogenetic approaches. The average numbers of candidate rearrangements per cancer sample are depicted along the left and right panels at various stages of the workflow.</p

Validated gene fusions and rearrangements.

a<p>Gene fusions initially nominated by breakpoint analysis and subsequently validated by paired-end RNA-seq (or 5′ RACE for <i>CEP85L/ROS1</i>) and RT-PCR.</p>b<p><i>ABL1</i> and <i>EGFR</i> locus rearrangements were previously reported in the respective cell lines <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003464#pgen.1003464-Heisterkamp1" target="_blank">[96]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003464#pgen.1003464-Hunts1" target="_blank">[98]</a>; however associated fusion transcripts were not identified.</p