Limits...
Transcriptome meta-analysis of lung cancer reveals recurrent aberrations in NRG1 and Hippo pathway genes.

Dhanasekaran SM, Balbin OA, Chen G, Nadal E, Kalyana-Sundaram S, Pan J, Veeneman B, Cao X, Malik R, Vats P, Wang R, Huang S, Zhong J, Jing X, Iyer M, Wu YM, Harms PW, Lin J, Reddy R, Brennan C, Palanisamy N, Chang AC, Truini A, Truini M, Robinson DR, Beer DG, Chinnaiyan AM - Nat Commun (2014)

Bottom Line: Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines.In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations.These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.

View Article: PubMed Central - PubMed

Affiliation: 1] Michigan Center for Translational Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA [2] Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA [3].

ABSTRACT
Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.

Show MeSH

Related in: MedlinePlus

Recurrent NRG1 rearrangements in lung cancer. A, Recurrent fusions involving NRG1 as a 3′ partner were detected in lung adenocarcinoma and lung squamous carcinoma in the three cohorts included in this study. Schematic representation of functional domains present in the NRG1 fusion proteins namely CD74-NRG1; RBPMS-NRG1 (LUAD); WRN-NRG1 (LUSC); SDC4-NRG1 (LUSC) and RAB2IL1-NRG1 (ovarian cancer from TCGA) compared to the wild-type NRG1 (Top). The receptor binding EGF domain is preserved in all fusions. TM, transmembrane domain; RRM- domain; IGc2- domain; SEC2P-domain. B, Analysis of RNASeq expression values revealed outlier NRG1 mRNA expression in all index cases (large blue dots) within each cohort. C, High NRG1 mRNA expression driven by the fusion event in the index tumor tissue compared to matched normal, in both an LUAD patient in the University of Michigan and Seoul cohorts. D, Boxplot showing outlier expression of NRG1 in H1793 in the University of Michigan lung cell line cohort. E, Two independent siRNAs mediated knockdown of NRG1 in H1793 cells as assessed by Q-PCR. F, Knock-down of NRG1 decreased cell proliferation as monitored by IncuCyte confluence analysis. G, Overexpression of NRG1 induces cell proliferation and migration. Cell proliferation by WST-1 assay (left panel) and cell counting (middle panel) on BEAS-2B cells stably transfected with Lac-Z or CD74-NRG1 fusion. Both assays demonstrated that cells expressing the CD74-NRG1 fusion had significantly higher proliferation rate at day 3 and 5 (Student’s t-test P<0.001 for both time-points) as compared to Lac-Z. The right panel represents a cell migration assay after 24 hours. BEAS-2B cells expressing CD74-NRG1 fusion showed a higher migration rate as compared to Lac-Z (Student’s t-test P=0.0014).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4274748&req=5

Figure 6: Recurrent NRG1 rearrangements in lung cancer. A, Recurrent fusions involving NRG1 as a 3′ partner were detected in lung adenocarcinoma and lung squamous carcinoma in the three cohorts included in this study. Schematic representation of functional domains present in the NRG1 fusion proteins namely CD74-NRG1; RBPMS-NRG1 (LUAD); WRN-NRG1 (LUSC); SDC4-NRG1 (LUSC) and RAB2IL1-NRG1 (ovarian cancer from TCGA) compared to the wild-type NRG1 (Top). The receptor binding EGF domain is preserved in all fusions. TM, transmembrane domain; RRM- domain; IGc2- domain; SEC2P-domain. B, Analysis of RNASeq expression values revealed outlier NRG1 mRNA expression in all index cases (large blue dots) within each cohort. C, High NRG1 mRNA expression driven by the fusion event in the index tumor tissue compared to matched normal, in both an LUAD patient in the University of Michigan and Seoul cohorts. D, Boxplot showing outlier expression of NRG1 in H1793 in the University of Michigan lung cell line cohort. E, Two independent siRNAs mediated knockdown of NRG1 in H1793 cells as assessed by Q-PCR. F, Knock-down of NRG1 decreased cell proliferation as monitored by IncuCyte confluence analysis. G, Overexpression of NRG1 induces cell proliferation and migration. Cell proliferation by WST-1 assay (left panel) and cell counting (middle panel) on BEAS-2B cells stably transfected with Lac-Z or CD74-NRG1 fusion. Both assays demonstrated that cells expressing the CD74-NRG1 fusion had significantly higher proliferation rate at day 3 and 5 (Student’s t-test P<0.001 for both time-points) as compared to Lac-Z. The right panel represents a cell migration assay after 24 hours. BEAS-2B cells expressing CD74-NRG1 fusion showed a higher migration rate as compared to Lac-Z (Student’s t-test P=0.0014).

Mentions: Remarkably, we noted functionally recurrent gene fusion where the common 3′gene neuregulin 1 (NRG1) was fused to various 5′ partners (Fig. 6A and Supplementary Table 8) CD74-NRG1, RBPMS-NRG1, WRN-NRG1 and SDC4-NRG1, in both LUAD and LUSC samples. Importantly, CD74-NRG1 fusion variant was recently identified by three independent groups20, 21, 22. While CD74-NRG1, SDC4-NRG1 and RBPMS-NRG1 fusion events resulted in the production of chimeric proteins, the WRN-NRG1 fusion results in the overexpression of full length NRG1 regulated by the WRN gene promoter. As a member of EGF ligand family, NRG1 transduces its signal through the HER/ErbB family receptor tyrosine kinases33, 34. NRG1 functional domains include kringle-like, immunoglobulin-like domain and the EGF domain located in the C-terminal region33. Notably the EGF domain is essential for receptor interaction35 and preserved in all the NRG1 fusions identified (Fig. 6A). All NRG1 fusion index samples were found in samples without known driver mutations and displayed NRG1 outlier expression in the tumor but not matching normal tissue (Fig. 6B and 6C). Strikingly similar to the pattern described above for the known receptor kinases fusions, we noticed NRG1 outlier expression in both index fusion samples (n=4) and an independent set of known driver aberration negative cases (n=10) (Supplementary Table 8). Among the lung cancer cell line RNASeq data, H1793 exhibited the highest NRG1 transcript expression (Fig. 6D and Supplementary Fig. 2). At 70% knock down with two independent NRG1 siRNAs (Fig. 6E) H1793 cell proliferation rate was affected as assessed using cell growth assays (Fig. 6F). Conversely upon stable overexpression of the CD74-NRG1 fusion protein in normal lung BEAS-2B cells we observed significant increase in cell proliferation, migration (Fig. 6G and Supplementary Fig. 7A) and an altered morphology relative to LacZ controls (Supplementary Fig. 7B and 7C). CD74-NRG1 overexpression induces epithelial to mesenchymal transition (EMT) as evidenced by increased VIM and SNAIL protein expression and decreased CDH1 level by Western blot analysis (Supplementary Fig. 7D and 9). We next performed gene expression profiling of CD74-NRG1 and LacZ control cells to identify affected biological pathways. Significant analysis of microarrays (SAM) showed overexpression of several EMT markers such as VIM, ZEB1, ZEB2, FZD7, TWIST1, VCAN, and CHD2 and under-expression of RGS2 and CDH1 among others further supporting the EMT phenotype in CD74-NRG1-positive cells (Supplemental Data 7). Vimentin, ZEB1 and ZEB2 were overexpressed more than 4-fold, while CDH1 and RGS2 were among the most under-expressed genes (Supplementary Fig. 7E and 8A). Gene set enrichment analysis (GSEA) identified down-regulation of cell adhesion (Supplementary Fig. 8B) and up-regulation of the SRC and ERBB pathways (Supplementary Fig. 8C and 8D) in CD74-NRG1 cells. We examined both total and phosphorylated ERBB3, a receptor known to bind NRG1, and observed a substantial decrease in total ERBB3 upon overexpression of CD74-NRG1 which was also reflected in its phosphorylated form as compared to LacZ control (Supplementary Fig. 8E and 9). Despite the observed decrease in total ERBB3 in the fusion expressing cells, phospho-ERBB3 was still detectable (Supplementary Fig. 8E and 9). Total ERBB3 decrease upon exposure to NRG1 has been previously demonstrated in MCF-736 and also in H568 lung cells upon CD74-NRG1 overexpression20. In addition we observed increased levels of phospho-ERK (1.95-fold) and phosphoJNK1 (5.5-fold) relative to LacZ control (Supplementary Fig. 8E and 9) potentially promoting the oncogenic phenotype in NRG1 fusion overexpressing cells. Finally, we examined other cancer types for NRG1 fusions and discovered one additional RAB2IL1-NRG1 fusion in the TCGA ovarian cancer RNASeq data. As observed in lung cancer, the functional EGF domain is retained in RAB2IL1-NRG1 and the fusion index case exhibited outlier NRG1 expression (Fig. 6A and 6B). Altogether, NRG1 is perturbed (NRG1 fusions and/or outlier expression) in 3.9% (15/386) of driver unknown samples, supporting a causal role for NRG1 in this lung cancer patient subpopulation.


Transcriptome meta-analysis of lung cancer reveals recurrent aberrations in NRG1 and Hippo pathway genes.

Dhanasekaran SM, Balbin OA, Chen G, Nadal E, Kalyana-Sundaram S, Pan J, Veeneman B, Cao X, Malik R, Vats P, Wang R, Huang S, Zhong J, Jing X, Iyer M, Wu YM, Harms PW, Lin J, Reddy R, Brennan C, Palanisamy N, Chang AC, Truini A, Truini M, Robinson DR, Beer DG, Chinnaiyan AM - Nat Commun (2014)

Recurrent NRG1 rearrangements in lung cancer. A, Recurrent fusions involving NRG1 as a 3′ partner were detected in lung adenocarcinoma and lung squamous carcinoma in the three cohorts included in this study. Schematic representation of functional domains present in the NRG1 fusion proteins namely CD74-NRG1; RBPMS-NRG1 (LUAD); WRN-NRG1 (LUSC); SDC4-NRG1 (LUSC) and RAB2IL1-NRG1 (ovarian cancer from TCGA) compared to the wild-type NRG1 (Top). The receptor binding EGF domain is preserved in all fusions. TM, transmembrane domain; RRM- domain; IGc2- domain; SEC2P-domain. B, Analysis of RNASeq expression values revealed outlier NRG1 mRNA expression in all index cases (large blue dots) within each cohort. C, High NRG1 mRNA expression driven by the fusion event in the index tumor tissue compared to matched normal, in both an LUAD patient in the University of Michigan and Seoul cohorts. D, Boxplot showing outlier expression of NRG1 in H1793 in the University of Michigan lung cell line cohort. E, Two independent siRNAs mediated knockdown of NRG1 in H1793 cells as assessed by Q-PCR. F, Knock-down of NRG1 decreased cell proliferation as monitored by IncuCyte confluence analysis. G, Overexpression of NRG1 induces cell proliferation and migration. Cell proliferation by WST-1 assay (left panel) and cell counting (middle panel) on BEAS-2B cells stably transfected with Lac-Z or CD74-NRG1 fusion. Both assays demonstrated that cells expressing the CD74-NRG1 fusion had significantly higher proliferation rate at day 3 and 5 (Student’s t-test P<0.001 for both time-points) as compared to Lac-Z. The right panel represents a cell migration assay after 24 hours. BEAS-2B cells expressing CD74-NRG1 fusion showed a higher migration rate as compared to Lac-Z (Student’s t-test P=0.0014).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4274748&req=5

Figure 6: Recurrent NRG1 rearrangements in lung cancer. A, Recurrent fusions involving NRG1 as a 3′ partner were detected in lung adenocarcinoma and lung squamous carcinoma in the three cohorts included in this study. Schematic representation of functional domains present in the NRG1 fusion proteins namely CD74-NRG1; RBPMS-NRG1 (LUAD); WRN-NRG1 (LUSC); SDC4-NRG1 (LUSC) and RAB2IL1-NRG1 (ovarian cancer from TCGA) compared to the wild-type NRG1 (Top). The receptor binding EGF domain is preserved in all fusions. TM, transmembrane domain; RRM- domain; IGc2- domain; SEC2P-domain. B, Analysis of RNASeq expression values revealed outlier NRG1 mRNA expression in all index cases (large blue dots) within each cohort. C, High NRG1 mRNA expression driven by the fusion event in the index tumor tissue compared to matched normal, in both an LUAD patient in the University of Michigan and Seoul cohorts. D, Boxplot showing outlier expression of NRG1 in H1793 in the University of Michigan lung cell line cohort. E, Two independent siRNAs mediated knockdown of NRG1 in H1793 cells as assessed by Q-PCR. F, Knock-down of NRG1 decreased cell proliferation as monitored by IncuCyte confluence analysis. G, Overexpression of NRG1 induces cell proliferation and migration. Cell proliferation by WST-1 assay (left panel) and cell counting (middle panel) on BEAS-2B cells stably transfected with Lac-Z or CD74-NRG1 fusion. Both assays demonstrated that cells expressing the CD74-NRG1 fusion had significantly higher proliferation rate at day 3 and 5 (Student’s t-test P<0.001 for both time-points) as compared to Lac-Z. The right panel represents a cell migration assay after 24 hours. BEAS-2B cells expressing CD74-NRG1 fusion showed a higher migration rate as compared to Lac-Z (Student’s t-test P=0.0014).
Mentions: Remarkably, we noted functionally recurrent gene fusion where the common 3′gene neuregulin 1 (NRG1) was fused to various 5′ partners (Fig. 6A and Supplementary Table 8) CD74-NRG1, RBPMS-NRG1, WRN-NRG1 and SDC4-NRG1, in both LUAD and LUSC samples. Importantly, CD74-NRG1 fusion variant was recently identified by three independent groups20, 21, 22. While CD74-NRG1, SDC4-NRG1 and RBPMS-NRG1 fusion events resulted in the production of chimeric proteins, the WRN-NRG1 fusion results in the overexpression of full length NRG1 regulated by the WRN gene promoter. As a member of EGF ligand family, NRG1 transduces its signal through the HER/ErbB family receptor tyrosine kinases33, 34. NRG1 functional domains include kringle-like, immunoglobulin-like domain and the EGF domain located in the C-terminal region33. Notably the EGF domain is essential for receptor interaction35 and preserved in all the NRG1 fusions identified (Fig. 6A). All NRG1 fusion index samples were found in samples without known driver mutations and displayed NRG1 outlier expression in the tumor but not matching normal tissue (Fig. 6B and 6C). Strikingly similar to the pattern described above for the known receptor kinases fusions, we noticed NRG1 outlier expression in both index fusion samples (n=4) and an independent set of known driver aberration negative cases (n=10) (Supplementary Table 8). Among the lung cancer cell line RNASeq data, H1793 exhibited the highest NRG1 transcript expression (Fig. 6D and Supplementary Fig. 2). At 70% knock down with two independent NRG1 siRNAs (Fig. 6E) H1793 cell proliferation rate was affected as assessed using cell growth assays (Fig. 6F). Conversely upon stable overexpression of the CD74-NRG1 fusion protein in normal lung BEAS-2B cells we observed significant increase in cell proliferation, migration (Fig. 6G and Supplementary Fig. 7A) and an altered morphology relative to LacZ controls (Supplementary Fig. 7B and 7C). CD74-NRG1 overexpression induces epithelial to mesenchymal transition (EMT) as evidenced by increased VIM and SNAIL protein expression and decreased CDH1 level by Western blot analysis (Supplementary Fig. 7D and 9). We next performed gene expression profiling of CD74-NRG1 and LacZ control cells to identify affected biological pathways. Significant analysis of microarrays (SAM) showed overexpression of several EMT markers such as VIM, ZEB1, ZEB2, FZD7, TWIST1, VCAN, and CHD2 and under-expression of RGS2 and CDH1 among others further supporting the EMT phenotype in CD74-NRG1-positive cells (Supplemental Data 7). Vimentin, ZEB1 and ZEB2 were overexpressed more than 4-fold, while CDH1 and RGS2 were among the most under-expressed genes (Supplementary Fig. 7E and 8A). Gene set enrichment analysis (GSEA) identified down-regulation of cell adhesion (Supplementary Fig. 8B) and up-regulation of the SRC and ERBB pathways (Supplementary Fig. 8C and 8D) in CD74-NRG1 cells. We examined both total and phosphorylated ERBB3, a receptor known to bind NRG1, and observed a substantial decrease in total ERBB3 upon overexpression of CD74-NRG1 which was also reflected in its phosphorylated form as compared to LacZ control (Supplementary Fig. 8E and 9). Despite the observed decrease in total ERBB3 in the fusion expressing cells, phospho-ERBB3 was still detectable (Supplementary Fig. 8E and 9). Total ERBB3 decrease upon exposure to NRG1 has been previously demonstrated in MCF-736 and also in H568 lung cells upon CD74-NRG1 overexpression20. In addition we observed increased levels of phospho-ERK (1.95-fold) and phosphoJNK1 (5.5-fold) relative to LacZ control (Supplementary Fig. 8E and 9) potentially promoting the oncogenic phenotype in NRG1 fusion overexpressing cells. Finally, we examined other cancer types for NRG1 fusions and discovered one additional RAB2IL1-NRG1 fusion in the TCGA ovarian cancer RNASeq data. As observed in lung cancer, the functional EGF domain is retained in RAB2IL1-NRG1 and the fusion index case exhibited outlier NRG1 expression (Fig. 6A and 6B). Altogether, NRG1 is perturbed (NRG1 fusions and/or outlier expression) in 3.9% (15/386) of driver unknown samples, supporting a causal role for NRG1 in this lung cancer patient subpopulation.

Bottom Line: Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines.In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations.These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.

View Article: PubMed Central - PubMed

Affiliation: 1] Michigan Center for Translational Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA [2] Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA [3].

ABSTRACT
Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.

Show MeSH
Related in: MedlinePlus