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. 2014 May 8;5:3830.
doi: 10.1038/ncomms4830.

Integrated Exome and Transcriptome Sequencing Reveals ZAK Isoform Usage in Gastric Cancer

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Free PMC article

Integrated Exome and Transcriptome Sequencing Reveals ZAK Isoform Usage in Gastric Cancer

Jinfeng Liu et al. Nat Commun. .
Free PMC article

Abstract

Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.

Conflict of interest statement

All authors were employed by Genentech, Inc. during the time the study was done.

Figures

Figure 1
Figure 1. Somatic mutations in gastric cancer.
(a) Number of somatic protein-altering mutations per tumour. Epstein-Barr virus (EBV) status (according to the presence of EBV reads in RNAseq data) and microsatellite status are shown below. EBV status for two samples is not available due to the absence of RNAseq data for the samples. (b) Mutation spectrum varies between MSI and MSS samples. (c) Recurrently mutated genes for MSS (left) and MSI (right) samples by MuSiC analysis. Each circle represents a gene and the size of the circle is proportional to the mutation count for that gene. The genes are represented in alphabetical order from left to right on the x axis. Genes with a statistically significant q-value are labelled.
Figure 2
Figure 2. Altered pathways in gastric cancer and potential targeted therapies.
(a) Barplot showing the fraction of samples containing an aberration in curated pathways. Top 10 most altered pathways are shown. Aberrations are defined as mutations or copy number changes in any of the pathway member genes. (b) Matrix showing the distribution of pathway aberrations per sample ordered from most frequently aberrant pathway (top) to least frequently aberrant pathway (bottom). Colours of the matrix indicate whether the pathway was affected by mutation (blue), copy number change (magenta) or both (orange). The colour bar on top shows MSI (green) or MSS (orange) status. (c) Potential targeted therapies that can be applied to this patient population, based on the status of amplification, known activating mutations or loss-of-function mutations of key cancer genes.
Figure 3
Figure 3. Differential ZAK isoform usage between normal and tumour samples.
(a) ZAK gene model and protein domain structure. Thick bars: coding exons; thin bars: UTR. TV2 lacks the last nine exons and has a long terminal coding and non-coding exon. Blue and red indicate unique TV1 and TV2 sequences. Transcript variant 1 (TV1) encodes a longer protein product with a sterile alpha motif domain. (b) ZAK TV1 fraction is significantly higher in gastric tumours (left) and colon tumours (right), compared with normal adjacent tissues. The fraction of TV1 was measured by the ratio between the number of reads uniquely assignable to TV1 and the number of reads mapped to the entire ZAK gene. To account for smooth muscle contamination in normal tissues, we fit a linear model with smoothelin expression as predictor and the log isoform fraction as response, and used the residuals of the model as the ‘adjusted isoform fraction’. Dots represent samples. Grey lines connect matched tumour and normal samples. The boxes in the box-and-whisker plots represent the interquartile range between the first and third quartiles; the dashed lines (whiskers) extend to the most extreme data points, which is no more than 1.5 times the interquartile range from the box. (c) ZAK isoform fractions derived from RNAseq data correlate with quantitative PCR (qPCR) measurements. For nine gastric cancer cell lines in our study, we quantified the ratio between ZAK total expression and ZAK TV1 expression using qPCR, and compared the measurements with the isoform fraction we derived from the RNAseq data. The two measurements have significant correlation (Pearson’s correlation coefficient r=0.91, P-value=0.00077). (d) ZAK isoform expression in six TCGA data sets where there are >10 normal samples. Normal samples are represented by blue dots and tumour samples by red dots. ZAK TV1 fraction is significantly higher (adjusted P-value <0.001 and fold change >2) in breast and bladder cancer data (marked by the green asterisks).
Figure 4
Figure 4. Experimental validation of ZAK function in cancer.
(a) Immunoblots of ZAK TV1 and TV2 expressions show that protein level of TV1 is higher in gastric tumours and cell lines, compared with normal stomach tissues. ZAK TV1 was detected with Bethyl α-ZAK antibody and TV2 with Sigma α-ZAK antibody (see Methods). (b) ZAK TV1, but not TV2, can stimulate multiple transcriptional programs related to cancer pathways. Transcription reporter assay in 293 cells transfected with empty vector, TV1 or TV2 along with the indicated firefly luciferase reporter construct (AP1, NFkB and TCF/LEF). Activity is normalized to cell number using CellTiter-Glo. Immunoblot shows relative ZAK isoform expression from 293 cells transfected with the indicated construct. ZAK was detected with Sigma α-ZAK antibody. (c) Depletion of ZAK from gastric cancer cell lines inhibits cell growth. In cell lines where ZAK knockdown led to reduced viability, there was consistently high TV1 expression, while TV2 expression was marginal and variable (for example, IM-95m cell line, see for example, panel a). Cell viability analysis was carried out 6 days after infection of gastric cancer cell lines with independent ZAK shRNAs. Cell number is normalized to shNTC-infected cells. Immunoblot indicates the level of ZAK-TV1 depletion 4 days after infection. ZAK was detected with Bethyl α-ZAK antibody.

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