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. 2018 Oct 15;24(20):5123-5132.
doi: 10.1158/1078-0432.CCR-18-0752. Epub 2018 Jun 26.

Characterization of Alternative Splicing Events in HPV-Negative Head and Neck Squamous Cell Carcinoma Identifies an Oncogenic DOCK5 Variant

Chao Liu  1   2 , Theresa Guo  3 , Guorong Xu  4 , Akihiro Sakai  1 , Shuling Ren  1   2 , Takahito Fukusumi  1 , Mizuo Ando  1 , Sayed Sadat  1 , Yuki Saito  1 , Zubair Khan  3 , Kathleen M Fisch  4 , Joseph Califano  5   6
Affiliations

Characterization of Alternative Splicing Events in HPV-Negative Head and Neck Squamous Cell Carcinoma Identifies an Oncogenic DOCK5 Variant

Chao Liu et al. Clin Cancer Res. .

Abstract

Purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide, and alternative splicing is considered to play important roles in tumor progression. Our study is designed to identify alternative splicing events (ASEs) in human papillomavirus (HPV)-negative HNSCC.Experimental Design: RNA sequencing data of 407 HPV-negative HNSCC and 38 normal samples were obtained from The Cancer Genome Atlas (TCGA), and splice junctions were discovered using MapSplice. Outlier analysis was used to identify significant splicing junctions between HPV-negative HNSCC and normal samples. To explore the functional role of the identified DOCK5 variant, we checked its expression with qRT-PCR in a separate primary tumor validation set and performed proliferation, migration, and invasion assays.Results: A total of 580 significant splicing events were identified in HPV-negative HNSCC, and the most common type of splicing events was an alternative start site (33.3%). The prevalence of a given individual ASE among the tumor cohort ranged from 9.8% and 64.4%. Within the 407 HPV-negative HNSCC samples in TCGA, the number of significant ASEs differentially expressed in each tumor ranged from 17 to 290. We identified a novel candidate oncogenic DOCK5 variant confirmed using qRT-PCR in a separate primary tumor validation set. Loss- and gain-of-function experiments indicated that DOCK5 variant promoted proliferation, migration, and invasion of HPV-negative HNSCC cells, and patients with higher expression of DOCK5 variant showed decreased overall survival.Conclusions: Analysis of ASEs in HPV-negative HNSCC identifies multiple alterations likely related to carcinogenesis, including an oncogenic DOCK5 variant. Clin Cancer Res; 24(20); 5123-32. ©2018 AACR.

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Conflict of interest statement

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

This study has no potential conflict of interest that should be disclosed.

Figures

Figure 1
Figure 1. Pipeline for identification of significant ASEs through RNA-seq analysis.
Initially, from MapSplice output, 4,660,670 raw junctions, representing splice variant isoforms, were present within TCGA HPV negative HNSCC cohort. These junctions were normalized and then filtered to exclude junctions without variation or those on X, Y and mitochondrial (MT) chromosomes. Then potential splice variants were identified on the basis of possible alternative splicing patterns, and 18,504 potential candidates remained. Outlier statistics were applied to identify candidates with differential expression between tumors and normal samples, of these, 969 splice variants were identified. Finally, these junctions were validated by IGV visualization, and 580 junctions, which mapped to 501 unique genes were confirmed.
Figure 2
Figure 2. DOCK5 variant is highly expressed in HPV negative HNSCC
(A) IGV visualization of DOCK5 variant shows an alternative end site with expression unique to tumors (top), in which ten representative samples with reads coverage were shown; Schematic diagram of DOCK5 variant shows the alternative end site (middle); Amino acid sequence of DOCK5 variant shows the alternative end site, and the amino acid length of DOCK5 variant was much shorter than DOCK5 WT (bottom). (B) Unique DOCK5 junction expression and overall DOCK5 gene expression in TCGA HPV negative HNSCC data is shown. Expression of the DOCK5 splice junction is identified mostly in tumors. The corresponding overall DOCK5 gene expression (RSEM normalized) shows that these differences in DOCK5 splice variant expression are not fully explained by overall gene expression. Each vertical bar represents one sample sorted by expression level of DOCK5 variant (Normal samples: blue; Tumors: red) (C) Biologic validation of DOCK5 variant expression in primary tumor tissues. Quantitative PCR for identification of the DOCK5 variant shows significantly increased expression in tumors compared with normal UPPP tissues (P < 0.001), while wild-type DOCK5 gene expression is similar between tumors and normal tissues (NS), and the ratio of DOCK5 variant to wild-type is higher in in tumors than UPPP tissues (P < 0.001). (D) Log rank test shows that the patients with higher expression of DOCK5 variant have decreased overall survival (P = 0.0374), but no significant changes of disease free survival (P = 0.2750). The patients with no expression of DOCK5 variant (n = 127) are defined as lower expression of DOCK5 variant group. ***: P < 0.001, NS: not significant.
Figure 3
Figure 3. DOCK5 variant promotes proliferation of HPV negative HNSCC cells
(A) qRT-PCR results show the decreased expression of both DOCK5 wild-type (WT) and DOCK5 variant (Var) when treated with pooled siRNA for DOCK5. Three siRNAs specifically targeting DOCK5 variant show inhibition of the alternatively spliced DOCK5, with minimal inhibition of the wild-type gene. Specifically, the right panel shows the ratio of DOCK5 variant to wild-type expression, demonstrating that this ratio is significantly decreased by these three ASE siRNAs in both BHY and JHU011 cell lines. (B) Cell viability is measured after knockdown the expression of overall DOCK5 (si Pool) and DOCK5 variant (Var si1, si2, si3) in BHY and JHU011 cells. Proliferation ratio represented the cell numbers relative to day zero. Significant growth inhibition is seen with specific silencing of DOCK5 in both cell lines compared with silencing of the whole DOCK5 gene and control group (Blank, NC). (C) Colony formation assay shows the colonies in DOCK5 variant knockdown group are much fewer and smaller than control groups in BHY and JHU011 cells. (D) qRT-PCR results show that following transfection of lentivirus of DOCK5 variant or wild-type in UM-SCC17B and Detroit562 cells, the expression of DOCK5 variant or wild-type were successfully upregulated. (E) Cell viability assay shows significant growth increase by overexpression of DOCK5 variant, not DOCK5 wild-type. (F) Colony formation assay demonstrates that the colonies in DOCK5 variant overexpression group are more and bigger than empty vector groups and DOCK5 WT overexpression group in UM-SCC17B and Detroit562 cells. ***: P < 0.001, **: P < 0.01, *: P < 0.05, NS: not significant.
Figure 4
Figure 4. DOCK5 variant promotes migration and invasion of HPV negative HNSCC cells
(A) Cell scratch assay demonstrates that cell migration ability was inhibited by knockdown of DOCK5 variant in BHY and JHU011 cells. (B) Cell scratch assay demonstrates that cell migration ability was enhanced by overexpression of DOCK5 variant in UM-SCC17B and Detroit562 cells. (C) Transwell migration assay shows that the number of migrating cells is decreased by knockdown of DOCK5 variant. (D) Transwell invasion assay shows that the number of invading cells is significantly reduced by knockdown of DOCK5 variant. (E) Transwell migration assay shows that the number of migrating cells is increased by overexpression of DOCK5 variant. (F) Transwell invasion assay shows that the number of invading cells is significantly increased by overexpression of DOCK5 variant. ***: P < 0.001, **: P < 0.01.
Figure 5
Figure 5. DOCK5 variant activates p38 and Erk MAPK pathway
(A) The expression of p-p38, p-Erk and p-MEK1/2 is decreased following knockdown the expression of DOCK5 variant in both BHY and JHU011 cells. (B) Overexpression of DOCK5 variant up-regulated the expression of p-p38, p-Erk and p-MEK1/2 in both UM-SCC17B and Detroit562 cells.
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