Dysregulation of splicing proteins in head and neck squamous cell carcinoma

Abstract

Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC.

Keywords: GeneSpring; SR proteins; in vitro labeling; mRNA processing; phosphorylation; spliceosome.

Figure 1.

Schematic workflow used to study the proteome and phosphoproteome in HNSCC.

Figure 2.

Summary statistics of the analysis. (A) Distribution of phosphoserine, phosphothreonine and phosphotyrosine sites (B) Heat map of the phosphorylation changes in a subset of kinases identified in HNSCC cell lines compared to OKF6/TERT1. (C) Motifs that were found to be enriched in the hyperphosphorylated kinase sites in the data set.

Figure 3.

Pathway analysis revealed dysregulation of proteins associated with splicing machinery in HNSCC. Gene spring version 12.6.1 was used for pathway analysis. Molecules that mapped to the mRNA processing pathway from the phosphoproteomic data are highlighted in green, molecules that mapped from the proteomic data are highlighted in orange and molecules that are common in both data sets are highlighted in yellow. The fold changes are represented using heat strips.

Figure 4.

Representative MS and MS/MS spectra of phosphopeptides identified in HNSCC cells (A) serine/arginine-rich splicing factor 2 (SRSF2) (B) SRSF protein kinase 2 (SRPK2) (C) Western blot analysis shows overexpression and hyperphosphorylation of SRPK2 in a panel of HNSCC cell lines compared to OKF6/TERT1. (D) Western blot analysis shows a decrease in SRPK2 expression in HNSCC cell lines transfected with SRPK2 siRNA. β-actin was used as a loading control.

Figure 5.

Inhibition of SRPK2 reduces the colony forming ability of HNSCC cells. Colony formation assay following (A) siRNA mediated knockdown of SRPK2 or control siRNA in a panel of HNSCC cell lines, as indicated. Colonies formed were visualized after staining with methylene blue. (B) A graphical representation of the colony forming ability of the HNSCC cells upon SRPK2 silencing *p < 0.05. (C) Colony forming ability of the HNSCC cells upon inhibition of SRPK2 using SRPIN340 or control (DMSO), in the indicated panel of HNSCC cells (D) A graphical representation of the colony forming ability of HNSCC cells upon SRPK2 inhibition *p < 0.05.

Figure 6.

Inhibition of SRPK2 reduces the invasive ability of HNSCC cells. Invasion assays were carried out using in a transwell system using Matrigel-coated filters and the number of cells that migrated to the lower chamber was counted. Cells that migrated are visualized following methylene blue staining in a panel of HNSCC cell lines as indicated. (A) HNSCC cell lines were transfected with either control (Scrambled) or SRPK2 siRNA and invaded cells were photographed (B) A graphical representation of the invasive ability of the HNSCC cells upon SRPK2 silencing *p < 0.05. (C) HNSCC cells were treated with a small molecule inhibitor of SRPK2 (SRPIN340) or vehicle control (DMSO) and invaded cells were photographed. (D) A graphical representation of the invasive ability of the HNSCC cells upon SRPK2 inhibition *p < 0.05.