Dysregulation of splicing proteins in head and neck squamous cell carcinoma

Abstract

ABSRTRACTSignaling 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.

Cigarette smoke and chewing tobacco alter expression of different sets of miRNAs in oral keratinocytes

“…Our data indicates dysregulation of proteins which play a key role in mRNA processing and splicing in tobacco treated cells. We and others have shown in the past that aberrant activation of spliceosome complex-associated proteins has been shown to influence cellular transformation leading to development of cancer, including HNSCC 64 , 65 .Our proteomic data shows significant overexpression of splicing proteins serine and arginine rich splicing factor 2 (SRSF2) and splicing factor 3b (SF3B1) (1.9 and 1.5 fold overexpressed respectively; p-value ≤ 0.05) in tobacco treated cells. Increased expression of SRSF2 is associated with progression and poor prognosis in human hepatocellular carcinoma 66 .…”

Identification of potential biomarkers of head and neck squamous cell carcinoma using iTRAQ based quantitative proteomic approach

“…Immortalized, non-transformed normal oral keratinocytes OKF6/TERT1 was a kind gift from Dr. James Rheinwald at Brigham and Women׳s Hospital in Boston, MA [1] . As described earlier, FaDu and JHU-O28 were cultured in RPMI-1640 culture media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution [2] . CAL 27 and OKF6/TERT1 were cultured in keratinocyte serum-free media (KSFM) (Life Technologies, Grand Island, NY) supplemented with bovine pituitary extract (25 mg/ml), calcium chloride (0.4 mM), epidermal growth factor (0.2 ng/ml) and 1% penicillin/streptomycin solution.…”

Phosphoproteomic analysis identifies CLK1 as a novel therapeutic target in gastric cancer

“…Since AKT is a known upstream regulator of CLK1 [36][37][38], we also studied the effect of inhibition of AKT on the expression of CLK1, SRSF2 and pSRPK2. Our data indicates that inhibition of AKT led to a decreased expression of CLK1, SRSF2 and pSRPK2 (p value < 0.05).…”