Comparative proteomics approach to screening of potential diagnostic and therapeutic targets for oral squamous cell carcinoma

Abstract

This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomics approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified with eight of these being reported for the first time in oral squamous carcinoma. Of the eight newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia as well as clinical stage, lymph node involvement, and recurrence, which are known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, not only being closely related to cell proliferation and apoptosis but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of oral squamous carcinoma. Further this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.

Fig. 1.

Proteomics analysis of OSCC tissues and corresponding normal tissues using 2-DE gels. Whole cell lysates (1 mg) from OSCC (A) and normal (B) tissues were separated by 2-DE and visualized by Coomassie Blue staining. Arrows indicate identified protein spots significantly and consistently altered between carcinoma tissue and control normal tissues.

Fig. 2.

Results of RACK1 as the representative of protein identification using ESI-Q-TOF-MS/MS. A, B, and C, output of the database searching by the MASCOT program using MS/MS data used in the identification of RACK1. The matched peptides were shown in bold red. D, MS/MS spectrum of parent ions with m/z values of 655.2098.

Fig. 3.

Validation of RACK1 in OSCC tissues and cells relative to normal tissues and normal keratinocytes. A, a, 2-DE gel map of RACK1 in OSCC tissues and corresponding normal tissues; arrows indicate RACK1 spots. Lower part, three-dimensional image of RACK1 expression by PDQuest software. A, b, chart indicating RACK1 expression in 2-DE. B, representative image of quantitative measurement of RACK1 protein in 10 pairs of OSCC tissues and corresponding normal tissues by Western blotting analysis using RACK1-specific antibody. C, representative image of mRNA levels of RACK1 between OSCCs and their corresponding normal tissues measured by semiquantitative reverse transcription PCR. D, protein levels of RACK1 in Tca8113 and HOK16E6E7 cells revealed by Western blotting analysis using RACK1-specific antibody. E, mRNA levels of RACK1 in Tca8113 and HOK16E6E7 cells measured by semiquantitative RT-PCR. Error bars represent ±S.D. of the mean.

Fig. 4.

Expression of RACK1 in human oral squamous carcinoma. Immunohistochemical analysis showed the expression of RACK1 to increase gradually with the progression of carcinogenesis from normal epithelium to premalignant lesion to invasive carcinoma. The staining of immunoreactivity was expressed as a product of the intensity and the proportion of cells staining positive. The positive staining of epithelium cells was expressed as yellow-brown granules with weak (thin arrows) to moderate-strong (thick arrows) intensity. RACK1 immunoreactivity was readily detected in the cytoplasm and occasionally in the nucleus. A, in normal epithelium, the positive staining was rarely detected or was limited to the most active basal or parabasal layer of the epithelium. With increasing severity of epithelial hyperplasia, the positive staining spread into other layers, and staining intensity was enhanced. B, for OLK without dysplasia, the immunostaining profile was similar to that of normal epithelium. C, for OLK with dysplasia, the positive staining spread sporadically into other epithelial layers with weak to moderate staining. D, for infiltrated OSCC, the positive staining spread to almost all the cancer nest with moderate to strong staining. E, RACK1 expression in recurrent OSCC lesion in the neck region exhibited the strongest immunoreactivity.

Fig. 5.

The mRNA and protein expression of RACK1 after specific siRNA transfection in Tca8113 cells. The Tca8113 cells were transfected with siRACK1-231 and the negative control siRNAs (siNC) as described under “Materials and Methods.” The levels of mRNA were determined by semiquantitative RT-PCR (A and C), and the protein levels were determined by Western blotting (B and D). Error bars represent ±S.D. of the mean.

Fig. 6.

Effects of gene silencing of RACK1 on the apoptosis of Tca8113 cells. Three apoptosis-related assays were carried out. A, 48 h after the transfection, Tca8113 cells were analyzed by using flow cytometry. A dot plot display of Annexin V-FITC fluorescence versus propidium iodide (PI) fluorescence is shown in logarithmic scale. Annexin V-positive cells were regarded as apoptotic cells. Upper left quadrant, necrosis cells; lower left quadrant, vital cells; lower right quadrant, early apoptosis cells; upper right quadrant, late apoptosis cells. B, colony formation. 6 h after transfection, Tca8113 cells were supplemented with fetal bovine serum-containing medium and allowed to grow for additional 10 days. The colonies were then fixed with methanol and stained with crystal violet. C, cell viability of Tca8113 cells harvested 24, 48, and 72 h post-transfection after treatment with siRACK1-231 and three different controls. siNC, siRNA for negative control. Error bars represent ±S.D. of the mean.