Identification of transgelin-2 as a biomarker of colorectal cancer by laser capture microdissection and quantitative proteome analysis

Abstract

To search for potential protein markers of colorectal cancer (CRC), the changes in protein expression levels between microdissected tumor cells and normal mucosa epithelia were analyzed by an acetylation stable isotopic labeling method coupled with linear quadrupole ion trap fourier transform mass spectrometry (LTQ-FTMS). In total, 137 proteins were up-regulated or down-regulated significantly in cancer by at least two-fold. Based on gene ontology analysis, the largest part of differential proteins were unknown for both subcellular localization and biological process. In particular, the significant up-regulation of transgelin-2 (TAGLN2) in CRC was validated by Western blot analysis and further evaluated by immunohistochemistry in paired tumor and normal mucosa samples from 120 consecutive CRC patients, 20 adenomas, and eight synchronous hepatic metastases of CRC. TAGLN2 expression was frequently observed in cancer cells, precancerous lesions, and hepatic metastases, whereas in normal epithelia expression was rarely observed. The overexpression of TAGLN2 was associated with lymph node and distant metastasis, advanced clinical stage (P < 0.001), and shorter overall survival in CRCs. Cox regression analysis indicated that high tumor-TAGLN2 expression represents an independent prognostic factor. Consequently, over-expression of TAGLN2 may serve as a new biomarker for predicting progression and prognosis of CRC.

Figure 1

Quantification and validation of protein over‐expression in pooled colorectal cancer (CRC) samples. (a) The expression level of transgelin‐2 (TAGLN2) was quantified to be four‐fold higher by calculating the areas under the monoisotopic peaks of the heavy isotopic (D3) versus light isotopic (D0) peptides. (b) Western blot analysis confirmed the over‐expression of TAGLN2 in tumor samples. The results of the Western blot for solute carrier family 12 member 2 (SLC12A2) and ras‐related protein Rab‐10 (RAB10) were also consistent with the D0/D3 acetylation analysis.

Figure 2

Expression of transgelin‐2 (TAGLN2) in 20 paired samples of colorectal cancer (CRC) (T) and normal mucosa (N). The intensity of each band was measured using the ImageQuant image analysis system and normalized against the signal from β‐actin. (a) The expression of TAGLN2 was significantly up‐regulated in tumor tissues compared with normal mucosa. (b) The difference of TAGLN2 expression between (T) and (N) was evaluated using the Satterthwaite’s approximate t‐test.

Figure 3

Immunohistochemical staining of transgelin‐2 (TAGLN2) in tissue specimens. (a,b) Negative staining or weak staining in normal mucosa epithelia; (c) positively stained villous adenoma and abutting negative normal mucosa epithelia; (d) positive staining in hepatic metastasis of colorectal cancer (CRC); (e) positively stained CRC tumor cells and abutting negative normal mucosa epithelia; (f,g,h) positive staining in tumor cells: weak (f), moderate (g), and strong staining (h). Some microvessel walls in the stroma also stained positive for TAGLN2 (a, b, d, arrowheads). Magnification, ×200 (a,b,d,f,g,h); ×100 (c,e).

Figure 4

Kaplan–Meier survival curves for colorectal cancer (CRC) patients according to the expression levels of transgelin‐2 (TAGLN2). The P‐value was determined using log‐rank testing.