Discovery of SLC3A2 cell membrane protein as a potential gastric cancer biomarker: implications in molecular imaging

Abstract

Despite decreasing incidence and mortality, gastric cancer remains the second leading cause of cancer-related deaths in the world. Successful management of gastric cancer is hampered by lack of highly sensitive and specific biomarkers especially for early cancer detection. Cell surface proteins that are aberrantly expressed between normal and cancer cells are potentially useful for cancer imaging and therapy due to easy accessibility of these targets. Combining two-phase partition and isobaric tags for relative and absolute quantification methods, we compared the relative expression levels of membrane proteins between noncancer and gastric cancer cells. About 33% of the data set was found to be plasma membrane and associated proteins using this approach (compared to only 11% in whole cell analysis), several of which have never been previously implicated in gastric cancer. Upregulation of SLC3A2 in gastric cancer cells was validated by immunoblotting of a panel of 13 gastric cancer cell lines and immunohistochemistry on tissue microarrays comprising 85 matched pairs of normal and tumor tissues. Immunofluorescence and immunohistochemistry both confirmed the plasma membrane localization of SLC3A2 in gastric cancer cells. The data supported the notion that SLC3A2 is a potential biomarker that could be exploited for molecular imaging-based detection of gastric cancer.

Figure 1

Relative levels of SLC3A2 in MKN7 (iTRAQ 116) with respect to HFE145 (iTRAQ 114) cells as determined by electrospray ionization tandem mass spectrometry. The upper panel shows a representative tandem mass spectrum of a SLC3A2 peptide (GENSWFSTQVDTVATK) while the lower panel shows the relative peak heights of the iTRAQ labels.

Figure 2

Comparison of whole cell versus 2-phase partition analysis. (a) Comparison of the distribution of all the proteins detected in the membrane and whole cell proteomics data sets across various subcellular compartments. Classification of proteins into subcellular compartments is done via Gene Ontology. (b) Determination of the congruence of iTRAQ ratios between the entire data sets generated from whole cell and membrane analyses in terms of their relative expression between normal and gastric cancer cells. Correlation analysis was performed using the log transformation values of the available iTRAQ ratios from 392 common proteins using SPSS v10.0. Pearson correlation coefficient between whole cell lysate and membrane sample was 0.572 (p < 0.0001).

Figure 3

Classification of proteins identified through membrane proteomics into their molecular functions and biological processes. This is done via PANTHER (Protein Analysis through Evolutionary Relationships) Classification System (www.pantherdb.org/).

Figure 4

(a) Western blot analyses of the expression of selected candidates (ERBB2, ICAM1, PLCG1, SLC3A2, LAMP2, and SLC7A5) in HFE145 noncancer and MKN7 gastric cancer cells. (b) Immunoblotting of SLC3A2 in the HFE145 noncancer gastric cell versus a panel of 13 gastric cancer cell lines. (c) Immunoblotting of SLC3A2 in HFE145 noncancer and 3 gastric cancer cell lines following pretreatment with Tunicamycin.

Figure 5

Immunofluorescence of SLC3A2 in the HFE145 noncancer gastric cell, and MKN7, MKN28 and SCH cancer cells. Images were all captured at 40× magnification. SLC3A2 was observed to be localized mainly to the plasma membrane (indicated by arrows pointing to signals along the edges or borders of cells) and overexpressed in gastric cancer cell lines compared with noncancer cells.

Figure 6

Immunohistochemistry of SLC3A2 in tissue microarrays of clinical gastric samples. A total of 85 matched normal and cancer tissues plus addition 10 normal tissues were analyzed. (a) Distribution plot of the IHC scores of SLC3A2 in individual normal and gastric cancer samples. Statistically significant higher expression of SLC3A2 was observed in gastric cancer samples compared to normal gastric tissues (p < 0.01, independent sample t test). (b) Representative IHC images (10× magnification) of SLC3A2 in 2 matched gastric cancer and normal tissues as well as a table showing the frequency of upregulation and down-regulation of SLC3A2 in clinical gastric specimens. Insets contain magnified images (40×) that show clear cell surface expression of SLC3A2 in gastric cancer cells.