Identification of secernin 1 as a novel immunotherapy target for gastric cancer using the expression profiles of cDNA microarray

Abstract

Despite the discovery of multiple TAAs, only a limited number is available for clinical application, particularly against epithelial malignancies. In this study we searched for novel TAAs using expression profiles of gastric cancer examined with cDNA microarray, and identified the SCRN1 gene as a candidate. SCRN1 was confirmed to be expressed in five out of seven gastric cancers with semiquantitative RT-PCR. With Northern blot analysis, it was detected abundantly in the testis and ovary, but it was barely detectable in 14 other normal human adult organs. Colony formation assay revealed that its augmented expression is associated with promoted cell growth. As these expression profiles and functional features of SCRN1 appeared to be compatible with the characteristics of the hypothesized ideal TAAs, we examined whether SCRN1 protein contains antigenic epitope peptides restricted to HLA-A*0201. We synthesized the candidate peptides derived from SCRN1, and tried to induce CTLs with each peptide. The CTL clones were successfully induced with a peptide SCRN1-196 (KMDAEHPEL), and they lyzed not only the peptide-pulsed targets but also the tumor cells expressing both SCRN1 and HLA-A*0201 endogenously. These results strongly suggest that SCRN1-196 is an epitope peptide restricted to HLA-A*0201. Furthermore, we synthesized an anchor-modified peptide SCRN1-9 V (KMDAEHPEV), in which leucine at position 9 was substituted for valine to increase the binding affinity to the HLA-A*0201 molecules. The CTL clones induced by SCRN1-9 V also recognized tumor cells expressing its natural SCRN1 protein endogenously. These results strongly suggest that SCRN1 is a novel TAA and these peptides, both native and modified, may be applicable for cancer vaccines to treat gastric cancer.

Figure 1

Expression of SCRN1 in gastric cancers. (a) Expression of SCRN1 in gastric cancers measured by cDNA microarray. Relative expression ratio (cancer/non‐cancer) of SCRN1 was shown in the 11 primary gastric cancers examined by cDNA microarray. Upregulated expression (Cy3/Cy5 intensity ratio >2.0) was observed in nine of the 11 gastric cancers that passed through the cut‐off filter. (b) Expression of SCRN1 in seven gastric cancer tissues (T) and the corresponding normal tissues (N), as measured by semiquantitative RT‐PCR. Expression of GAPDH serves as an internal control.

Figure 2

Expression of SCRN1 in normal human tissues and gastric cancer cell lines, and its effect on cell growth. (a) Northern blot analysis of SCRN1 on multiple tissue. The transcript of SCRN1 is approximately 4.9 kb in size. Expression of β‐actin served as a control. (b) Expression of SCRN1 in gastric cancer cell lines by semiquantitative RT‐PCR. Expression of GAPDH serves as a control. (c) Colony formation assays showed that SCRN1 promoted cell growth in NIH3T3 cells. Cells transfected with mock or plasmids expressing SCRN1 were fixed, and subsequently stained with Giemsa solution. Similar results were obtained in three experiments.

Figure 3

Cytotoxicities of cytotoxic T lymphocyte (CTL) clones induced with SCRN1‐196 peptide. Cytotoxicities of CTLs were examined using a 4 h ⁵¹Cr release assay. (a) Cytotoxicities to T2 cells (HLA‐A*0201) pulsed with SCRN1‐196 (closed square) or control (open square) peptides. (b) Cytotoxicities to SNU475 (both SCRN1 and HLA‐A*0201 positive), Kato III cells (SCRN1 negative, HLA‐A*0201 positive), and MKN‐45 cells (SCRN1 positive, HLA‐A*0201 negative). CTL Clone‐23 induced with SCRN1‐196 showed potent cytotoxicities against not only T2 cells pulsed with SCRN1‐196 but also SNU475 cells. There was no significant cytotoxicity against Kato III cells or MKN45 cells, neither of which express HLA‐A*0201 and SCRN1 simultaneously. (c) A cold target inhibition assay. ⁵¹Cr‐labeled SNU475 cells were used as hot targets at an effector/target (E/T) ratio of 20. T2 cells pulsed with SCRN1‐196 (closed square) or control (open square) peptides were used as a cold target. The number of target cells is fixed to 1 × 10⁴/well. We added T2 cells to SNU475 cells in each microculture at various cold target/hot target ratios, and examined cytotoxic activity. T2 cells inhibited cytotoxic activity against SNU475 cells only when pulsed with SCRN1‐196 peptides. (d) Antibody blocking assay. The cytotoxicity against SNU475 cells was inhibited by anti‐CD8 monoclonal antibody or anti‐HLA‐class I monoclonal antibody. Specific lysis of SNU475 by CTLs in the presence of an isotype‐matched control Ig was 23%. E/T ratio: 20.

Figure 4

Cytotoxicities of cytotoxic T lymphocyte (CTL) clones induced with SCRN1–9 V, modified from native SCRN1‐196 peptide. Cytotoxicities of the CTL Clone‐16, which were induced with SCRN1–9 V, were tested against (a) peptide‐pulsed T2 cells (HLA‐A*0201) and (b) tumor cell lines using 4 h ⁵¹Cr release assay. CTL Clone‐16 recognized not only SCRN1–9 V (closed triangle) but also the parental peptide SCRN1‐196 (closed square). This CTL clone killed SNU475 cells expressing both the SCRN1 gene and the HLA‐A*0201 molecule. E/T, effector/target.