Abstract
Background/Aim: In previous work, we found that prostate stem cell antigen (PSCA) gene, encoding a glycosylphosphatidylinositol-anchored protein, is a presumable tumor suppressor in gastric cancer and gallbladder cancer (GBC). The introduction of PSCA cDNA into GBC cell lines significantly suppressed tumorigenecity of cells in mice. The PSCA protein is thought to be involved in some form of intracellular signaling that remains to be elucidated. Materials and Methods: Using microarrays, we conducted gene-expression profiling on tumors generated by a GBC cell line TGBC-1TKB, with and without expression of PSCA, which was implanted into mice. Genes whose expression was down-regulated by PSCA were selected, and their down-regulation was confirmed by real-time PCR. Results: We identified several immune-related genes down-regulated by PSCA, including interleukin 8 (IL8), IL1 receptor antagonist (IL1RN) and S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9). Conclusion: PSCA signaling may suppress tumor growth in vivo by modulating immunological characteristics of GBC cells.
- Prostate stem cell antigen
- GPI-anchored protein
- intracellular signaling
- gallbladder cancer
- tumor suppressor
Originally, prostate stem cell antigen gene (PSCA), encoding a glycosylphosphatidylinositol (GPI)-anchored membrane protein, was identified as a gene up-regulated in prostate cancer (1), and its up-regulation was later demonstrated in other types of cancers, including urinary bladder cancer, renal cell carcinoma, pancreatic cancer, non-small cell lung cancer, hydatidiform mole, and ovarian mucinous tumor, where PSCA is thought to act in tumor progression (2-8).
On the other hand, PSCA may act as a tumor suppressor. We previously conducted a genome-wide association study to identify gastric cancer susceptibility genes and found an association between gastric cancer and PSCA (9). The gene was down-regulated in gastric cancer and had a cell growth-inhibitory effect on cultured gastric cancer cells (9). We also observed down-regulation of the gene in gallbladder cancer (GBC) and cell growth-inhibitory activity of the gene on GBC cells in vitro and in mice (10). This activity was attenuated by non-synonymous single nucleotide polymorphism (SNP) rs2294008 that changes the first codon for methionine to threonine, resulting in truncation of the first nine amino acids (11).
It is thought that PSCA protein is located in a lipid raft on the outer surface of the cell membrane, which is a special microdomain enriched in glycosphingolipids, cholesterol and other lipidated proteins, and that it has some function in intracellular signal transduction as a GPI-anchored protein (12), although its biological function has not been precisely revealed.
In the present study, in order to identify genes whose expression is modified by PSCA signaling that acts to inhibit cell growth, we conducted gene-expression profiling of GBC cells implanted into mice, of which tumor formation was suppressed by PSCA (10, 11).
Materials and Methods
Tumor tissues. TGBC-1TKB tumors expressing PSCA sense or antisense strand were obtained by implantation of TGBC-1TKB, a cell line established from poorly differentiated tubular adenocarcinoma (13, 14), into SCID mice in our previous study (11), and, in same manner, G-415 tumors expressing PSCA sense or antisense strand were obtained by implantation of G-415, a cell line established from undifferentiated-type of GBC (15, 16), in our previous study (10). TGBC-1TKB and G-415 cells were provided by RIKEN BioResource Center (Tsukuba, Japan) and by Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer of Tohoku University (Sendai, Japan), respectively. The cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Wako Pure Chemical Industries, Osaka, Japan). The cell lines with/without stable PSCA expression were prepared by introducing sense or antisense cDNA of human PSCA into the cells (9), followed by G418 selection (Geneticin, WAKO, at 250 μg/ml and 130 μg/ml for TGBC-1TKB and G-415, respectively, in DMEM). After checking the PSCA expression status, we subcutaneously injected the cell lines without PSCA expression (Control) and the cell lines with PSCA expression, respectively, into 3 sites (1×106 cells per injection site) in the back skin per mouse (CB17 / Icr-Prkdc <scid> / CrlCrlj Genotype: scid / scid). For each cell line, the assay was performed on 2 mice for sense strand, and 2 mice for antisense strand, from which one mouse was selected for control in this study. Mice implanted with TGBC-1TKB and G-415 were sacrificed at 91 and 35 days after implantation (10, 11), respectively, and the tumors were surgically excised and frozen immediately after sacrification.
TaqMan Gene Expression Assay ID
Microarray expression analyses and real-time reverse transcriptase-polymerase chain reaction (PCR). Total RNA was extracted from tumor tissues with ISOGEN (Wako). Each RNA sample was derived from three tumors generated in each mouse (10, 11). Microarray expression analyses were performed with GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA), following a standard protocol recommended by the manufacturer. After converting about 2 μg of total RNA to the first-strand cDNA with High-Capacity cDNA Reverse Transcription Kit (Life Technologies Japan, Tokyo, Japan), quantitative RT-PCR was performed by TaqMan Gene Expression Assay (Life Technologies Japan, Applied Biosystems Assay ID for each gene is shown in Table I). RT-PCR was conducted for 40 cycles of 95°C for 15 s and 60°C for 60 s, by ABI PRISM 7900HT Sequence Detection System (Life Technologies Japan). The relative transcript level was calculated using the Ct value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript as reference.
Results
Expression analyses revealed down-regulation of 17 genes in PSCA-expressing tumors generated in mice. We previously conducted tumor-formation assays in mice using the GBC cell line TGBC-1TKB expressing sense- or antisense-strand of PSCA cDNA, which revealed that the sense strand harboring the T allele of rs2294008 significantly suppressed tumor growth compared to the anti-sense strand (11). In this study, we isolated RNA from the tumors generated in the mice (one mouse implanted with GBC cells expressing PSCA antisense strand and two mice implanted with GBC cells expressing sense strand), and performed gene-expression profiling using microarrays. As a result, we found 18 genes down-regulated in the tumors generated by the cells expressing the sense strand. These genes expressed one-fifth or less transcripts compared to those in the tumors generated by the cells expressing antisense strand: peptidase inhibitor 3, skin-derived (PI3); S100 calcium-binding protein A9 (S100A9); S100A8; S100P; interleukin 8 (IL8); Interleukin-1 receptor antagonist (IL1RN); keratin 6A (KRT6A); serpin peptidase inhibitor, clade B, member 1 (SERPINB1); SERPINB2; MAX dimerization protein 1 (MXD1); gap junction protein, beta 6 (GJB6); calcium/calmodulin-dependent protein kinase II inhibitor 1 (CAMK2N1); sciellin (SCEL); alpha-2-macroglobulin-like 1 (A2ML1); kallikrein-related peptidase 8 (KLK8); lipocalin 2 (LCN2); kynureninase (KYNU); and FLJ20073 encoding hypothetical protein (Table II).
We performed real-time PCR analyses on these genes, except for FLJ20073 as no probe was available for the TaqMan Gene Expression Assay, which revealed their down-regulation (Figure 1). The gene-expression profiling using microarrays showed some of the genes were expressed in TGBC-1TKB cells expressing the PSCA antisense strand, that were maintained in petri dishes before implantation (Table II); however, real-time PCR revealed that the 17 genes were not down-regulated in vitro in TGBC-1TKB cells expressing the PSCA sense strand compared to those expressing the PSCA antisense strand (data not shown).
As we previously performed a tumor-formation assay in mice using another GBC cell line, G-415, and observed the tumor-formation inhibitory effect of PSCA on this cell line (10), we examined expression of the 17 genes in the tumors (one tumor expressing the antisense strand and two expressing the sense strand) obtained in the previous study. As a result, real-time PCR demonstrated the apparent down-regulation of at least 13 out of the 17 genes: PI3, S100A9, IL8, IL1RN, KRT6A, S100A8, MXD1, GJB6, SCEL, S100P, LCN2, KYNU and SERPINB1 (Figure 2).
Discussion
We previously demonstrated that PSCA has an inhibitory effect on both in vitro cell proliferation and in vivo tumor formation of TGBC-1TKB and G-415 GBC cell lines (10, 11). Because it is a GPI-anchored protein, the PSCA protein is thought to have a role in intracellular signal transduction (2, 12). This study demonstrated that 17 genes are down-regulated by the presence of PSCA in the GBC cells, and they may be involved in the inhibition of tumor formation in mice. Some of the genes seemed to be expressed in cultured TGBC-1TKB cells (Table II) and their down-regulation was not observed in vitro (data not shown). The expression of the other genes was not observed in vitro but was induced in vivo. The down-regulation of the 17 genes seem to be a consequence of interaction between PSCA protein on the cell surface and some physiological factor in the mouse, such as a hormone, cytokine, immune cells etc., and the expression of these genes may be modified by PSCA signaling pathways. Our in vivo functional studies with gene-expression profiling offered novel information on PSCA signaling.
Down-regulation of 17 genes in tumors generated by implantation of Prostate stem cell antigen (PSCA)-expressing TGBC-1TKB cells into mice. Down-regulation of the genes was confirmed by real-time polymerase chain reaction (PCR), with statistical significance between the antisense-expressing cells (Anti) and each of the sense-expressing cell lines (S1 and S2) (p<0.05). Bar: Standard deviation.
Down-regulation of 13 out of 17 genes in tumors generated by implantation of Prostate stem cell antigen (PSCA)-expressing G-415 cells into mice. Down-regulation of the genes was demonstrated by real-time PCR, with statistical significance between the antisense-expressing cells (Anti) and each of the sense expressing cell lines (S1 and S2) (p<0.05) except for S2 in the assay for CAMK2N, SERPINB2 and A2ML1 (indicated by asterisks). The Ct value for KLK8 was not determined because of low or no expression of the gene in the tumors. Bar: Standard deviation.
On the whole, this study demonstrated that immune- or inflammation-related genes are down-regulated in tumors by PSCA. Accumulating evidence suggests that IL8, a cytokine, promotes cell proliferation, survival and migration of cancer cells via its autocrine and paracrine activities, and it elicits an angiogenic response in endothelial cells (17). IL1RN encodes an endogenous inhibitor for an IL1 receptor. Although its role in carcinogenesis has not been disclosed, the association of IL1RN gene polymorphism with a variety of cancer types, including gastric cancer, has been demonstrated (18), and it was reported that the expression level of IL1RN is correlated with metastasis of gastric cancer (19). SERPINB1 is an endogenous inhibitor of serine proteases, important for homeostatic expansion of IL17+ γδ and CD4+ Th17 cells, and for neutrophil survival (20, 21). S100A8 and S100A9 proteins exist as homo- and heterodimers (S100A8/A9) and the heterodimer mediates several pro-tumor responses (22). Remarkably, the S100A8/A9 dimer secreted from tumor cells recruits myeloid-derived suppressor cells that protect tumor cells from an anticancer immunological response in the cancer microenvironment (23, 24). PI3 protein has an immunomodulatory property and inhibits an inflammatory response (25). It was recently reported that PI3 expression is correlated with poor prognosis of high-grade serous ovarian carcinoma and basal-like breast cancer (26). LCN2, encoding an iron-trafficking protein, was recently recognized as an innate immune protein, and its involvement is suggested in several diseases such as encephalomyelitis and Alzheimer's disease (27, 28). The protein also contributes to carcinogenesis by promoting epithelial–mesenchymal transition and by increasing iron metabolism required for vigorous cell proliferation (29). In addition to this study, there is a study demonstrating the involvement of PSCA in the regulation of immune-related genes. The study revealed that silencing of PSCA in a bladder cancer cell line using short hairpin RNA up-regulates genes of the IL1 signaling pathway (30).
Genes down-regulated in prostate stem cell antigen gene (PSCA)-expressing cell lines and tumors.
The finding in PSCA knock-out mice is informative when considering PSCA function in the context of immune reaction (31). Mice showed no malignancies in the organ where PSCA was expressed, even with ageing or exposure to sublethal radiation. However, when the mice were cross-bred to TRAMP mice, a mouse model of prostate cancer with prostate-restricted expression of SV40 large T antigen, the PSCA−/−TRAMP mice developed prostate tumors in the same way as PSCA+/+TRAMP mice; however, the former had a higher rate of metastasis. These findings suggest the contribution of PSCA to inhibition of metastasis, which may be through modifying the immunological characteristics of cancer cells.
Some of these 17 genes are known to be involved in cancer progression. S100P proteins form a dimer in a calcium-dependent manner and bind to proteins, such as Ezrin (EZR) and Receptor for advanced glycation endproducts (RAGE), which have a role in cancer progression (32). Expression of KLK8 has been found in several types of cancer including lung cancer (33), but its contribution to carcinogenesis is yet to be elucidated. A recent study revealed that SERPINB2 contributes to brain metastasis of lung and breast cancer (34).
On the contrary, some of the genes have been reported for their tumor-suppressive function. MXD1 is known as a tumor suppressor regulating oncogenic activity of MYC gene (35). CAMK2N1, it was reported, inhibits prostate cancer through reducing cellular proliferation, arresting cells in G0/G1 phases and inducing apoptotic cell death (36). KRT6A encodes cytokeratin and loss of its expression is correlated to poor prognosis of breast cancer (37).
The remaining genes are those whose relation to carcinogenesis has not been reported. GJB6 encodes a gap junction protein and its mutation results in deafness (38). SCEL protein is one of the components for the confined envelope which is an insoluble protein structure formed under the plasma membrane of epithelial cells (39). A2ML1 encodes a member of the alpha-macroglobulin superfamily, which acts as an inhibitor for several proteases, and the protein was recently identified as an antigen of autoimmune paraneoplastic pemphigus (40). KYNU is an enzyme that catalyzes the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic and 3-hydroxyanthranilic acids, respectively, and is involved in glioma pathophysiology (41).
Our previous studies demonstrated that PSCA is a potential tumor suppressor in gastric cancer, one of the major types of cancers worldwide, as well as in GBC. Investigation of PSCA signaling has the potential to provide important information to help reveal the mechanism of carcinogenesis and to create effective strategies for cancer prevention.
Acknowledgements
This work was supported by a JST grant for the Personalized Medicine Project and by JSPS KAKENHI (no. 23501327). We thank Ms. Misuzu Okuyama for technical support.
- Received January 28, 2015.
- Revision received February 16, 2015.
- Accepted February 17, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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