Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

AIMS: Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. METHODS: We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. RESULTS: We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes. CONCLUSION: This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue.

Elevated expression of notch1 is associated with metastasis of human malignancies.

The expression level of Notch1 has been studied in many primary tumor types, but has not been widely investigated in metastatic lesions from human malignancies. Using immunohistochemistry (IHC), the expression level of Notch1 was evaluated and compared between primary and metastatic tumors in 12 different cancers. The mean IHC score of Notch1 was significantly increased in metastatic hepatocellular carcinoma (HCC; 5.4 ± 0.7) and in metastatic renal cell carcinoma (RCC; 5.0 ± 2.3) compared with primary HCC (3.1 ± 0.7, P = .035) and RCC (1.3 ± 0.6, P = .049), respectively. Similarly, the expression level of Notch1 showed an increasing trend in the metastatic malignancies in the larynx, prostate, and stomach compared with corresponding primary malignancies (P values are .055, .072, and .074, respectively). The results demonstrate elevated expression of Notch1 in some metastatic tumors, suggesting that Notch1 may play an important role in the development or maintenance of metastatic lesions, and targeting of Notch1 might be a therapeutic approach against tumor metastasis.

The C-terminal common to group 3 POTES (CtG3P): a newly discovered nucleolar marker associated with malignant progression and metastasis.

A gene family expressed in prostate, ovary, testis and placenta (POTEs) is newly defined and primate-specific. POTE genes have 13 paralogs, which are dispersed in 8 chromosomes and divided into three groups. The proteins encoded by these genes contain three domains: An N-terminal, ankyrin repeats and a C-terminus. Previous studies suggest that POTE proteins are localized in the inner aspect of cellular membrane and are considered as cancer-testis antigens, because they expressed widely in cancers, but in limited benign tissues. In this study, we will study the subcellular distribution of all POTE proteins and their associations with the progress and metastasis of malignancies. By performing Immunohistochemistry, Immunocytochemistry and immunofluorescence assay on tissue microarray slides containing tissues with different pathology and origins or on cell lines, we found that the epitopes of N- and C-terminals of all detected POTEs were widely expressed in benign and malignant tissues. Among these epitopes, C-terminal common to group 3 POTEs (CtG3P) was the only portion localized in nucleoli. The nucleolar IHC scores for CtG3P was lowest in benign tissues (4.47 ± 3.43), significantly higher in localized malignancies (5.32 ± 3.36, p = 3.63E-02), and highest in metastatic malignancies (7.90 ± 2.29, p = 8.13E-12). The CtG3P was better in differentiation of benign from malignant changes, and/or in differentiation of localized from metastatic cancers as compared with Ki-67 and AgNORs. In addition, transient transfection of siRNA against mRNA of group 3 POTEs influences the growth and survival of MCF-7 cells in vitro in a dose dependent manner.

Elevated Jagged-1 and Notch-1 expression in high grade and metastatic prostate cancers.

BACKGROUND: Emerging evidence has suggested that Notch signaling pathway may be involved in the development, progression and metastasis of prostate cancer (PCa). In the present study, we investigated the expression levels of Jagged-1 and Notch-1 in human prostate tumors and their associations with PCa progression and metastasis. METHODS: Immunohistochemistry (IHC) for Jagged-1 and Notch-1 was performed on tissue microarray (TMA) slides containing 286 formalin-fixed and paraffin-embedded (FFPE) tissue specimens with various prostatic pathologies, including benign changes, high grade prostatic intraepithelial neoplasia (HGPIN), low- and high-grade PCas as well as metastatic PCa. RESULTS: Cytoplasmic and membranous IHC scores for Jagged-1 in both metastatic PCa and high grade PCa were significantly higher than those in low grade PCa and in benign prostatic tissues. Similarly, cytoplasmic IHC scores of Notch-1 in both metastatic PCa and high grade PCa were significantly elevated compared with those observed in low grade PCa and in benign prostatic tissues. A statistically significant correlation was identified between the expression of Jagged-1 and Notch-1 in human prostatic tissues. Furthermore, significantly more highly expressed Jagged-1 in membrane was observed in Caucasian patients with high-grade or metastatic PCa (vs. African Americans) and in PCa patients with positive surgical margins (vs. negative surgical margins). CONCLUSION: Our results provide strong evidence that up-regulation of Jagged1-Notch1 signaling plays a role in PCa progression and metastasis and suggest that Jagged-1 and Notch-1 may be useful markers in distinguishing indolent and aggressive PCas.