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Identification of differentially expressed genes in a renal cell carcinoma tumor model after endostatin-treatment
Jens van Wijngaarden1, Karien de Rooij, Ermond van Beek
1Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands.
Abstract:
Endostatin is a cleavage product of collagen XVIII that has shown to inhibit tumor-angiogenesis in experimental tumor models. At present, the exact molecular mechanism of action of endostatin is not completely elucidated. In this study, we wanted to identify specific target genes of endostatin. For this purpose, the human renal cell carcinoma RC-9 was subcutaneously implanted in nude mice and treated with endostatin. Tumor growth was inhibited by endostatin after 4 days of treatment. Using immunohistochemistry and the hypoxia marker pimonidazole, we demonstrate disintegration of blood vessels and hypoxia and anoxia as a result of the treatment. Hereafter, we applied the polymerase chain reaction (PCR)-based subtractive suppression hybridization (SSH) method, together with the mirror orientation selection (MOS) technique to identify specifically induced and suppressed genes after endostatin-treatment. We found eight genes to be specifically induced and 11 to be suppressed by the endostatin-treatment. Among other genes, core binding factor a-1/osteoblast-specific factor-2 (cbfa1/osf2) was found to be specifically suppressed by endostatin. Unexpectedly, cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor, not only in the experimental RC-9 tumor model, but in sections of human breast cancer as well. Since an effect of antiangiogenic therapy on granulocytes has been reported before, this might lead to new insights in the role of granulocytes in antiangiogenic therapy in general. In conclusion, the SSH-PCR implemented with the MOS-technique is a powerful tool to identify differentially expressed genes. Using these techniques, we have identified several target genes of endostatin, of which cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor.
Insights
Endostatin inhibits tumor growth by disrupting blood vessels. This study identified specific genes, including cbfa1/osf2, regulated by endostatin, revealing its potential impact on granulocytes in anti-angiogenic therapy.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Endostatin, a collagen XVIII cleavage product, inhibits tumor angiogenesis.
- The precise molecular mechanisms of endostatin action remain unclear.
- Identifying endostatin's target genes is crucial for understanding its anti-cancer effects.
Purpose of the Study:
- To identify specific genes induced or suppressed by endostatin treatment.
- To elucidate the molecular targets of endostatin in renal cell carcinoma.
- To investigate the role of endostatin in modulating tumor microenvironment and immune cells.
Main Methods:
- Subcutaneous implantation of human renal cell carcinoma (RC-9) in nude mice.
- Treatment with endostatin, followed by immunohistochemistry and hypoxia marker analysis.
- Polymerase chain reaction (PCR)-based subtractive suppression hybridization (SSH) with mirror orientation selection (MOS) to identify differentially expressed genes.
Main Results:
- Endostatin treatment inhibited tumor growth and caused blood vessel disintegration, hypoxia, and anoxia.
- SSH-PCR identified eight induced and eleven suppressed genes in response to endostatin.
- Core binding factor a-1/osteoblast-specific factor-2 (cbfa1/osf2) was significantly suppressed by endostatin.
- cbfa1/osf2 was found to be specifically expressed in tumor-associated granulocytes in both experimental and human breast cancer models.
Conclusions:
- SSH-PCR combined with MOS is an effective method for identifying differentially expressed genes.
- Endostatin targets specific genes, including cbfa1/osf2, which is expressed in granulocytes.
- These findings suggest a novel role for granulocytes in endostatin's anti-angiogenic therapy, warranting further investigation.