Regulation of chemokine receptor expression profiles in head and neck cancer

Autor: Da-Liang Ou, 歐大諒
Rok vydání: 2007
Druh dokumentu: 學位論文 ; thesis
Popis: 95
Part 1 Nasopharyngeal carcinoma is an epithelial cancer that metastasizes predictably to cervical lymph nodes or distant organs. To assess whether the chemokine receptors of NPC cells play important roles in metastasis and are associated with history of radiotherapy. We analyzed the significance of various chemokine receptors (CCR1-10, CXCR1-6, XCR1, and CX3CR1) in five NPC cell lines (TW01, TW04, HONE1, BM1, and AS1) and 52 NPC tumor biopsies from 48 NPC patients. We found that only CCR7, CCR9, CXCR4 and CXCR6 showed substantial amount of messenger RNA in these NPC cell lines. Quantitative real-time reverse transcription-PCR revealed that heterogeneous expression of CCR7, CCR9, CXCR4 and CXCR6 mRNA in above NPC cell lines, subsequent flow cytometric analysis, chemotaxis assay and actin polymerization assay indicated surface receptors CCR7, CXCR4 and CXCR6 were functional in NPC cells. Surprisingly, CCR9 protein was expressed in cytoplasm but not on membrane of the NPC cells. Negative immunoreactivity for CCR7, CXCR4, and CXCR6 was demonstrated in almost all nasopharyngeal (NP) specimens from patients with primary NPC (n = 12) and in those with regional metastatic NPC (n = 15). However, expression of two or three of these chemokine receptors was demonstrated in NP specimens from patients with liver metastasis. Strong positivity was demonstrated for all three of these chemokine receptors in almost all of the regional and distant metastasis specimens. Significant differences in the expression of CCR7, CXCR4, and CXCR6 were found between primary tumours and metastases (p < 0.001, p < 0.001, and p < 0.002, respectively). This observation was further confirmed by laser capture microdissection of freshly frozen tumours from primary (n = 5) and metastatic (n = 8) NPC sites (p = 0.05, 0.03, and 0.03 for CCR7, CXCR4, and CXCR6, respectively). In addition, significant differences in CXCR4 expression were demonstrated between de novo and post-radiotherapy groups (1/22 vs. 5/8; p < 0.003). It appears reasonable to conclude, therefore, that CCR7, CXCR4, and CXCR6 are expressed and active in human NPC metastases, while CXCR4 expression is associated with history of radiotherapy. Furthermore, we investigated whether CXCR4 on NPC cells (TW01, HONE1, BM1, and AS1) were undergone epigenetic regulation. Then, methylation-specific PCR (MSP) was performed to confirm methylation status of CXCR4 gene promoter. We found that the 5’ CpG islands of CXCR4 gene was unmethylated in AS1 cells, whereas promoter hypermethylation was detected in 3 NPC cell lines. Finally, NPC Cells were treated with a histone deacetylase inhibitor (Trichostatin A) and a demethylating agent (5-Aza-2’-deoxycytidine). Then, quantitative real-time RT-PCR, immunohistochemistry, and Western blot analysis were used to assess CXCR4 mRNA and protein expression induced by TSA and 5-Aza in NPC cell lines. We found that CXCR4 expression was increased in NPC cell lines treated with TSA, or both TSA and 5-Aza. These findings demonstrated that epigenetic regulation of CXCR4 in NPC cell lines. Part 2 Twist has been found to be correlated with metastasis in various cancers including hepatocarcinoma, breast and prostate cancers. However, the role of Twist in head and neck squamous carcinoma (HNSCC) remains unknown. The purpose of this study is to investigate the expression and possible role of Twist in HNSCC. We examined CH1 tissue microarrays (TMAs) which composed of tumors from 50 patients with head and neck tumor. Immunohistochemical (IHC) stain analysis of CXCR4, CXCR6, CCR7, Twist, E-cadherin, Fibronectin and Osteopontin were performed, and the relationship between the staining pattern and clinicopathological features were studied. Out of the 50 cases of head and neck cancer, 20 (40.0%), 25 (50.0%), 25 (50.0%), 20 (40.0%), 25 (50.0%), 15 (30.0%) and 36 (72.0%) showed positive staining in the tumors cells for the Twist, CXCR4, CXCR6, CCR7, E-cadherin, Fibronectin and Osteopontin proteins, respectively. Further, Twist expression was positively associated with differentiation status (p= 0.027), lymph node metastasis (p= 0.032) and disease progression (p= 0.029). Besides, CCR7 and CXCR4 expression were also associated with lymph node metastasis. Further analysis revealed that the expression of Twist was positively correlated with CXCR4 (Spearman, r=0.408, p= 0.003) and CCR7 (r=0.417, p= 0.003) in tissue microarray experiment. Furthermore, down-regulation of Twist through short hairpin RNA (sh-RNA) was prepared in head and neck squamous carcinoma HONE1 cells. Quantitative RT-PCR, Western blotting and Immunocytochemical staining analysis were used to evaluate the effect of Twist sh-RNA. We found that knockdown of Twist by short hairpin RNA (sh-RNA) in HONE1 cells showed decreased mRNA and protein expression of CXCR4 and CCR7. This study demonstrated that the transcription factor Twist could regulate the CXCR4 and CCR7 expression in squamous cell carcinoma, which might lead to lymph node metastasis potential.
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