Gene Diagnosis and Therapy of Salivary Gland Tumors Targetted for FGFR genes

针对FGFR基因的唾液腺肿瘤的基因诊断和治疗

• 批准号: 11557161
• 负责人: OKAMOTO Tetsuji
• 金额: $ 8.58万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557161/
• 关键词: Salivary Gland Tumors; KGFR; FGFR2-IIIb; FGFs; MAPK; Apoptosis; Differentiation; Gene Diagnosis; Gene Therapy; FGF; FGF受容体; 唾液腺腫瘍; MAPK

We have previously reported that normal human salivary gland-derived epithelial cells exclusively express keratinocyte growth factor receptor (KGFR). In the process of malignant transformation of human salivary gland tumors KGFR gene expression disappeared concomitant with the de novo expression of the fibroblast growth factor receptor 1 (FGFR 1) and FGFR4 genes.In the present study, we introduced wild-type KGFR cDNA or chimeric KGFR/FGFR 1 cDNA, which encoded the extracellular domain of KGFR and the intracellular domain of FGFR 1, into the human salivary adenocarcinoma cell line HSY.Following results were obtained.1. The growth of HSY_<R2-IIIb> in serum-free medium was significantly decreased compare to that of HSYzeo. FGF-2 exhibited no growth stimulation on HSY_<R2-IIIb> although FGF-1 stimulated the growth slightly. In addition, growth of HSY_<R2-IIIb> was stimulated by KGF for initial 3 days of culture, but then inhibited.2. TUNEL positive cells, DNA ladder formation and increase … More of CPP32/Caspase-3 activity were observed in HSY_<R2-IIIb> upon stimulation with KGF.3. FGF-1 and FGF-2 stimulated the phosphorylation of both MEK1/2 and p38 MAPK in HSY and HSYzeo. In contrast, FGF-1, FGF-2 and KGF stimulated MEK1/2 phosphorylation but not P38 and JNK/SAPK in HSY_<R2-IIIb>.4. Growth of HSY_<R2-IIIb> tumors in athymic mice were dramatically decreased compare to that of HSYzeo. Some clones of HSY_<R2-IIIb> lost their tumorigenicity. By histological examination, HSY_<R2-IIIb> tumors exhibited differentiated morphology such as acinar-like and duct-like structure.5. KGFR gene therapy starting at one week after tumor inoculation completely cured HSY tumors and that starting at 2-4 weeks significantly inhibited the growth although the tumors did not disappear. Forty eight hours after gene therapy, about 70 % of turmor cells exhibited KGFR expression in nucleus and cytoplasm, and at 4th week from gene therapy, about 20 % of tumor cells still express KGFR protein in cytoplasm and cell membrane.6. The KGFR tyrosine kinase suppressed the activity of FGF Receptor Substrate 2 (FRS2) and inhibited the growth of HSY by inducing differentiation and apoptosis in vitro and in vivo.Our results provided a novel and significant insight into the mechanism of KGFR tumor-suppression, and suggest that KGFR gene therapy might be a viable alternative to inhibiting human salivary adenocarcinoma growth. Less

我们之前报道过，正常人唾液腺来源的上皮细胞专门表达角质形成细胞生长因子受体（KGFR）。在人唾液腺肿瘤恶变过程中，KGFR基因表达消失，同时成纤维细胞生长因子受体1（FGFR 1）和FGFR4基因从头表达。在本研究中，我们引入了野生型KGFR cDNA或嵌合KGFR/FGFR 1 cDNA，其编码KGFR的胞外结构域和胞内结构域 将FGFR 1导入人唾液腺癌细胞系HSY中，得到以下结果： 1．与HSYzeo相比，HSY_<R2-IIIb>在无血清培养基中的生长显着下降。 FGF-2对HSY_<R2-IIIb>没有表现出生长刺激，尽管FGF-1稍微刺激生长。此外，在培养的前3天，HSY_<R2-IIIb>的生长受到KGF的刺激，但随后被抑制。2．在用 KGF.3 刺激后，在 HSY_<R2-IIIb> 中观察到 TUNEL 阳性细胞、DNA 阶梯形成和 CPP32/Caspase-3 活性增加。 FGF-1 和 FGF-2 刺激 HSY 和 HSYzeo 中 MEK1/2 和 p38 MAPK 的磷酸化。相反，在HSY_<R2-IIIb>.4中，FGF-1、FGF-2和KGF刺激MEK1/2磷酸化，但不刺激P38和JNK/SAPK。与HSYzeo相比，无胸腺小鼠中HSY_<R2-IIIb>肿瘤的生长显着降低。 HSY_<R2-IIIb>的一些克隆失去了致瘤性。组织学检查HSY_<R2-IIIb>肿瘤呈现腺泡样、导管样结构等分化形态。5．肿瘤接种后1周开始的KGFR基因治疗完全治愈了HSY肿瘤，2-4周开始的KGFR基因治疗显着抑制了生长，但肿瘤没有消失。基因治疗后48小时，约70%的肿瘤细胞在细胞核和细胞质中表达KGFR蛋白，基因治疗后第4周，约20%的肿瘤细胞在细胞质和细胞膜中仍表达KGFR蛋白。 6． KGFR 酪氨酸激酶抑制 FGF 受体底物 2 (FRS2) 的活性，并通过诱导体外和体内分化和凋亡来抑制 HSY 的生长。我们的研究结果为 KGFR 肿瘤抑制机制提供了新颖且重要的见解，并表明 KGFR 基因治疗可能是抑制人类唾液腺癌生长的可行替代方案。较少的

项目成果

Furue, M., Okamoto, T., Asajima, M.and Sato, J.D.: "Effects of hepatocyte growth factor and activin A on the three dimensional growth and morphogenesis of rat submandibulargland epithelial cells enbedded in collagen gels."In Vitro Cell.Dev.Biol.. 35. 131-

Furue, M.、Okamoto, T.、Asajima, M. 和 Sato, J.D.：“肝细胞生长因子和激活素 A 对胶原凝胶中包埋的大鼠下颌下腺上皮细胞的三维生长和形态发生的影响。”体外细胞。

Furue, M., Okamoto, T.and Asajima, M.: "Effect of transforming growth factor-β(TGF-β) on morphogenesis in rat salivary gland-derived RSMG-1 cells."Tissue Culture Research Communications. 18. 339-343 (1999)

Furue, M.、Okamoto, T. 和 Asajima, M.：“转化生长因子-β (TGF-β) 对大鼠唾液腺来源的 RSMG-1 细胞形态发生的影响。”《组织培养研究通讯》18。339 -343 (1999)

HAYASHIDOU, Y., et al.: "Fibroblasts enhance the ability of oral aquamous cell carcinoma cells to activate matrix metalloproteinase-2 by inducing the expression of membrane type 1 matrix metalloproteinase."Cancer Letter. (in press). (2000)

HAYASHIDOU, Y. 等人：“成纤维细胞通过诱导膜 1 型基质金属蛋白酶的表达，增强口腔水状细胞癌细胞激活基质金属蛋白酶 2 的能力。”Cancer Letter。

Furue,M.,Okamoto,T.,Asajima,M.and Sato,J.D.: "Effects of hepatocyte growth factorand activin A on the three dimensional growth and morphogenesis of rat submandibulargland epithelial cells embedded in collagen gels."In Vitro Cell.Dev.Biol.. 35. 131-135 (20

Furue, M.、Okamoto, T.、Asajima, M. 和 Sato, J.D.：“肝细胞生长因子和激活素 A 对胶原凝胶中包埋的大鼠下颌下腺上皮细胞的三维生长和形态发生的影响。”In Vitro Cell.Dev

Ogata,K.,Michimukai,E.,Sakamoto,A.,Ozaki,T.,Okamoto,T.: "Nevoid basal cell carcinoma syndrome with a palmer epidermoid cyst and jaw cyst."British J.Dermatology. (印刷中).

Ogata, K.、Michimukai, E.、Sakamoto, A.、Ozaki, T.、Okamoto, T.：“伴有帕尔默表皮样囊肿和颌囊肿的痣样基底细胞癌综合征”（出版中）。 。