Wang Wan-Jen, Ho Pei-Chuan, Nagarajan Ganesan, Chen Yu-An, Kuo Hsiang-Ling, Subhan Dudekula, Su Wan-Pei, Chang Jean-Yun, Lu Chen-Yu, Chang Katarina T, Lin Sing-Ru, Lee Ming-Hui, Chang Nan-Shan
Laboratory of Molecular Immunology, Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan.
Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan 70101, Taiwan.
Cancers (Basel). 2019 Nov 19;11(11):1818. doi: 10.3390/cancers11111818.
Membrane hyaluronidase Hyal-2 supports cancer cell growth. Inhibition of Hyal-2 by specific antibody against Hyal-2 or pY216-Hyal-2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal-2. Alternatively, WWOX undergoes self-polymerization and localizes in the cell membrane. Proapoptotic pY33-WWOX binds Hyal-2, and TGF-β induces internalization of the pY33-WWOX/Hyal-2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14-WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface-exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3-dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7-21 peptide, or truncation to 5-amino acid WWOX7-11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time-lapse microscopy revealed that WWOX7-21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33-WWOX, downregulation of prosurvival pERK, prompt increases in Ca influx, and disruption of the IkBα/WWOX/ERK prosurvival signaling. In contrast, pS14-WWOX7-21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib-mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14-WWOX significantly enhanced the ceritinib-induced apoptosis. Together, the -terminal epitopes WWOX7-21 and WWOX7-11 are potent in blocking cancer growth in vivo. WWOX7-21 and WWOX7-11 peptides and pS14-WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.
膜透明质酸酶Hyal-2支持癌细胞生长。用抗Hyal-2或pY216-Hyal-2的特异性抗体抑制Hyal-2可导致体内癌症生长受抑和预防。通过免疫电子显微镜观察，肿瘤抑制因子WWOX部分通过Hyal-2锚定在细胞膜上。或者，WWOX会发生自聚合并定位于细胞膜。促凋亡的pY33-WWOX与Hyal-2结合，转化生长因子-β（TGF-β）诱导pY33-WWOX/Hyal-2复合物内化至细胞核从而导致细胞死亡。相反，当WWOX中pY33下调而pS14上调时，pS14-WWOX在体内支持癌症生长。在此，我们研究了膜结合型WWOX是否通过表面暴露的表位（尤其是在S14区域）接收细胞外信号，这些信号可抑制和预防癌症生长。通过使用模拟的三维结构并生成特异性抗体，确定了WWOX在氨基酸#7至21以及#286至299处的表位。合成的WWOX7-21肽或截短至5个氨基酸的WWOX7-11可显著抑制和预防小鼠黑色素瘤和皮肤癌细胞的生长与转移。延时显微镜观察显示，WWOX7-21肽可通过色瑞替尼强力增强4T1乳腺癌干细胞球的破裂和死亡。这是由于促凋亡的pY33-WWOX迅速上调、促生存的pERK下调、钙离子内流迅速增加以及IkBα/WWOX/ERK促生存信号通路被破坏。相反，pS14-WWOX7-21肽在体内显著增加癌症生长，并在体外保护癌细胞免受色瑞替尼介导的凋亡，这是由于ERK磷酸化延长所致。此外，抗pS14-WWOX的特异性抗体显著增强了色瑞替尼诱导的凋亡。总之，N端表位WWOX7-21和WWOX7-11在体内有效阻断癌症生长。WWOX7-21和WWOX7-11肽以及pS14-WWOX抗体在体内抑制和预防癌症生长方面具有治疗价值。
