內皮唾酸蛋白(CD248)，亦稱為腫瘤內皮細胞標誌一(tumor endothelial marker 1/ TEM1或endosialin)，是一種在細胞表面上高度唾液酸醣化之穿膜醣蛋白。其表現於纖維母細胞、周細胞以及部分種類之腫瘤細胞。根據文獻報導，CD248在腫瘤進展扮演重要的角色，其會影響纖維母細胞的貼附、爬行以及增生能力，皆是與轉移息息相關之步驟。黑色素瘤為一具有高度轉移能力且極度惡性之腫瘤，但目前導致黑色素瘤高轉移能力之原因與機制仍未清楚，故無法發展有效的治療藥物。為了探究黑色素瘤細胞上內源性CD248之角色與功能，我們利用功能喪失的實驗，證實B16F10 cells內源性CD248可能參與於癌細胞轉移，例如細胞貼附於纖連蛋白之能力與細胞爬行能力，並增強其相關之訊息傳遞路徑(FAK、ERK1/2與Akt)，且亦影響腫瘤細胞構組仿血管結構的能力，以上結果皆顯示內源性CD248可能於黑色素瘤轉移中扮演重要角色。根據此現象，針對黑色素瘤轉移部份，欲達到減緩並治療之效果，我們使用了CD248重組蛋白作為一治療策略。我們假設外加的CD248重組蛋白可以與黑色素瘤細胞上內源性CD248產生競爭關係，進而影響且抑制其內源性原有相關功能，進而達到減緩轉移惡性程度的情形。如我們的假設，外加CD248重組蛋白會與細胞上內源性CD248競爭纖連蛋白結合位，使B16F10 cells其貼附於纖連蛋白之能力下降。另外，CD248重組蛋白使B16F10 cells細胞爬行能力下降，並抑制其相關之訊息傳遞路徑(FAK、ERK1/2與Akt)，且亦阻礙腫瘤細胞構組仿血管結構的能力。利用功能增益實驗確認CD248重組蛋白仍能抑制因細胞過度表現CD248而提高之爬行能力。最後，我們利用小鼠轉移動物模式，觀察到CD248重組蛋白使得黑色素瘤於肺部轉移數目下降，且使組織中CD248表現量較低以及形成較少仿血管構造。綜合上述，CD248重組蛋白可成為一治療黑色素瘤之替代性方法。

CD248 is an orphan receptor expressed by fibroblasts and several tumor cells. Our preliminary study showed CD248 plays a key role on cell adhesion and migration on fibroblasts. Melanoma is a highly metastatic skin cancer with high mortality rate. Thus, understanding the molecular mechanism underlining metastatic properties might contribute to the therapeutic strategy. In this study, knockdown of CD248 in B16F10 cells resulted in a decrease of cell-fibronectin interaction, chemotactic migration, and vasculogenic mimicry activity, and involved in signaling pathways associated with cell adhesion and migration such as FAK, ERK1/2 and Akt signaling pathways, suggesting that CD248 plays important roles in tumor metastatic behaviors. Then we proposed that recombinant CD248 protein containing extracellular domains (rCD248) could inhibit melanoma metastasis by competing with endogenous CD248 on melanoma cells. In consistent with our hypothesis, I demonstrated that rCD248 treatment significantly reduced cell-fibronectin interaction, chemotactic migration, and vasculogenic mimicry activity in vitro. Additionally, we also observed that signaling pathways associated with cell adhesion and migration such as FAK, ERK1/2 and Akt signaling pathways were also reduced by rCD248. Furthermore, overexpression of CD248 in HEK-293T cells resulted in an increase of chemotactic migration, and rCD248 treatment could inhibit this consequence. Moreover, rCD248 treatment dramatically decreased pulmonary metastasis as evident by reduction tumor nodules in the lung in vivo. Collectively, our results suggest that rCD248 could inhibit the metastasis of melanoma by competing with the endogenous CD248. Application of rCD248 may provide an alternative method to inhibit tumor metastasis.

1.Andrés Valdivia, Gabriel Mingo, Varina Aldana, et al. Fact or Fiction, It Is Time for a Verdicton Vasculogenic Mimicry? Frontiers in Oncology Volume 9 Article 680 (2019)
2.Akash Nanda, Baktiar Karim, Zhongsheng Peng, et al. Tumor endothelial marker 1 (Tem1) functions in the growth and progression of abdominal tumors. PNAS vol. 103 no. 9 3351–3356 (2006)
3.Benjamin Geiger, Alexander Bershadsky, Roumen Pankov, et al. Transmembrane extracellular matrix-cytoskeleton crosstalk. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 2 793-805 (2005)
4.Bruce R. Lester, James B. McCarthy, et al. Tumor cell adhesion to the extracellular matrix and signal transduction mechanisms implicated in tumor cell motility, invasion and metastasis. Cancer and Metastasis Reviews 11: 3144 (1992)
5.Brian Tomkowicz, Katherine Rybinski, Brian Foley, et al. Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. PNAS vol. 104 no. 46 17965–17970(2007)
6.Eiji Kiyohara, Nicholas Donovan, Ling Takeshima, et al. Endosialin Expression in Metastatic Melanoma Tumor Microenvironment Vasculature: Potential Therapeutic Implications. Cancer Microenvironment 8:111–118(2015)
7.Hong Ge, Hui Luo, et al. Overview of advances in vasculogenic mimicry – a potential target for tumor therapy. Cancer Management and Research 10 2429–2437(2018)
8.Jacob Pitcovski, Ehud Shahar, Elina Aizenshtein, et al. Melanoma antigens and related immunological markers. Critical Reviews in Oncology/Hematology S1040-8428(17)30199-3(2017)
9.Jason R. Todd, Karen A. Ryall, Simon Vyse, et al. Systematic analysis of tumour cell-extracellular matrix adhesion identifies independent prognostic factors in breast cancer. Oncotarget, Vol. 7, No. 39(2016)
10.Jingxin Zhang, Lili Qiao, Ning Liang, et al. Vasculogenic mimicry and tumor metastasis. JBUON 21(3): 533-541(2016)
11.Margarida Maia, Astrid DeVriese, Tom Janssens, et al. CD248 facilitates tumor growth via its cytoplasmic domain. BMC Cancer 11:162 (2011)
12.Michael A. Davies, MD, et al. The Role of the PI3K-AKT Pathway in Melanoma. Cancer J 18: 142Y147 (2012)
13.Kabir A. Khan , Jack L. McMurray, Fiyaz Mohammed, et al. C-type lectin domain group 14 proteins in vascular biology, cancer and inflammation. The FEBS Journal 286 3299–3332(2019)
14.Praveen Agrawal, Barbara Fontanals-Cirera, Elena Sokolova, et al. A systems biology approach identifies FUT8 as a driver of melanoma metastasis. Cancer Cell. 31(6): 804–819.e7. (2017)
15.Ruixia Huang, Einar K. Rofstad, et al. Integrins as therapeutic targets in the organ-specific metastasis of human malignant melanoma. Journal of Experimental & Clinical Cancer Research 37:92(2018)
16.S. KONTSEKOVA, K. POLCICOVA, M. TAKACOVA, et al. Endosialin: Molecular and Functional Links to Tumor Angiogenesis. Neoplasma. 63(2):183-92, 2 (2016)
17.Sandra P. D’Angelo, Omid A. Hamid, Ahmad Tarhini, et al. A phase 2 study of ontuxizumab, a monoclonal antibody targeting endosialin, in metastatic melanoma. Invest New Drugs (1):103-113. (2018)
18.Satyajit K.Mitra, Daniel A.Hanson, David D. Schlaepfer, et al. FOCAL ADHESION KINASE: IN COMMAND AND CONTROL OF CELL MOTILITY. Nature VOLUME 6(2005)
19.William E. Damsky, Jr., Lara E. Rosenbaum, Marcus Bosenberg, et al. Decoding Melanoma Metastasis. Cancers, 3, 126-163(2011)
20.Yanet Valdez, Margarida Maia and Edward M. Conway, et al. CD248: Reviewing its Role in Health and Disease. Current Drug Targets, 13, 432-439 (2012)
21.Yi-Kai Hong, Yao-Chou Lee, Tsung-Lin Cheng, et al. Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors. Journal of Investigative Dermatology 139, 2204-2214 (2019)
22.Yun-Yan Hsu, Guey-Yueh Shi, Kuan-Chieh Wang, et al. Thrombomodulin promotes focal adhesion kinase activation and contributes to angiogenesis by binding to fibronectin. Oncotarget, Vol. 7, No. 42 (2016)
23.Zong-Jun-Lin Liu, Yu-Juan Zhou, Rui-Lin Ding, et al. In vitro and in vivo apatinib inhibits vasculogenic mimicry in melanoma MUM-2B cells. PLoS ONE 13(7) (2018)