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Rationale:Aberrant glycosylation has been known to regulate cancer cell trafficking via promoting intravascular adhesion during metastasis. Clinicopathological studies have also shown a strong correlation between aberrant glycosylation and invasive/metastatic potentials of human cancers. However, the major glycosylation enzymes that drive lung cancer metastases and the signaling networks involved in the process remain incompletely understood. Methods:We identified fucosyltransferase 4 (FUT4) as a strong predictor of patient prognosis from two independent cohorts of patients with NSCLC (TCGA and NTUH). We analyzed functional roles of FUT4 through over-expression and knock-down studies using lung cancer cell lines in vitro. Metastatic and homing potentials of FUT4-overexpressed and/or knockdown cells were evaluated in vivo in immunodeficient mice. Genome-wide RNA-seq and MS-based immune-precipitation proteomics were performed to study molecular signaling networks. Results: High expressions of FUT4 were associated with poor survivals in lung adenocarcinomas (p= 0.000681). FUT4-overexpressed lung cancer cells showed significantly increased migration, invasion, and adhesion abilities in vitro, as well as enhanced homing ability to the lungs in vivo. FUT4-overexpressedcells displayed typical characteristics of epithelial-to-mesenchymal transition (EMT) in a protein level-dependent manner. Lewis X and AAL antigens were significantly up-regulated in FUT4-overexpressedcells, which facilitated binding to E-, L- and P-selections. GSEA of RNA-seq data revealed the enrichment of metastasis-related signaling, especially in EGF and TGF-β signalings. Molecularly, FUT4 enhanced EGFR/TGFBR signaling through glycosylation of TGFBR receptors, which lead to increased binding affinity between ligands and its receptors. Conclusion: FUT4 promoted metastasis through enhancing mesenchymal phenotype mediated by glycosylated EGFR/TGFBR signaling, and through increasing binding affinity of cancer cells to selectins. High levels of FUT4 in tumor tissues significantly correlate with poor prognosis in NSCLC patients. Our study not only provided new insights into the role of FUT4 on molecular signaling as a potential therapeutic target, but also identified FUT4 as a prognostic marker in NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Hsuan-Hsuan Lu, Yi-Hsiu Juan, Rueyhung Roc Weng, Shu-Yung Lin, Yen-Wei Chen, Hsin-Han Hou, Zheng-Ci Hung, Yi-Jhen Huang, Tsai-Yu Yang, Yi-Chieh Wu, Giovanni Audrey Oswita, Jin-Yuan Shih, Hsing-Chen Tsai, Chong-Jen Yu. Fucosyltransferase 4-mediated aberrant glycosylation and cell signaling networks promote lung cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1111.

Emerging evidence indicates that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. ADAM15 is up-regulated in multiple cancers and the enzymatic activities of its extracellular metalloprotrease domain promote cancer proliferation and migration by mediating the ErbB signaling pathway. In lung cancer, the enzymatic activity of ADAM15 extracellular domain is reported to be independent of ErbB signaling, indicating an alternative mechanism in promoting lung cancer progression. However, the role of ADAM15 in non-small cell lung cancer (NSCLC) and its correlation to patient prognosis is not well understood. This study demonstrated the longest ADAM15 isoforms, isoform 6 (i6), which contained the most cytoplasmic Src homology 3 (SH3) binding motifs, was significantly up-regulated in progressive NSCLC cell. Comparison of i6 and isoform 1 (i1) revealed that the cytoplasmic domain was essential for NSCLC cell proliferation and aggressiveness. ADAM15 i6, but not i1, mediated the activation the activation of the Src homolog 2 domain containing (Shc) through physical interaction with growth factor receptor-bound protein 2 (Grb2). ADAM15 i6 overexpression promoted NSCLC cell growth in a xenograft mouse model, while ADAM15 or Grb2 knockdown resulted in tumor shrinkage. ADAM15 was overexpressed in primary NSCLC tissues compared to adjacent normal tissues. Patients with ADAM15 i6 high-expressing lung tumors had shorter survival times. Thus, a novel mechanism of the ADAM15 cytoplasmic domain is identified in NSCLC tumor progression. This may help shed light on the molecular mechanisms of ADAM proteins and facilitate development of novel therapy for NSCLC. Citation Format: Hsin-Han Hou. A disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) proliferation through cytosolic domain, not extra-cytosolic metalloprotease activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 542.

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, has been shown to be upregulated in multiple cancers, including gastric, lung, breast, and prostate cancers, and the enzymatic activities of its extracellular metalloprotrease domain promote breast cancer proliferation and migration through mediating ErbB signaling pathway. The patients with ADAM15 high-expressing lung tumors have shorter survival time and ADAM15 has been proved to enhance synovial fibroblasts anti-apoptosis ability via focal adhesion kinase signaling pathway. We firstly demonstrated other than extracellular enzymetic activity, the longest isoform of ADAM15 (ADAM15 i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, significantly upregulated in primary lung cancer tissues and promoted NSCLC proliferation via growth factor receptor-bound protein 2 (Grb2) and Src homolog 2 domain containing (Shc) association. In this study, we further explore the roles of ADAM15 cytosolic domain in NSCLC apoptosis resistance. Overexpression of ADAM15 i6 promoted CL1-0 cell anti-apoptosis ability according to the trypan blue inclusion assay. Ablation of nephrocystin (NPHP1) attenuated the ADAM15 i6-promoted anti-apoptosis ability. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will shed light on the molecular mechanisms of ADAM proteins, and facilitate development of novel therapy in NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) anti-apoptosis ability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2017-538

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, has been shown to be upregulated in multiple cancers, including gastric, lung, breast, and prostate cancers, and the enzymatic activities of its extracellular metalloprotrease domain promote breast cancer proliferation and migration through mediating ErbB signaling pathway. The patients with ADAM15 high-expressing lung tumors have shorter survival time and enzymatic activity of ADAM15 extracellular domain activates MMP-9 and promotes NSCLC migration and invasion. We firstly demonstrated other than extracellular enzymetic activity, the longest isoform of ADAM15 (ADAM15 i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, significantly upregulated in primary lung cancer tissues and promoted NSCLC proliferation via growth factor receptor-bound protein 2 (Grb2) and Src homolog 2 domain containing (Shc) association. In this study, we further explore the roles of ADAM15 cytosolic domain in NSCLC invasion and migration. Overexpression of ADAM15 i6 promoted CL1-0 cell invasion and migration according to the transwell and wound healing assay. Ablation of Ras protein activator (RASA)1 and protein tyrosine kinase (PTK)6 attenuated the ADAM15 i6-promoted invasion and migration respectively. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will shed light on the molecular mechanisms of ADAM proteins, and facilitate development of novel therapy in NSCLC. Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) invasion and migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1166.

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, was reported to over-express in multiple cancers, including gastric, lung, breast, and prostate cancers. It has been shown that the enzymatic activities of its extracellular metalloprotease domain mediate ErbB signaling pathway and promote breast cancer proliferation and migration. Nevertheless, in non-small cell lung cancer (NSCLC), the role of ADAM15 and its correlation to patient prognosis is still not well understood. Moreover, unlike in breast cancer, the enzymatic activities of the ADAM15 extracellular domain appeared to be independent of ErbB signaling and lung cancer proliferation, indicating an alternative mechanism of ADAM15 in promoting lung cancer progression. Here, we demonstrated that ADAM15 was over-expressed in primary NSCLC tissues compared to the adjacent normal tissues. Patients with ADAM15 high-expressing lung tumors had shorter survival times. Furthermore, we examined different isoforms of ADAM15 and discovered that, isoform 6 (i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, was significantly upregulated in primary lung cancer tissues. Comparison of i6 and isoform 1 (i1), which has no cytoplasmic SH3 binding motifs, revealed that the cytoplastmic domain is essential for lung cancer proliferation, as well as aggressiveness in an in vitro lung cancer migration model. We also showed that ADAM15 i6, but not i1, mediated ErbB signaling pathway through physical interaction with growth factor receptor-bound protein 2 (Grb2), and activation of Src homolog 2 domain containing (Shc). Most importantly, overexpression of ADAM15 i6 promoted NSCLC cell growth in a xenograft mouse model while knockdown of i6 resulted in tumor shrinkage. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will not only shed light on the molecular mechanisms of ADAM proteins, but also facilitate development of novel therapy in NSCLC. Citation Format: Hsin-Han Hou. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4948. doi:10.1158/1538-7445.AM2015-4948

Lung cancer is the leading cause of cancer-related deaths in worldwide, including in Taiwan. Lung cancer can typically be grouped into two large categories: small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which account for 15% and 85% of lung cancers, respectively. Despite the continuing endeavors by scientists and clinicians, the cure rate remains low and there is urgent to develop a novel target for NSCLC therapy. The a disintegrin and metalloprotease (ADAM)-15 is a transmembrane protease mediating a wide variety of activities including proteolysis, adhesion, cell fusion and signaling. The expression of ADAM-15 is significantly up-regulated in NSCLC compared to normal tissue. However, the mechanisms of ADAM-15-modulated tumorigenesis in NSCLC are remained unclear. The external enzymetic activity of ADAM15 doesn’t involve in the common pathways in tumoregenesis of NSCLC, such as HER3-EGFR signaling. ADAM15 isoforms are different in cytoplasmic tails, which may drive different biological behavior through intracellular signaling. Therefore, we hypothesized that the diverse cytosolic domain of ADAM15 isoforms plays a crucial role in tumoregenesis of NSCLC. In this study, using real-time quantitative PCR measuring the mRNA expression of 22 ADAMs in NSCLC, we identified a correlation of higher expression of ADAM15 in NSCLC of non-smokers (p=0.002, compared with ex- and current smokers) and in adenocarcinoma (p=0.038, compared with squamous cell ca). Comparing to non-invasive CL1-0 cells and matched normal tissue, CL1-5 cells and NSCLC tissue expressed more the longest ADAM-15 isoform with the largest amount of Src homology (SH)3-binding domain. The association ADAM-15 with SH3-motif containing protein, growth factor receptor-bound (Grb) 2, promoted the ability of proliferation in NSCLC in vitro and in vivo. In conclusion, we suggested that the cytosolic domain of ADAM-15 promotes proliferation of NSCLC via enhance of Grb2-associated signaling pathways, and the inhibition of ADAM-15 may be a potential therapeutic target for NSCLC. Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintegrin and metalloprotease (ADAM)-15 promotes proliferation of non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2014-3317