Liu et al. Cancer Cell Int (2021) 21:287 https://doi.org/10.1186/s12935-021-01991-z Cancer Cell International PRIMARY RESEARCH Open Access Vesicle transporter GOLT1B mediates the cell membrane localization of DVL2 and PD-L2 and promotes colorectal cancer metastasis Tengfei Liu†, Binbin Liu†, Yiting Liu, Xingzhi Feng, Xuefei Jiang, Jiahui Long, Qianling Gao and Zihuan Yang* Abstract Background: Colorectal cancer (CRC) is the third most diagnosed and second leading cause of cancer death worldwide. Hallmark proteins processing is usually dysregulated in cancers. Finding key regulatory molecules is of great importance for CRC metastasis intervention. GOLT1B is a vesicle transport protein which is involved in cytosolic proteins trafcking. However, its role in cancer has never been addressed. Methods: CRC cell lines and subcutaneous xenograft animal model were utilized to investigate the biological func- tion of GOLT1B. Patients samples were used to validate the correlation between GOLT1B and clinical outcome. In vivo targeted delivery of GOLT1B-siRNA was investigated in PDX (Patient derived tumor xenograft) model. Results: We found that GOLT1B was highly expressed in CRC, and was an independent prognostic marker of overall survival (OS) and progression free survival (PFS). GOLT1B could promote CRC metastasis in vitro and in vivo. GOLT1B overexpression could increase DVL2 level and enhance its plasma membrane translocation, which subsequently activated downstream Wnt/β-catenin pathway and increase the nuclear β-catenin level, hence induce epithelial- mesenchymal transition (EMT). In addition, GOLT1B could also interact with PD-L2 and increase its membrane level. Co-culture of GOLT1B-overexpresed CRC cells with Jurkat cells signifcantly induced T cells apoptosis, which might further promote cancer cell the migration and invasion. Further, targeted delivery of GOLT1B siRNA could signifcantly inhibit tumor progression in GOLT1B highly expressed PDX model. Conclusion: Taken together, our fndings suggest that the vesicle transporter GOLT1B could promote CRC metastasis not only by assisting DVL2 translocation and activating Wnt/β-catenin pathway, but also facilitating PD-L2 membrane localization to induce immune suppression. Targeted inhibition of GOLT1B could be a potential therapeutic strategy for CRC treatment. Keywords: GOLT1B, CRC , Metastasis, PDX, DVL2, PD-L2 Introduction Colorectal cancer is now the third most common malig- nant tumor worldwide, and distant metastasis is the main cause of its death [1, 2]. Clarifying the key molecules related to CRC metastasis and prognosis and their regu- *Correspondence: [email protected] latory mechanisms is the key to the treatment of CRC. †Tengfei Liu and Binbin Liu contributed equally to this work Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Protein localization is very important for the mainte- Diseases, Guangdong Institute of Gastroenterology, The Sixth Afliated nance of their normal function. For example, DVL2 are Hospital of Sun Yat-Sen University, Guangzhou 510655, Guangdong, recruited to the membrane receptor complex to initi- China ate downstream Wnt signal cascade [3, 4]. Programmed © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. Cancer Cell Int (2021) 21:287 Page 2 of 15 death-ligand 2 (PD-L2) expressed on the surface of can- McCoy’s 5a Medium Modifed (Gibco, USA) medium cer cells could induce immunosuppression through inter- containing 10% fetal bovine serum (Gibco, USA), RKO action with PD-1 in immune cells [5, 6], and blocking this was cultured with MEM (Gibco, USA) medium contain- interaction gave rise to an impressive clinical beneft in ing 10% fetal bovine serum (Gibco, USA), Jurkat T lym- various of cancers [7–14]. Aberrant localization of hall- phocyte cell line were purchased from the China Center mark proteins can alter their function so that their abil- for Type Culture Collection (Shanghai, China). Cells were ity to suppress cancer or induce cancer metastasis is maintained in RPMI1640 medium supplemented with changed. Terefore, the mislocalization of such proteins 10% fetal bovine serum and placed in a humidifed incu- could serve as novel therapeutic targets of cancer. bator at 37 °C with 5% CO2. Golgi apparatus is a crucial cell component responsi- ble for transporting, modifying, and packaging proteins Primers and antibodies into vesicles for delivery to targeted locations. Stud- All the primers used are listed in the supplemental data ies have shown that Golgi-related genes play important (Additional fle 1: Table S1). T-PER tissue protein extrac- roles in regulating cancer occurrence and development. tion reagent (Termo, Rockford, IL, USA), protease Golgi phosphorylated protein 2 (GOLPH2) dysregula- inhibitor group III and phosphatase inhibitor group II tion has been reported in many cancers, including oral (Millipore, Germany) were used for protein preparation. squamous cell carcinoma, hepatocellular carcinomas, Proteins were quantifed using BCA kits (Termo, USA). and esophageal cancer [15–17]. Golgi phosphorylated Antibodies used are anti-GOLT1B (Afnity Biosciences, protein 3 (GOLPH3) promotes the development of CRC DF9071), anti-DVL2 (CST, #3224), anti-β-catenin (CST, and induce resistance to 5-FU by mediating the mTOR #25362), anti-GSK3β (CST, #9832), anti-pGSK3β(ser9) pathway [18–20]. Golgi membrane protein 1 (GOLM1) (CST, #5558), anti-TCF1/TCF7 (CST, #2203), anti-TCF4/ expression is correlated with early recurrence, metas- TCFL2 (CST, #2569), anti-PD-L2 (CST, #82723), anti- tasis, and poor survival of HCC patients [21, 22]. Golgi fag (Sigma, F1804), anti-PCNA (Proteintech Group, vesicle transporter 1A (GOLT1A) has been shown to # 10205–2-AP) and anti-GAPDH (Proteintech Group, regulate tamoxifen sensitivity in breast cancer and pro- #10494–1-AP). mote cell proliferation in lung cancer [23, 24]. However, the function of GOLTIB, which belongs to the same fam- Patient samples ily, has never been addressed. Little is known about the Paired normal tissue (5 cm from the tumor boundary), mechanism of Golgi-related proteins in regulating cancer paracancerous (2 cm from the tumor boundary) and metastasis. tumor tissues were obtained from 8 CRC patient from In the present study, we found that the vesicular the Sixth Afliated Hospital of Sun Yat-sen University. transporter GOLT1B, also known as Golgi transport 1 Tissue microarray (TMA) of primary CRC from 224 homolog B, was overexpressed in CRC. High GOLT1B patients were constructed. Freshly resected tumor tissue is signifcantly correlated with poor prognosis. In vitro from 3 CRC patients were used to construct PDX model. and in vivo experiments showed that GOLT1B could pro- None of these patients received adjuvant chemotherapy mote CRC cells migration and invasion. As to the mecha- or radiotherapy before surgury. Studies were approved by nism, GOLT1B could interact with DVL2 and facilitate the Human Medical Ethics Committee of the Sixth Afli- its transportation to the cell membrane, where it can ated Hospital of Sun Yat-sen University. bind to the cytoplasmic C-terminus of frizzled receptor, leading to the activation of Wnt signaling and epithelial- Plasmid construction and siRNA transfection mesenchymal transition (EMT). In addition, we found Te cDNA ORF of human GOLT1B was amplifed and that GOLT1B can also interact with PD-L2 and induce cloned into the pcDNA 3.1(+) plasmid by homologous T-cell apoptosis. Our results suggest that GOLT1B is recombination using In-fusion HD cloning kit (Clo- very important in regulating protein transportation and netech, Tokyo, Japan). Te constructed plasmid was used localization. GOLT1B may be a predictive marker and for transient transfection. Te plasmid was transfected therapeutic target for metastatic CRC. into CRC cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions and Materials and methods the cells were collected 48 h after transfection for experi- Cell lines and cell culture ments. In this study, the full-length human GOLT1B Human CRC cell lines (HCT116, RKO) were purchased cDNA ORF was constructed by homologous recombina- from the American Type Culture Collection (ATCC). Te tion into the lentivirus expression vector pCDH-CMV- STR genotyping data of the two cell lines are consistent MCS-EF1-copGFP (SBI Pharmaceuticals, Tokyo, Japan) with the ATCC database. HCT116 was cultured with for the construction of stable cell lines. HEK 293T cells Liu et al. Cancer Cell Int (2021) 21:287 Page 3 of 15 were co-transfected with pCDH-GOLT1B/pCDH-Vector, Cell migration and invasion were determined by tran- pCMV-δ8.91 and pCMV-VSVG using Lipofectamine swell assay. Breify, CRC cells resuspended in serum-free 3000 to produce lentiviruses. Te medium was changed medium were added to the upper well of transwell cham- 24 h after transfection and virus supernatants were col- bers and cultured for 48 h. For invasion assay, Matrigel lected 48 h and 72 h after transfection. After ultracen- (Corning, USA) was diluted at 1:10 with serum-free cul- trifugation, the supernatant was concentrated and added ture medium and coated to the upper surface of the tran- to luciferase-expressing HCT116.