Am J Cancer Res 2020;10(11):3705-3720 www.ajcr.us /ISSN:2156-6976/ajcr0117564 Original Article GOLM1 upregulates expression of PD-L1 through EGFR/STAT3 pathway in hepatocellular carcinoma Jiuliang Yan1*, Binghai Zhou1,2*, Lei Guo1*, Zheng Chen1*, Bo Zhang1*, Shuang Liu3, Wentao Zhang1, Mincheng Yu1, Yongfeng Xu1, Yongsheng Xiao1, Jian Zhou1, Jia Fan1, Hui Li1, Qinghai Ye1 1Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion (Fudan University), Ministry of Education, Shanghai 200032, People’s Republic of China; 2Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, People’s Republic of China; 3Department of Neurosurgery, Zhongshan Hospital, Fudan University, Shanghai 200032, People’s Republic of China. *Equal contributors. Received July 5, 2020; Accepted October 25, 2020; Epub November 1, 2020; Published November 15, 2020 Abstract: GOLM1, a type II transmembrane protein, is associated with tumor progression, metastasis and immu- nosuppression. However, the relationship between GOLM1 and the immunosuppressive molecule PD-L1 in HCC remains largely unclear. Here, we revealed that GOLM1 acts as a novel positive regulator of PD-L1, whose abnormal expression plays a crucial role in cancer immune evasion and progression. We found that GOLM1 is overexpressed and positively correlated with PD-L1 expression in HCC. Mechanistically, we found that GOLM1 promotes the phos- phorylation of STAT3 by enhancing the level of EGFR, which in turn upregulates the transcriptional expression of PD-L1. Taken together, we demonstrated that GOLM1 acts as a positive regulator of PD-L1 expression via the EGFR/ STAT3 signaling pathway in human HCC cells. This study provides a new insight into the regulatory mechanism of PD-L1 expression in HCC, which may provide a novel therapeutic target for HCC immunotherapy. Keywords: GOLM1, hepatocellular carcinoma, PD-L1, EGFR, STAT3 Introduction tumors [8-11]. Among these immune check- points, the research on programmed cell death Hepatocellular carcinoma (HCC) is the fifth protein 1 and its ligand (PD-1/PD-L1) has most common cancer and the second leading attracted the most widespread attention [12]. cause of cancer-related deaths in the world [1]. However, the anti-PD-1 therapy approved for Although great progress has been made in the HCC treatment only achieved a 20% respon- diagnosis and treatment of HCC, its overall se rate [13]. Treatment with pembrolizumab prognosis is still poor, with a 5-year survival or nivolumab failed to reach the primary end- rate of approximately 12% [2]. At present, treat- points of the KEYNOTE-240 and CheckMate- ments for early HCC include surgical resection, 459 HCC clinical trials [14, 15]. Thus, exploring liver transplantation and local radiofrequency the regulatory mechanism of immune check- (RF) ablation [3], but their efficacy is still limit- points and identifying novel therapies to ed. Molecular targeted therapies, such as the improve HCC patients’ long-term outcome is small-molecule multikinase inhibitor sorafenib urgently needed. (first-line use) [4], regorafenib (second-line use) [5] and lenvatinib (first-line use) [6] have been Golgi membrane protein 1 (GOLM1, also known used for the treatment of advanced HCC. as Golgi protein 73, GP73 or Golgi phosphopro- However, these drugs only extend the median tein 2, GOLPH2), a type II transmembrane pro- overall survival of patients with advanced HCC tein, is associated with tumor progression [16, by no longer than 4 months [7]. Immune check- 17], metastasis [18-21] and immunosuppres- point blockade (ICB) has brought considerable sion [22, 23]. It has been reported that the clinical benefits in the treatment of different expression level of GOLM1 can be increased GOLM1 upregulates expression of PD-L1 in HCC not only in viral infections [24-27] but also in Materials and methods several types of cancer, such as HCC, lung ade- nocarcinoma and prostate cancer [28-30]. Our Antibodies and reagents previous study revealed that GOLM1 is one of the leading genes associated with HCC metas- The antibodies listed below were used in tasis. We found that GOLM1 expression was Western blotting, immunohistochemical: anti- closely related to early cancer recurrence, me- GOLM1 (ab109628, Abcam; ab92612, Abcam), tastasis and poor prognosis in HCC patients. anti-PD-L1 (#13684T, Cell Signaling Technolo- Detailed studies revealed that GOLM1, as a gy; #64988S, Cell Signaling Technology), anti- specific mediator (cargo adaptor), selectively STAT3 (#9139S, Cell Signaling Technology), interacts with epidermal growth factor receptor anti-p-STAT3 (Tyr705) (#9145S, Cell Signaling (EGFR) to assist EGFR/RTK anchoring on the Technology), anti-EGFR (ab52894, Abcam), trans-Golgi network (TGN) and recycling back to anti-p-EGFR (phospho Y1068) (ab40815, Ab- the plasma membrane, leading to the contin- cam). BP-1-102 (#T3708) was purchased from ued activation of downstream kinases, and Topscience. Gefitinib (ab142052) was pur- finally mediating HCC metastasis [18]. However, chased from Abcam. the relationship between GOLM1 and the HCC cell lines immunosuppressive molecule PD-L1 in HCC remains largely unclear. The HCC cell lines HCC-LM3, Huh7, Hep3B, Bel- 7402 and Hepa1-6 were obtained from Liver Studies in various tumor types have demon- Cancer Institute and Zhongshan Hospital, strated that levels of PD-L1 expression can pre- Fudan University, Shanghai, China. These cell dict therapeutic responses to monotherapies lines were cultured in Dulbecco’s modified blocking the PD-1/PD-L1 axis [31-33]. These Eagle’s medium (DMEM) (Thermo Fisher levels of PD-L1 expression are regulated in Scientific, Waltham, MA, USA) containing 10% highly complicated manners and can be affect- fetal bovine serum (FBS) (Gibco) and 1% peni- ed by transcriptional control and post-transla- cillin/streptomycin (Gibco). All cell lines were tional regulation [34]. One of the most critical incubated in a humidified incubator containing transcription factors in human cancer cells is 5% CO at 37°C. the signal transducer and activator of transcrip- 2 tion 3 (STAT3), which can directly act on the Tissue microarray and immunohistochemistry PD-L1 promoter to enhance PD-L1 expression [35]. Some studies have suggested that EGFR Tissue microarrays containing 239 matched is involved in the activation of STAT3 [36-38]. pairs of primary HCC samples and adjacent Activated EGFR recruits and phosphorylates nontumor liver tissues were constructed as STAT3 at Tyr705, then phosphorylated STAT3 described previously [42, 43]. In brief, tumor enters the nucleus to promote the expression specimens were collected from HCC patients of some cancer-related genes [39-41]. Due to who underwent surgical resection from Janua- the important regulation of GOLM1 on EGFR, ry 2005 to December 2006 in Liver Surgery we speculate that there might be a regulatory Department of Zhongshan Hospital, Fudan relationship in the GOLM1/EGFR/STAT3/PD-L1 University, Shanghai, China. The Research axis. Ethics Committee of Zhongshan Hospital app- roved the research protocol and all patients Herein, we found that GOLM1 promoted PD-L1 signed the informed consent forms. Paraffin- transcriptional expression by enhancing STAT3 embedded implanted tumors were cut into Tyr705 phosphorylation. Specifically, we found 5-μm sections. Immunohistochemical (IHC) that GOLM1 enhanced levels of EGFR, hence staining of the HCC samples was performed as the levels of phosphorylated EGFR, which phos- described previously [44]. Briefly, each sample phorylated STAT3, promoting the expression of was stained with the indicated antibodies and PD-L1 in HCC. Our results identified that GOLM1 then incubated with an avidin-biotin-peroxidase is a positive regulator of PD-L1 expression, complex. The chromogen 3-amino-9-ethylcar- which provides a new insight in the relationship bazole was used to visualize the target protein. between GOLM1 and immune response. The expression levels of GOLM1 and PD-L1 in 3706 Am J Cancer Res 2020;10(11):3705-3720 GOLM1 upregulates expression of PD-L1 in HCC tissues were evaluated by the H-score method performed using SYBR-Green PCR Master mix through combining the value of the immunore- (Yeasen Biotechnology Co., Ltd.) according action intensity and the percentage of stained to the manufacturer’s instructions. The prim- cells [45]. H-score = ∑π (i + 1), where i is the ers’ sequences used were as follows: human intensity of the stained cells (0 for negative GOLM1 forward, 5’-CCGGAGCCTCGAAAAGAG- staining, 1 for weak staining, 2 for modera- ATT-3’ and reverse, 5’-ATGATCCGTGTCTGGAG- te staining, and 3 for strong staining) and π is GTC-3’; human PD-L1 forward, 5’-TCACTTG- the value corresponding to the percentage GTAATTCTGGGAGC-3’ and reverse, 5’-CTTTG- of stained cells with different intensities (0, < AGTTTGTATCTTGGATGCC-3’; human GAPDH for- 5% positive cells; 1, 6-25% positive cells; 2, ward, 5’-ACAGTCCATGCCATCACTGCC-3’ and 26-50% positive cells; 3, 51-75% positive cells; reverse, 5’-GCCTGCTTCACCACCTTCTTG-3’. 4, > 75% positive cells). Tumor tissues with H-scores greater than the median of all scor- Lentivirus transfection ed tumor tissues were classified as high ex- pression. Cells were seeded into the 6-well cell culture plates and were cultured for 24 hours to 50% The encyclopedia of RNA interactomes confluence in DMEM containing 10% FBS (ENCORI) database at 37°C. Then