LAPTM4B up-regulation promoted immune cell infiltration in pan-cancer
We acquired diverse tumor data from TCGA and identified LAPTM4B up-regulation in 12 of 33 tumor types, including Liver Hepatocellular Carcinoma (LIHC) (p < 0.0001) (Fig. 1A). We employed different algorithms to analyze immunological characteristics (Fig. 1B). MDSCs outperformed other immune cells, suggesting their pivotal role in LAPTM4B-mediated tumorigenesis (Fig. 1B, C).
A Differential expression of LAPTM4B in various tumor tissues and adjacent normal tissues. B Correlation between immune cell infiltration and LAPTM4B expression in tumors obtained through different computational methods. C The relationship between mdscs’ markers and LAPTM4B expression across different tumor types.
LAPTM4B up-regulation predicted pan-cancer prognosis
To evaluate how LAPTM4B expression affects cancer patient survival, we conducted survival analyses. The OS rates of 33 LAPTM4B-overexpressing cancers were evaluated (Fig. 2A). Kaplan-Meier survival curves were generated for cancer types including Uveal Melanoma (UVM), Adrenocortical Carcinoma (ACC), Breast Cancer (BRCA), Head and Neck Squamous Cell Carcinoma (HNSC), Kidney Chromophobe (KICH), LIHC, Mesothelioma (MESO), Sarcoma (SARC), and Skin Cutaneous Melanoma (SKCM) (Fig. 2B–J). Kaplan–Meier survival curves for LAPTM4B-overexpressing pan-cancer were examined for PFS, DFI, and DSS (Supplementary Figs. 1–3). LAPTM4B up-regulation had a detrimental influence on patient OS, especially in HCC.
A Forest plot of overall survival in different tumor types. B Impact of high LAPTM4B expression on survival probability in uveal melanoma (UVM) patients. C Impact of high LAPTM4B expression on survival probability in adrenocortical carcinoma (ACC) patients. D Impact of high LAPTM4B expression on survival probability in breast cancer (BRCA) patients. E Impact of high LAPTM4B expression on survival probability in head and neck squamous cell carcinoma (HNSC) patients. F Impact of high LAPTM4B expression on survival probability in kidney chromophobe (KICH) patients. G Impact of high LAPTM4B expression on survival probability in liver hepatocellular carcinoma (LIHC) patients. H Impact of high LAPTM4B expression on survival probability in mesothelioma (MESO) patients. I Impact of high LAPTM4B expression on survival probability in sarcoma (SARC) patients. J Impact of high LAPTM4B expression on survival probability in skin cutaneous melanoma (SKCM) patients.
LAPTM4B up-regulation associated with aggressive clinicopathological characteristics and poor prognosis in HCC patients
To mimic liver cancer development, a liver cancer mouse model was established. Mouse liver tissue immunohistochemistry revealed LAPTM4B up-regulation, which was associated with more MDSCs and cell adhesion molecules whereas fewer T cells (Supplementary Figure 4). Earlier survival analysis demonstrated an adverse impact of LAPTM4B up-regulation on four survival outcomes in HCC patients (Supplementary Fig. 5). To validate LAPTM4B expression in HCC, we examined LAPTM4B expression in HCC based on Gene Expression Omnibus (GEO) database. LAPTM4B expression significantly increased in HCC tissues compared with non-carcinoma tissues (Fig. 3A). PCR assays were performed on 92 paired samples of HCC tumor and adjacent normal tissues from The Affiliated Hospital of Qingdao University for validation; thus, LAPTM4B expression significantly increased in HCC tissues (P < 0.001; Fig. 3B).
A LAPTM4B expression in hepatocellular carcinoma (HCC) tumors and adjacent tissues in the Gene Expression Omnibus (GEO). B Correlated Expression of LAPTM4B mRNA. C Single-cell sequencing results of LAPTM4B expression in tumor (a) and adjacent normal (b) tissues from TCGA database. D Western Blot Analysis of LAPTM4B Expression in 42 Pairs of HCC Tumor and Adjacent Tissues. E a. Immunofluorescence staining of LAPTM4B in HCC tumor and adjacent tissues; b. Dot plot representing LAPTM4B expression levels in tumor and adjacent tissues from 187 HCC patients at Eastern Hepatobiliary Surgery Hospital.
These observations were verified through single-cell sequencing. LAPTM4B-positive cells were significantly accumulated in HCC tissues (Fig. 3Ca), but not in non-carcinoma tissues (Fig. 3Cb). LAPTM4B protein expression was verified on samples in 42 patients from The Affiliated Hospital of Qingdao University by Western-blotting assay, and LAPTM4B expression was significantly elevated in tumor tissues (Fig. 3D).
Tissue microarrays comprising 187 tissue samples from Eastern Hepatobiliary Surgery Hospital Affiliated to Naval Medical University were constructed. LAPTM4B expression was significantly different between tumor and normal tissues (p = 8.16e-09; Fig. 3E). Clinical and follow-up data were analyzed to monitor patient survival status. Patients with AFP > 20 ng/ml, age>50, tumor diameter>6 cm, early recurrence (+), and advanced BCLC stage exhibited LAPTM4B up-regulation (P < 0.05; Supplementary Fig. 6A). Therefore, LAPTM4B up-regulation predicted poorer clinical outcomes. Apart from LAPTM4B, variables like BMI and disease stage were unfavorable for patient OS and DFI (Supplementary Fig. 6B–E).
Therefore, LAPTM4B expression significantly increased in HCC tissues compared to non-carcinoma tissues, which predicted poor prognosis.
ETV1 transcription activated LAPTM4B expression
LAPTM4B expression depended on ETV1 in HCC (Fig. 4A). ETV1, also called ETS Related Protein 81 (ER81), belongs to Polyomavirus Enhancer Activator 3 (PEA3) subfamily (ETV1, ETV4, ETV5), with an N-terminal acidic transactivation domain [16]. ETV1 predicts HCC metastasis and poor prognosis [17].
A Data from the Cistrome DB database indicates that ETV1 is the primary transcriptional regulatory factor for LAPTM4B. B Gene expression motif plot highlighting the binding motif of ETV1 to the LAPTM4B promoter. C Dual-Luciferase reporter gene assay. a, Reduced activity of LAPTM4B after decreasing ETV1 expression with shRNA; b, Inhibition by sequences P1, P2, P3, depicted in the bar chart in c. D ETV1 binds to the endogenous promoter of LAPTM4B. Gene sequence analysis was performed to predict the positions of putative ETV1 binding sites within the LAPTM4B promoter, and primers for ChIP assays were designed. Chromatin from Huh7 cells was cross-linked, sonicated, and immunoprecipitated (IP) using ETV1 antibody or rabbit IgG. Specific primers targeting LAPTM4B promoter regions −842 bp to −809 bp, −637 bp to −622 bp, and −175 bp to −160 bp were used for qPCR to measure the promoter DNA quantity associated with IP chromatin. E ETV1 binds to the LAPTM4B promoter region −175 bp to −160 bp. Nuclear extracts were prepared from cells transfected with pHis-ETV1, and EMSA was performed using a FAM-labeled DNA probe synthesized from LAPTM4B promoter sequence −175 bp to −160 bp. Unlabeled probes (50x) or 1 μg of ETV1 antibody was added to the reaction to demonstrate the specificity of ETV1/DNA complex formation. EMSA was also conducted using a FAM-labeled mutated probe (Mut pro). F After silencing ETV1 using shRNA, Western blot experiments were conducted, and the expression of ETV1 and LAPTM4B decreased in Huh7, HepG2, and Hep3B cells. G The co-expression relationship between ETV1 and LAPTM4B was observed in CCLE, GTEx, LIHC, and TCGA datasets.
To validate how ETV1 regulates LAPTM4B transcription, motifs and binding sequences were identified as P1-P3 (Fig. 4B). In dual-luciferase reporter gene assay, ETV1 activated −175 bp to −160 bp region of LAPTM4B promoter in Huh7 cells (Fig. 4C). Upon ChIP assays, ETV1 bound to −500 bp to −200 bp region of LAPTM4B promoter (Fig. 4D). EMSA indicated that FAM-labeled DNA probes synthesized from LAPTM4B promoter region formed DNA/protein complexes with ETV1. Adding unlabeled probes (50×) or ETV1-specific antibodies interfered with ETV1/DNA complex formation or formed supershifted complexes. However, FAM-labeled mutant probes could not form ETV1/DNA complexes (Fig. 4E).
Western-blotting was conducted for ETV1 and LAPTM4B expression in liver cancer cells. ETV1 knockdown decreased LAPTM4B expression (Fig. 4F), consistent with database-based validation results (Fig. 4G). Therefore, ETV1 was bound to a specific promoter region of LAPTM4B gene.
ETV1 was the potent transcription factor for LAPTM4B, which bound to LAPTM4B promoter region (-175bp to -160bp), facilitating LAPTM4B transcription.
LAPTM4B promoted LCSCs through Wnt1/c-Myc/β-catenin Pathway
Tumor heterogeneity is caused by cells exhibiting characteristics similar to stem/progenitor cells, often called cancer stem cells (CSCs) [18]. Due to distinct stem cell-like self-renewal and differentiation abilities, CSCs regenerate distinctive tumor features. HCC tumor growth is driven by CSCs [19]. These LCSCs facilitate HCC initiation, progression metastasis, recurrence, and conventional chemotherapy and radiotherapy resistance [20].
To investigate the mechanism of LAPTM4B in LCSCs, LAPTM4B was over-expressed in Hep3B and Huh7 cells. PCR and flow cytometry revealed significant up-regulation of LCSC markers in LAPTM4B-overexpressing relative to control groups (Fig. 5A, B). Three-dimensional cell growth and tissue formation were explored by spheroid assay, yielding noteworthy results in cells. LAPTM4B overexpression increased tumor diameter and quantity (Fig. 5C).
A mRNA levels of hepatocellular carcinoma stem cell markers in Hep3B and Huh7 cell lines overexpressing LAPTM4B. B Flow cytometric analysis of CD133 expression in Hep3b and Huh7 cell lines overexpressing LAPTM4B. C Tumor spheroid assay: observation of the diameter and quantity of spheroids in Hep3B and Huh7 cell lines overexpressing LAPTM4B. D validation of the correlation between LAPTM4B and MYC expression in TCGA, LIHC, GTEx, and CCLE datasets. E construction of LAPTM4B overexpressing cell lines and validation of pathway-related protein expression. F Based on the results from E, silencing Wnt1 using shRNA resulted in a corresponding decrease in the expression levels of c-Myc and β-catenin.
To identify the specific pathways through which LAPTM4B induces LCSC proliferation, pathway enrichment analysis was conducted (Supplementary Fig. 7A, B). In HCC, significantly enriched pathways included E2F_TARGETS, G2M_CHECKPOINT, and MYC_TARGET, exhibiting enrichment concurrent with LAPTM4B up-regulation (Supplementary Fig. 7C–E; p < 2.2e-16). The association of MYC_TARGET with LAPTM4B expression was validated in a public database, revealing a robust correlation (Fig. 5D). LAPTM4B-overexpressing cells were established for Western-blotting, unveiling increased c-Myc, β-catenin, and Wnt1 phosphorylation levels in LAPTM4B-transfected cells (Fig. 5E). Wnt1 was silenced with shRNA, which down-regulated c-Myc and β-catenin (Fig. 5F). Therefore, LAPTM4B-induced expression in LCSCs via Wnt1/c-Myc/β-catenin pathway.
LAPTM4B-induced MDSCs infiltration predicted poor prognosis
MDSCs are major immune suppressor cells primarily found under pathologies including chronic inflammation and cancer [21]. TME secretes various cytokines and chemokines to promote immature bone marrow cell generation and migration from bone marrow to the tumor site [22]. Human M-MDSCs are CD11b+ CD14+ CD33+ HLA-DRlow, G-MDSCs are CD11b+ CD15+ CD66b+ HLA-DRlow, while their murine counterparts are CD11b+ Ly6C+ and CD11b+ Ly6G+ Ly6Clow, respectively [23].
We introduced Asialo GM1 antibody in mice through tail vein injection to deplete immune cells, creating humanized immunodeficient mice that mimicked human immune system. Flow cytometry confirmed successful immunodeficient mouse construction, as evidenced by decreased CD4+/CD8+T cell expression (Fig. 6Da). Immunodeficient BABL/c mice were subcutaneously injected with 5*10^5 LAPTM4B-Lv and EGFP-Lv-infected Huh7 cells, and then with 1 × 106 human PBMCs for immune system reconstitution (Fig. 6A). The tumor growth size was recorded from 0-28 days, and we observed a significant change in tumor volume after 21 days (Fig. 6B; p < 0.0001). Tumors were removed and examined on day 28, revealing increased LAPTM4B-Lv tumor size (Fig. 6C). MDSCs expression increased in TME of LAPTM4B-Lv (Fig. 6Db).
A Schematic Representation of the Construction Model for Humanized Immunodeficient Mice: Starting from the day of tumor formation, intravenous tail injection of Asialo GM1 antibody was performed to deplete immune cells. Human peripheral blood mononuclear cells (PBMCs) were injected on days 7, 14, 21, and 28. Tumor specimens were collected for analysis on day 28. B Line Graph Showing Tumor Volume Changes in LAPTM4B Overexpressing Tumors Compared to the Control Group from Day −7 to Day 28. C Photographs illustrating tumor volume changes in LAPTM4B overexpressing group compared to the control group. D Flow Cytometric Analysis of CD4+ and CD8+ Cells in Hepatic Tissues of Immunodeficient Mice. Compared to the control group (left panel in a), the immunodeficient group (right panel in a) exhibited decreased expression of CD4+ and CD8+ cells. Flow cytometric analysis of MDSCs in mice. Compared to the control group (left panel in b), the immunodeficient group (right panel in b) showed elevated levels of MDSCs. E Tissue microarray staining shows decreased cd45 density and increased ly-6g density in high LAPTM4B expression group compared to low expression group. F Survival KM curve illustrates Overall Survival and Disease-Free Survival of patients with high and low CD45 expression.
We stained tissue microarrays of patients from Eastern Hepatobiliary Surgery Hospital. LAPTM4B-overexpressing patients showed decreased T cell marker CD45 expression and increased MDSCs marker Ly-6G expression (Fig. 6E). Upon follow-up analysis, CD45 down-regulation predicted reduced OS and DFS (Fig. 6F). LAPTM4B up-regulation induced MDSCs migration, suppressed immune cell function, and adversely affected patient survival.
LAPTM4B activated a suppressed TME via CXCL8 to promote MDSCs infiltration
To substantiate the molecular mechanism of LAPTM4B in inducing MDSC migration, four different databases were analyzed, suggesting CXCL8 as a candidate cytokine regulated by LAPTM4B (Fig. 7A). CXCL8, also called interleukin-8 (IL-8), is a multifunctional chemokine, regulating tumor proliferation, invasion, and migration, often via autocrine or paracrine pathways [24]. Tissue microarray analysis revealed CXCL8 overexpression in TME of LAPTM4B-overexpressing patients (Fig. 7B; p < 0.05).
A Co-expression correlation of CXCL8 and LAPTM4B across TCGA, LIHC, GTEx, and CCLE datasets. B Correlation between high and low expression of LAPTM4B and CXCL8 observed through tissue microarray staining (a) and visualized using violin plots (b). C Western blot experiments confirming the impact of siRNA interference targeting CXCL8 at varying transfection efficiencies. D Based on the results from C, selected transfection efficiencies were used to validate the correlation between LAPTM4B and CXCL8 expressions through Western blotting. E Migration experiments of MDSCs were conducted in both Huh7 and Hep3B cell lines using Transwell assays. The results were visualized through bar charts. F KM survival curves were generated for overall survival (OS) and disease-free survival (DFS) using data from 138 patients from the Eastern Hepatobiliary Hospital. The study aimed to observe the survival rates of patients with high and low expression levels of CXCL8. G Tissue microarray staining was performed to observe the correlation between high and low expression of LAPTM4B in patients and CD31 (a). The results were visualized using violin plots (b).
siRNAs with varying interference efficiencies were designed to silence CXCL8. 1Si-CXCL8 demonstrated the highest silencing efficiency (Fig. 7C), and was used for CXCL8-targeted investigation. LAPTM4B overexpression increased CXCL8 expression, promoting MDSC migration (Fig. 7D, E). CXCL8 silencing suppressed MDSCs migration compared with baseline, even after LAPTM4B overexpression (Fig. 7E). Therefore, LAPTM4B drove MDSCs migration toward tumor tissue primarily via CXCL8.
We conducted longitudinal follows-up of patients and tissue chip staining analyses. CXCL8-overexpressing patients had reduced OS and DFS (Fig. 7F). LAPTM4B up-regulation significantly increased CD31 density (p < 0.001; Fig. 7G). CD31, a 130 kDa membrane glycoprotein, is in immunoglobulin superfamily and instrumental in mediating homophilic/heterophilic adhesion. It is primarily localized at intercellular junctions of endothelial cells [25]. Therefore, LAPTM4B up-regulation might promote tumor angiogenesis. Thus, LAPTM4B-secreted CXCL8 drove MDSCs migration into tumor tissues.
PD-L1 antibody counteracted LAPTM4B-mediated HCC progression
PD-1, an immune checkpoint molecule on T cell surface, and its counterpart, PD-L1 (CD274) often overexpressed on cancer cell surface, form a binding interaction, which suppresses T cell proliferation and activation [26]. PD-1/PD-L1 pathway is vital for cancer immunotherapy, and targeting inhibitors make significant breakthroughs in treatment [27].
Through public database analysis, LAPTM4B expression was strongly positively correlated with CD274 (Fig. 8A). In LAPTM4B-overexpressing patients, CD274 (PD-L1) overexpression on tumor surface suggests that treatment with PD-L1 antibodies may have therapeutic efficacy. We collected intraoperative samples and radiological data from 21 HCC patients undergoing PD-L1 therapy at The Affiliated Hospital of Qingdao University. LAPTM4B-overexpressing patients were responsive to PD-L1 therapy. Following PD-L1 treatment, tumor size significantly decreased (Fig. 8B). Immunohistochemistry revealed that, patients responding effectively to PD-L1 therapy exhibited LAPTM4B up-regulation (Fig. 8C; Pearson r = −0.7906, p < 0.0001).
A Co-expression correlation of CD274 and LAPTM4B across TCGA, LIHC, GTEx, and CCLE datasets. B Impact of LAPTM4B expression on PD-L1 antibody therapy, sourced from MRI data of patients at the Affiliated Hospital of Qingdao University. C Immunohistochemistry of LAPTM4B expression in patients responding and not responding to PD-L1 antibody therapy (a), followed by visualization of immunohistochemistry results (b, c, d). D ETV1 binds to the promoter region of LAPTM4B, inducing LAPTM4B transcription, promoting the proliferation of liver cancer stem cells (LCSCs) through the Wnt1/c-Myc/β-catenin pathway. LAPTM4B secretes the cytokine CXCL8, inducing the migration of myeloid-derived suppressor cells (MDSCs), leading to tumor progression.
LAPTM4B up-regulation substantially worsened HCC patient prognosis. However, these patients are responsive to PD-L1 antibody therapy, highlighting sensitivity of LAPTM4B-overexpressing patients to targeted treatments and underscoring effectiveness of PD-L1 blockade on mitigating LAPTM4B overexpression-related effects in HCC.
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- Source: https://www.nature.com/articles/s41419-024-06542-8