Association of TMEM147 expression with clinical outcomes in patients with HCC
We evaluated TMEM147 expression in The Cancer Genome Atlas (TCGA) based on the RNA-seq data for liver hepatocellular carcinoma (LIHC). TMEM147 was highly expressed in HCC (Fig. 1A). TMEM147 expression levels were significantly elevated in HCC cell lines compared with normal liver cells, as demonstrated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting (Fig. 1B, C). We also determined the role of TMEM147 in an independent panel of primary HCC and adjacent normal tissues from patients with HCC by RT-qPCR and western blotting analysis. TMEM147 expression was upregulated in HCC tissues (Fig. 1D, E). To determine the clinical significance of TMEM147 expression in patients with HCC, we also analyzed its expression levels in a tissue microarray of 150 paired HCC samples with available clinical information. TMEM147 expression levels were compared between the HCC tumor samples and adjacent non-tumor tissues using immunohistochemistry (IHC). TMEM147 was overexpressed in tumor tissues compared with adjacent non-tumor tissues (Fig. 1F). IHC revealed high expression of TMEM147 in 71.3% (107/150) of HCC patients and low expression in 28.7% (43/150) of patients (Table 2). Moreover, TMEM147 expression levels were associated with tumor size (p =0.024), serum alpha-fetoprotein (AFP) (p = 0.007) and vascular invasion (p = 0.028). However, there was no significant association between TMEM147 expression level and sex, age, TNM stage and tumor number. Based on TCGA data, survival analysis using the Kaplan–Meier method revealed that patients with low TMEM147 expression had significantly better overall survival (OS) (p < 0.001, HR = 2.1, Fig. 1G) and disease-free survival (DFS) (p < 0.05, HR = 1.4 Fig. 1H). Consistently, survival analysis in our cohort of 150 patients indicated that patients with low TMEM147 expression had better OS (p < 0.05, HR=1.6, Fig. 1I). Overall, these results indicate that high TMEM147 expression was associated with a poorer prognosis in patients with HCC.
Effects of TMEM147 on HCC growth and metastasis in vitro
Given that TMEM147 was upregulated in HCC tissues and cell lines in our study, we investigated its role in HCC cells by gain- and loss-of-function studies. TMEM147-overexpressing (TMEM147U) and TMEM147-knockdown (TMEM147KD) lentivirus plasmids were transfected into SMMC-7721 and Hep3B cells and significantly upregulated and downregulated the expression levels of TMEM147, respectively, as shown by RT-qPCR and western blot (Fig. 2A, B). TMEM147-overexpressing HCC cells demonstrated significantly faster growth according to CCK-8 assay (Fig. 2C). We also determined the role of TMEM147 in cell proliferation by EDU assay. The EDU-positivity rate was higher in TMEM147-overexpressing cells and lower in TMEM147-knockdown cells (Fig. 2D). Similarly, TMEM147 overexpression increased HCC colony formation (Fig. 2E), while TMEM147 knockdown significantly impaired growth and reduced colony formation compared with negative control (NC) cells. We also explored the role of TMEM147 in HCC cell migration and invasion by transwell assays. TMEM147-overexpressing cells showed higher migration and invasion activities while TMEM147-knockdown cells showed lower activities compared with control cells (Fig. 2F, G).
Effect of TMEM147 on glycolysis in HCC cells
Gene Ontology (GO) enrichment analysis using TCGA database showed striking alterations in metabolic processes, including carbon metabolism and central carbon metabolism in cancer (Fig. 3A, Table 3), indicating a correlation between TMEM147 expression and enhanced carbon metabolism in HCC. Accordingly, aberrant glucose metabolism occurs in HCC and is known to be a vital metabolic process activating cancer cell proliferation and invasion. To verify the effect of TMEM147 on glycolysis, we examined the metabolic processes in TMEM147-overexpressing and -silenced HCC cells. Cellular glucose uptake and lactate production in the culture medium were significantly increased in TMEM147-overexpressing cells (Fig. 3B, C), and TMEM147 overexpression was also associated with increased cellular ATP levels (Fig. 3D). In contrast, TMEM147 knockdown had the opposite effects. We further examined the effects of TMEM147 on glucose metabolism by measuring the extracellular acid ratio (ECAR). Silencing of TMEM147 attenuated whereas overexpression of TMEM147 enhanced the glycolytic capability of HCC cells, indicating that TMEM147 mainly affected the glycolytic component of glucose metabolism (Fig. 3E). Correlation analysis using TCGA database also showed that TMEM147 expression was positively related to several metabolic enzymes involved in glycolysis, including GLUT1, PKM2, HK1, HK2, LDHA, LDHA (Supplementary file 1: Fig. S1). Consistently, GLUT1, PKM2, HK2, LDHA were significantly downregulated by TMEM147 knockdown and upregulated by TMEM147 overexpression, as shown by RT-qPCR and western blot (Fig. 3F, G).
Effect of TMEM147 on HCC growth in vivo
We further evaluated the effects of TMEM147 on tumorigenesis in vivo in a mouse tumor xenograft model, by subcutaneously injecting NC, and TMEM147 NC (TMEM147NC), overexpressing (TMEM147U), and knockdown (TMEM147KD) SMMC-7721 cells into nude mice. Six representative xenograft tumors from each group were photographed at 6 weeks (Fig. 4A). TMEM147U increased tumor growth and volume compared with the NC group, whereas TMEMKD had the opposite effects (Fig. 4B, C). TMEM147 expression was positively correlated with glycolysis (GLUT1, HK2, PKM2, LDHA) and proliferation markers (Ki-67, proliferating cell nuclear antigen), as shown by IHC staining (Fig. 4D). Further, the hepatic metastasis model was used to evaluate the role of TMEM-147 in HCC cells metastasis ability. The TMEM-147 knockdown HepG2 cells were inoculated into nude spleen and then the HepG2 could reflow into the liver, the results demonstrated that TMEM147 knockdown group exerted less metastasis ability (Fig. 4E).
Interaction between TMEM147 and EGFR and effect on MAPK signaling
We elucidated the molecular mechanisms of TMEM147 in promoting HCC progression by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. TMEM147 was correlated with MAPK activation by receptor tyrosine kinases (RTKs) (Supplementary file 2: Fig. S2). Considering that EGFR is one of the RTKs that activate the MAPK signal pathway, we hypothesized that TMEM147 might exert its biological function via the downstream EGFR/MAPK signaling pathway. We verified the interaction between TMEM147 and EGFR by immunoprecipitation assay and showed that TMEM147 could combine with EGFR (Fig. 5A), and further demonstrated their colocalization by immunofluorescence (Fig. 5B). Furthermore, EGFR expression and MAPK activity were both upregulated in TMEM147-overexpressing cells and inhibited in TMEM147-downregulated cells, as shown by western blot (Fig. 5C). To confirm the correlation between TMEM147 and EGFR, we added EGFR-silencing RNA (sh-EGFR) and an EGFR inhibitor (sorafenib) to TMEM147-overexpressing HCC cells. TMEM147 significantly promoted EGFR/MAPK activation, while this effect was reversed by either sh-EGFR or sorafenib (Fig. 5D). We verified the correlation between TMEM147 expression and EGFR/MAPK activity by analyzing the expression of TMEM147, EGFR, phosphorylated ERK, phosphorylated MEK, and phosphorylated RAF, and showed that TMEM147 expression was positively correlated with EGFR/MAPK pathway hub genes (Fig. 5E).
TMEM147 drives HCC tumorigenesis through EGFR
We verified EGFR as the functional interactor driving HCC tumorigenesis by examining the effects of EGFR shRNA and the EGFR inhibitor sorafenib. Downregulation and inhibition of EGFR attenuated the increased proliferation caused by TMEM147 overexpression, as shown by CCK8, EDU, and colony-formation assays (Fig. 6A-C). EGFR knockdown and inhibition also partly restrained the effect of TMEM147 overexpression on PC cell migration and invasion in transwell assays (Fig. 6D, E). To verify the role of EGFR in the TMEM147-mediated glycolysis effect, we demonstrated that TMEM147 overexpression induced glucose uptake, lactate production, and cellular ATP levels, and these effects could be rescued by EGFR downregulation or inhibition (Fig. 6F-H). Furthermore, EGFR downregulation or inhibition reduced the TMEM147-induced expression of key glycolysis enzymes at both the mRNA and protein levels, as shown by RT-qPCR and western blot (Fig. 6I, J).
Role of retromer in TMEM147-induced EGFR/ MAPK pathway
TMEM147 was previously shown to localize to endoplasmic reticulum membranes and the Golgi apparatus, as a regulator of M3R trafficking to the cell surface[21]. However, retromer is a highly conserved protein complex important for EGFR trafficking and subcellular organelle location from the endosomes to trans-Golgi network or plasma membrane[22, 23]. We therefore proposed that a hypothetical retromer complex might act as a platform for TMEM147-mediated EGFR subcellular organelle localization, trafficking, and activity regulation. Correlation analysis using TCGA database showed that TMEM147 expression was positively related to the retromer components, including VPS26, VPS29, VPS35(Fig. 7A). We therefore explored the possible involvement of retromer complex structures in TMEM47-induced EGFR/MAPK signaling. We determined if TMEM147 and retromer influenced EGFR trafficking to the membrane by EGFR recycling assay. EGFR recycling was increased compared with the NC, and this effect was reversed by downregulation of VPS26, VPS29, or VPS35 (Fig.7B). And the result was verified by immunofluorescence assay (Fig. 7C). Furthermore, EGFR degradation was slower in TMEM147-overexpressing cells and faster in VPS26-, VPS29-, and VPS35-downregulated HCC cells, according to protein stability assay (Fig. 7D). We subsequently evaluated the role of the TMEM147/retromer axis in the EGFR downstream MAPK pathway by western-blot assay. TMEM47 overexpression significantly activated the MAPK pathway and upregulated phosphorylated ERK, MEK, and MEK expression, while this effect was reversed by downregulation of VPS26, VPS29, or VPS35 (Fig. 7E).
Effects of TMEM147 on HCC proliferation, invasion, and glycolysis via retromer components
We determined if TMEM147 induced HCC proliferation, invasion, and glycolysis via the retromer components VPS26, VPS29, and VPS35 by examining the effects of VPS26, VPS29, and VPS35 shRNAs. VPS26, VPS29, and VPS35 downregulation attenuated the TMEM147-induced proliferation, detected by CCK8 and colony-formation assays (Fig. 8A, B). Furthermore, VPS26, VPS29, and VPS35 knockdown partly reversed the effects of TMEM147 overexpression on HCC cell migration and invasion in transwell assays (Fig. 8C, D). We investigated the role of the retromer components in the TMEM147-mediated glycolysis effect by bioinformatics analysis using TCGA database. VPS26, VPS29, and VPS35 expression were positively related to several metabolic enzymes involved in glycolysis, including GLUT1, PKM2, HK1, HK2, LDHA, and LDHA (Supplementary file 3: Fig. S3). TMEM147 overexpression induced glucose uptake and lactate production, while these effects were rescued by VPS26, VPS29, or VPS35 downregulation (Fig. 8E-F).