Background

Chordomas, derived from embryonic remnants of the notochord, are rare and slow-growing primary malignant tumors of the spine with an incidence rate of 1–4% of all bone cancers [1, 2]. The major treatment for chordomas is surgery and postoperative radiotherapy as chordomas are refractory to cytotoxic chemotherapeutics [3]. However, it is difficult to treat chordomas due to their local destructive behavior and their preference for growing near the critical skull base, lumbosacral nerves and blood vessels [4]. In addition, high recurrence (> 50% of patients) [5] and metastasis are major causes of treatment failure in chordomas [6, 7]. Herein, it is necessary to study the mechanism of chordoma tumorigenesis and progression and find the effective therapeutic targets for chordoma treatment.

Epidermal growth factor receptor (EGFR), a member of tyrosine kinase receptors, is generally overexpressed in chordomas [8]. EGFR affects tumor growth and metastasis [9]. Our previous study shows that phosphorylated EGFR plays important roles in chordomas progress [10]. EGFR phosphorylation triggers downstream signaling pathways such as signal transducer and activator of transcription 3 (STAT3) [11], AKT [12] and ERK1/2 [13] pathways. The activation of these signaling pathways further drives epithelial–mesenchymal transition (EMT) [14], which is a critical process in cancer metastasis [15, 16]. However, only a few studies demonstrate that EMT is involved in the pathogenesis of chordomas [17]. STAT3 activation also decreases the expression of TP53 and suppresses the TP53 signaling pathway [18], which also responds to stress signals and regulates the expression of its target genes leading to various cellular responses to prevent tumorigenesis [19, 20] as a transcription factor. At present, only Ma’ group explicitly proposes that the TP53 (alias: p53) signaling pathway plays a role in chordoma development [21]. It is necessary and crucial to clarify how the EGFR and TP53 signaling pathway make contributions to chordoma tumorigenesis and progression.

CKLF-like MARVEL transmembrane domain containing (CMTM)3 is a member of the CMTM family [22, 23]. It is located on 16q22.1, an important tumor suppressor locus with tumor suppressive properties [24]. CMTM3 is frequently reduced in multiple types of cancer, such as prostate cancer [25, 26] and renal cell carcinoma [42]. We overexpressed CMTM3 using adenovirus and investigated the whole transcriptome of JHC7 cells using Illumina RNA-Seq technology to screen the potential signaling pathway involved in CMTM3-suppressed cell proliferation. The results showed that there were 479 differently expressed genes (DEGs) between JHC7-MOCK and JHC7-CMTM3 cells with 232 upregulated genes and 247 downregulated genes (padj < 0.05, Fig. 5A, Additional files 2, 3). Among these DEGs, 68 genes were significantly upregulated and 18 genes were downregulated (Table 3, Fig. 5B, padj < 0.001).

Fig. 5
figure 5

CMTM3 induces changes in gene expression profiles. A The number of different expressed genes (padj < 0.05, MOI: 100). B A heat map summary of the gene expression values of JHC7-MOCK and JHC7-CMTM3 cells. Red indicates high and green indicates low gene expression values (padj < 0.001). C KEGG enrichment analysis. The TOP seven upregulated signaling pathways are shown of CMTM3-vs-MOCK in JHC7 cells (padj < 0.05). D The expression of TP53 was detected in CMTM3 overexpressed chordoma cell lines by Real-time PCR. E The expression of TP53 was detected in CMTM3 overexpressed JHC7 cells by western blot. F Up, the transfection efficiency of the CMTM3-siRNA was detected in JHC7 cells by western blot. 100 μg of total protein lysates were loaded; Down. the expression of TP53 in CMTM3 silenced JHC7 cells was detected by western blot. Data were representative of three independent experiments

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Table 3 68 upregulated and 18 downregulated genes in JHC7-CMTM3 cells vs. JHC7-MOCK cells (padj < 0.001)
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We observed that the expression of the well-known tumor suppressor gene TP53, a target of STAT3 was obviously unregulated by overexpression of CMTM3 (ranking first besides CMTM3). In addition, The KEGG pathway enrichment analysis of JHC7 cells showed that the upregulated TP53 was involved in most of the top significant enrichment signaling pathways (padj < 0.05), particularly the TP53 signaling pathway, MAPK signaling pathway and apoptosis in CMTM3 overexpressed JHC7 cells (Fig. 5C). Wherein, TP53 signaling pathway plays important roles in regulation of tumorigenesis and progression and the expression of genes involved in the TP53 signaling pathway was significantly upregulated in CMTM3 overexpressed JHC7 cells, including TP53, SESN2, GADD45A, TP73, GADD45B and CYCS (Table 3, Fig. 5B).

Then, we confirmed these results with JHC7, U-CH1 and MUG-Chor1 cells by Real-time PCR and revealed that TP53 expression was apparently upregulated in CMTM3 overexpressed cells (Fig. 5D). Western blot was further performed in JHC7 cells and the expression of TP53 was upregulated in CMTM3 overexpressed cells (Fig. 5E). We knocked down CMTM3 in JHC7 cells (Fig. 5F, up) and found that TP53 was downregulated in CMTM3 silenced cells (Fig. 5F, down) at the protein level. Our previous study demonstrated that CMTM3 suppressed migration and invasion, but not the proliferation of gastric cancer cells [31, 32]. To clarify whether TP53 upregulation modulated CMTM3 inhibited cell proliferation, we analyzed TP53 expression in gastric cancer cell line SGC-7901 and the gastric epithelial cell line GES-1 by CMTM3 knockdown system using lentivirus transduction [31]. As shown in Additional file 4, TP53 expression was not influenced by CMTM3 knocked down in SGC-7901 and GES-1 cells. These data suggested that CMTM3 might contribute to inhibiting cell proliferation by upregulating TP53 expression. It has been reported that TP53 may respond to stress signals [43] and we found that the expression of heat shock protein family A member 6 (HSPA6), a member of the heat shock protein family was (ranking second besides CMTM3) was upregulated (Fig. 5B, Table 3). Additional file 5 further confirmed that the expression of HSPA6 was upregulated by CMTM3 at the mRNA level by Real-time PCR. These results suggested that TP53 signaling pathway involved in the CMTM3-suppressed proliferation in chordoma cells.

Taken together, we propose that CMTM3 suppresses chordomas through EGFR/STAT3 mediated EMT progression and TP53 signaling pathway.

Discussion

At present, the major treatment for chordomas is surgery and postoperative radiotherapy and they are difficult to be treated due to their destructive behavior locally and their sites [4]. Besides, it is crucial to accurately assess the dose profile outside the tumor with radiation treatment [44]. Therefore, it is necessary to explore the mechanism of chordomas tumorigenesis and development, and find its effective therapeutic targets. It has been reported that CMTM3 acts as a tumor suppressor gene to inhibit numerous cancers. Our previous data also demonstrated that CMTM3 suppressed the metastasis of gastric cancer through the EGFR/STAT3/EMT signaling pathway by interacting with Rab5 [31, 32]. In this paper, we used CMTM3 overexpressed and CMTM3 silenced systems to explore the roles and mechanism of CMTM3 in chordomas. This is the first time we found that CMTM3 inhibited chordoma with regulating EGFR/STAT3 mediated EMT progression and the TP53 signaling pathway. This study investigates the function and mechanism of CMTM3 in chordomas and provides us with a potential target for the treatment of chordomas. In the future, we may characterize the tumor tissues and cells by Raman-enhanced spectroscopy probe [45] to identify CMTM3 for potential tumor markers or diagnosis.

EGFR plays critical roles in the progression of cancers. Strong EGFR expression is significantly associated with tumor growth and metastasis [8]. Adle-Biassette demonstrated that EGFR was expressed in 244/284 (85.9%) of chordomas [46] and Yilmaz found that intensely positive EGFR was revealed in 77.5% (38/49) of chordoma patients [47]. Our previous study also found that EGFR played important roles in chordoma tumorigenesis and development [10]. In this paper, we found that CMTM3 facilitated the degradation of EGFR, and reduced EGFR expression level and p-EGFR (Fig. 4). CMTM3 colocalized with EGFR, but CMTM3 was not coimmunoprecipitated with the anti-EGFR antibody by Co-IP assay (Additional file 1B, C), which suggested that CMTM3 did not interact with EGFR. To date, the clinical relevance of EGFR in chordoma development is still controversial because of its relatively low incidence rate. Inhibitors [48, 49] or antibodies [50] against EGFR may act as potential treatments for chordomas. However, clinical trials with EGFR inhibitors and antibodies have not established a clear therapeutic benefit in all chordoma patients [51, 52]. We believe it is necessary and interesting to explore whether CMTM3 participates in facilitating the sensitivity to EGFR-targeted drugs and antibodies.

As we mentioned, CMTM3 inhibited tumorigenesis and progression mostly in epithelial-derived cancers and few studies revealed the roles of CMTM3 in mesenchymal-derived cancers. To our knowledge, this is the first study to assess the effects and mechanism of CMTM3 in mesenchymal cell-derived tumors. Our previous studies revealed that CMTM3 inhibited cell migration, but did not affect cell proliferation in epithelial-derived gastric cancer. In addition, CMTM3 reduced the phosphorylation of ERK1/2 in gastric cancer cells [32]. In this paper, CMTM3 inhibited cell proliferation and migration, but did not influence ERK1/2 phosphorylation in mesenchymal-derived chordomas. Thus, the roles of CMTM3 might vary in different cell-derived tumors.

TP53 plays crucial roles in tumor initiation and progression [53, 54]. This is the first time we revealed that CMTM3 regulates the TP53 signaling pathway in tumorigenesis and tumor progression. In this study, CMTM3 inhibited cell proliferation and suppressed p-STAT3, which significantly upregulated TP53 expression and enriched the TP53 signaling pathway in chordoma cells. However, CMTM3 has no effects on gastric cancer cell proliferation and does not alter TP53 expression after silencing CMTM3 by lentivirus (Additional file 4). These results suggest that CMTM3 suppresses cancer cell proliferation through upregulation of TP53 expression and activation of the TP53 signaling pathway.

Although our findings demonstrate the roles and mechanism of CMTM3 in the tumorigenesis and development of chordomas, this study still has limitations. First, we revealed that CMTM3 suppressed chordoma cell proliferation, migration and invasion in vitro, and tumor growth in vivo. However, tumor metastasis was not observed in the tumor-bearing mice. We speculated that this is because of the slow-growing characteristics of chordomas and that 5 months were not enough for tumor metastasis or for the observation of the chordoma-bearing mice survival. Second, we observed that CMTM3 reduced EGFR protein level and suppressed p-EGFR, and the fold change of p-EGFR is higher than that of EGFR (Fig. 4). Nevertheless, we still could not conclude that CMTM3 activated p-EGFR directly because the inhibition of EGFR phosphorylation may be a reflection of the reduced EGFR protein level.

Conclusions

In summary, our study first shows the function and mechanism of CMTM3 in chordomas. We find that CMTM3 expression is reduced in chordoma tissues and inhibits proliferation, migration and invasion of chordoma cells in vitro and suppresses tumor growth in vivo. Furthermore, CMTM3 facilitates EGFR degradation and inhibits tumorigenesis and development of chordomas via EGFR/STAT3 regulated EMT signaling pathway and TP53 signaling pathway. CMTM3 may act as a promising molecular target for chordomas, which would be clinically beneficial.