Background
Colorectal cancer (CRC) is the second leading cause of tumor-related death and the third most common malignant tumor worldwide, only secondary to lung cancer and breast cancer [
1,
2]. The main cause of death from CRC is distant metastasis of the tumor. The distant metastasis of CRC leads to its advanced stage with low remission and survival rates in patients. Lymph node metastasis is the most common metastatic pathway of CRC [
3] and is also a key factor in predicting and determining the prognosis of patients [
4]. O’Connell et al. reviewed clinical and pathological data of more than 100,000 patients with CRC [
2]. Results showed that the 5-year stage-specific survivals were 93.2% for stage I, 84.7% for stage IIa, 72.2% for stage IIb, 83.4% for stage IIIa, 64.1% for stage IIIb, 44.3% for stage IIIc, and 8.1% for stage IV [
2] (AJCC sixth edition system). Leonard L et al.’s statistics showed that the 5-year survival rate was 65.4% in patients with lymph node metastasis and 94.4% in patients without lymph node metastasis [
5]. Therefore, the pathological examination of lymph nodes in CRC patients is crucial for post-operative treatment and patient prognosis [
4].
The early diagnosis of tumor lymph node metastasis depends on the detection of specific markers of lymphangiogenesis. VEGFR3 is a relatively specific receptor expressed on lymphatic endothelial cells and involved in the signal transduction of activation of lymphatic endothelial cells [
6]. However, VEGFR3 is also expressed on porous capillaries in endocrine organs as well as on monocytes and megakaryocytes. The other two lymphatic endothelial growth factors, VEGF-C and VEGF-D, promote lymphangiogenesis by binding to their receptor VEGFR-3 and induce anastomoses between lymphatic vessels and lymphatic vessels [
6]. Other potential lymph node metastasis markers such as lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) expressed on lymphatic vessel endothelium is considered to be a specific marker of lymphatic vessels. LYVE-1 was overexpressed in colon tumors compared with in unaffected colon tissues [
7]. However, these lymphatic vessel markers only indicate the lymphatic hyperplasia of the tumor rather than directly indicate lymph node metastasis, and thereby cannot be used as a clinical diagnostic indicator to assess patient prognosis. As a result, it is essential to search for more specific markers of lymph node metastasis.
Voltage-gated Na
+ channels (VGSCs) are macro-molecular protein complexes embedded in cell membranes, which are composed of a channel-forming alpha subunit and one or more smaller beta subunits [
8]. The VGSC α subunit family contains nine members, Nav1.1–Nav1.9, encoded by the
SCN1A–SCN11A genes [
8]. VGSCs are abundantly expressed in excitable cells such as neurons and cardiomyocytes, which are responsible for the generation of action potentials and the transmission of neural signals [
8]. However, VGSCs have been shown to be expressed in tumor cells from a variety of cancers, such as breast cancer, cervical cancer, colon cancer, melanoma, neuroblastoma, non-small cell lung cancer, ovarian cancer, and prostate cancer [
9]. Abnormally high expression of VGSC alpha subunits in cancer cells promotes migration and invasion of tumor cells [
9]. Nav1.1, Nav1.2, Nav1.3, Nav1.4, and Nav1.9 are expressed in ovarian cancer, non-small cell lung cancer, and prostate cancer [
10]. The Nav1.7 alpha subunit promotes gastric cancer progression through MET transcriptional regulator-mediated upregulation of sodium-hydrogen antiporter 1 [
10]. Other studies have shown that the expression of the Nav1.5 alpha subunit is closely related to a poor prognosis in breast cancer [
11], non-small cell lung cancer [
12], ovarian cancer [
13], and prostate cancer [
14]. The Nav1.5 alpha subunit is also a key regulator of the transcriptional regulatory network of genes that control CRC cell invasion [
15]. The Nav1.5 alpha subunit controls colon cancer metastasis via regulation of Wnt signaling, cell migration, ectodermal development, steroid metabolism, and cell cycle-dependent protein expression [
15]. Other studies have shown that Nav1.5 upregulates CRC-inducible gene expression through the MAPK signaling pathway, therefore promoting colon cancer metastasis [
16].
Nav1.6 is expressed in breast cancer, cervical cancer, lymphoma, melanoma, mesothelioma, non-small cell lung cancer, prostate cancer, and small cell lung cancer [
9]. Furthermore, the over-expression of Nav1.6 can promote the metastasis and invasion of cervical cancer [
9,
17]. Similarly, our study found that Nav1.6 was highly expressed in CRC tissues and positively correlated with lymph node metastasis. Additionally, in the surgically removed lymph nodes, we found that Nav1.6, not Nav1.5, was highly expressed in metastatic lymph nodes.
Lymph node metastasis requires tumor cells to flow in or settle in the marginal sinus of the lymph nodes. Tumor-associated macrophages (TAMs) or M2 macrophages play an important role in the development, metastasis, and prognosis of a variety of malignant, metastatic tumors [
18]. For example, in local lymph node metastasis of oral squamous cell carcinoma, M2 macrophage infiltration into the marginal sinus of the lymph node increased with malignancy [
19]. Chemokine receptor 2 (CCR2)-positive monocytes and macrophages are classified as TAMs, both of which are regulated by the CCR2/CCL2 axis [
20]. Another chemokine receptor CCR4 is an important chemokine receptor that regulates immune homeostasis and is thought to be involved in the progression of hematological malignancies [
21]. CCR4 expression is positively correlated with HER2 expression, tumor recurrence, and lymph node, lung, and bone metastasis of breast cancer [
21]. Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 axis in head and neck squamous cell carcinoma [
22]. Our result indicated that CCR2 and CCR4 were also highly expressed in CRC tissues and positively correlated with lymph node metastasis and over-expression of Nav1.6. This study suggests that Nav1.6 could be used as a potential biomarker for lymph node metastasis in patients with CRC. And the CCL2-CCR2 and CCL22-CCR4 axis may be the mechanism of high expression of Nav1.6 promoting lymph node metastasis of CRC.
Discussion
The pathological examination of lymph nodes in patients with CRC is critical for post-operative treatment and prognosis prediction [
4]. Early diagnosis of tumor lymph node metastasis relies on the use of specific sensitive lymphatic biomarkers and advanced techniques to make an accurate diagnosis of lymph angiogenesis and lymph node metastasis. However, the existing lymphatic metastasis markers are mostly lymphatic endothelial markers, which are not suitable for clinical diagnosis and evaluation of patient prognosis.
Abnormal expression of various sodium channel alpha subunits is closely related to tumor invasion and metastasis, but the relationship between sodium channel over-expression and tumor lymph node metastasis is still unclear [
9]. Previous studies have shown that the Nav1.5 alpha subunit plays a key role in the transcriptional regulatory network that controls colon cancer cell invasion [
15]. Our results showed that Nav1.1, Nav1.2, Nav1.4, Nav1.5, Nav1.6, Nav1.8, and Nav1.9 were highly expressed in CRC tissues. However, only a high expression of Nav1.1 and Nav1.6 rather than Nav1.5 was positively correlated with lymph node metastasis. Further immunohistochemistry shows that Nav1.5 and Nav1.6 were both lowly expressed in non-metastatic lymph nodes, while Nav1.6 was highly expressed in metastatic lymph nodes. These results suggested that a high expression of Nav1.1 and Nav1.6 may promote lymph node metastasis in colorectal cancer. Nav1.1 is mainly expressed in the central nervous system, especially the brain, whereas Nav1.6 is mainly expressed in the peripheral nervous system and epithelial cells, especially in the intestinal nerve center and intestinal epithelial cells [
8]. Therefore, Nav1.6 is more suitable for in-depth study as a potential CRC lymph node metastasis marker.
CCR2, CCR4, and their respective ligand-mediated pathways, CCR2\CCL2 axis and CCR4\CCL22 axis, are considered to be important mechanisms of lymph node metastasis [
21,
23]. In this study, we found that CCR2 and CCR4 were highly expressed in Nav1.6 high-expression CRC tissues and were positively correlated with lymph node metastasis. CCR2\CCL2 axis and CCR4\CCL22 axis may involve in the over-expression of Nav1.6 promoting lymph node metastasis. However, whether the upregulation of Nav1.6 expression promotes lymph node metastasis through CCR2\CCL2 axis and CCR4\CCL22 axis needs further study.
Tumor-infiltrating lymphocytes (TILs) are predictors of lymph node metastasis in many cancers such as early gastric cancers, melanoma, and rectal cancers [
24‐
26]. High TILs were significantly associated with an absence of lymph node metastasis [
24]. A high frequency of CD8-positive lymphocyte infiltration correlates with the lack of lymph node involvement in early rectal cancer [
26]. Among the 97 patients, the proportion of patients with a high frequency of CD8-positive lymphocyte infiltration was about 15%. This result indicated that these patients with a high frequency of CD8-positive lymphocyte infiltration may have a less probability of lymph node metastasis in the future.
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