JAML was highly expressed in the cancer tissues of patients with CRC, accompanied by decreased T-cell infiltration and poor prognosis.
The expression of JAML in 50 CRC patients was detected using immunohistochemistry, and the relationships between JAML expression and clinicopathological parameters were also analysed. Of the 50 patients, 28 (56%) were male and 22 (44%) were female. The patients ranged in age from 41 to 88 years (median age 66.5 years); 34 patients were ≥ 60 years (68%), and 6 patients were < 60 years (32%). JAML was stained brown‒yellow and was expressed mainly in the cytoplasm and cell membrane of cancer cells, with a small amount of expression in interstitial immune cells and almost no expression in normal intestinal gland cells adjacent to cancer cells (Fig. 1A). The IHC result was the product of the stained area and intensity fractions. A score less than or equal to three indicated no expression, a score greater than or equal to four indicated expression, a score less than or equal to six indicated low expression, and a score greater than or equal to eight indicated high expression. IHC analysis of CRC tissues revealed that the expression of JAML was significantly greater than that in adjacent tissues (p < 0.0001) (4 was the cut-off value for IHC results) (Fig. 1B). Subsequently, we studied the relationship between JAML in CRC and various CRC pathological parameters. We found that JAML was highly expressed in 50% (25/50) of patients with CRC, especially in patients with lymph node metastasis [81% (17/21) of patients with lymph node metastasis vs. 27.6% (8/29) of patients without lymph node metastasis (p < 0.001) and late TNM stage (p < 0.0001)] (Table 1).
Table 1
The relationship between the expression of JAML and various CRC pathological parameters
Varialbles | JAML expression |
High | Low | p |
Age(yr) |
≤ 60 | 9 | 11 | 0.564 |
> 60 | 16 | 14 |
Gender |
Male | 16 | 12 | 0.254 |
Female | 9 | 13 |
Primary tumour |
T1-T2 | 1 | 1 | 1 |
T3-T4 | 24 | 24 |
Regional lymph node involvement |
N0 | 8 | 21 | 0.00195 |
N+ | 17 | 4 |
Histological grade |
G2 | 15 | 14 | 0.774 |
G3 | 10 | 11 |
TNM stage groupings |
I-II | 7 | 21 | 0.000066 |
III-IV | 18 | 4 |
We examined the relationship between JAML expression and tumour-infiltrating lymphocytes in CRC tissues. We found that high expression of JAML was often accompanied by decreased infiltration of CD3+T cells; that is, the density of tumour-infiltrating CD3+T cells in the JAML− high group was lower than that in the JAML− low group (Fig. 1C–D). We further determined the proportions of CD4+, CD8+ and Foxp3+T-lymphocytes within the two groups. We found that the density of CD8+T-lymphocytes infiltration in the JAML− high group was lower than that in the JAML− low group (p < 0.05); however, there was no significant difference in the distribution of CD4+T and Foxp3+ T-lymphocytes between the two groups (Fig. 1E–G).
Patients in the JAML− high group had shorter overall survival than those in the in the JAML− low group (p = 0.0362, HR = 0.4295, 95% CI of 0.1908–0.9667) (Fig. 1H). Further survival analysis showed that the survival rate in the JAML− high CD3− low group was significantly lower than that in the JAML− low CD3− high group (p = 0.0003, HR = 0.1106, 95% CI = 0.03794–0.3224) (Fig. 1I). Mortality risk ratio analysis revealed that the expression of JAML, higher TNM stage and regional lymph node metastasis predicted poor prognosis (HR > 1, p < 0.05), and the expression of CD3 and CD8 predicted a better prognosis (HR < 1, p < 0.05) (Fig. 2A).
JAML promoted the proliferation, migration, and invasion of CRC cells in vitro
We speculated that the expression of JAML in CRC may affect the malignant biological behaviour of tumour cells. First, we examined the expression of JAML in CRC cell lines (HCT116, LOVO, DLD-1, SW480, and SW620) and colorectal epithelial cells (NCM460) (Fig. 3A, B). Compared with that in NCM460 cells, the expression of JAML in LOVO, DLD-1, SW480, and SW620 cells was greater, while its expression was relatively lower in DLD-1 and SW480 cells than in HCT116 and LOVO cells. Therefore, we transfected the JAML plasmid into DLD-1 and SW480 cells to increase JAML expression. Western blot analysis also showed that the expression of JAML in DLD-1 and SW480 cells was significantly upregulated after transfection with the JAML plasmid (Fig. 3C, D). A lentivirus (shJAML) was used to transfect HCT116 and LOVO cells to reduce JAML expression (Fig. 3E, F). We obtained DLD-1 and SW480 cells with increased JAML expression and HCT116 and LOVO cells with low JAML expression.
We then performed cell migration, invasion, and proliferation experiments to verify the effect of JAML on the malignant behaviour of tumour cells. Transwell migration and invasion experiments showed that overexpression of JAML significantly increased the migration and invasion of DLD-1 and SW480 cells (Fig. 3G, H, J, K), whereas low expression of JAML decreased the migration and invasion abilities of HCT116 and LOVO cells (Fig. 3G, I, J, L). The results of the EdU proliferation assay ultimately showed that the overexpression of JAML significantly increased the proliferative ability of DLD-1 and SW480 cells (Fig. 3M, N), while the low expression of JAML decreased the proliferative ability of HCT116 and LOVO cells (Fig. 3M, O). Taken together, these results suggested that JAML promoted the proliferation, migration, and invasion of CRC cells.
Overexpression of JAML in CRC cells activated the PI3K-AKT-mTOR signalling pathway in vitro
Western blotting was used to verify the relationship between changes in JAML expression and the expression of PI3K, AKT, and mTOR in CRC cells (Fig. 4A). We found that after increasing JAML expression in DLD-1 and SW480 cells, the expression of P-PI3KTYR467/199, P-AKTSER473, and P-mTORSer2448Ser2448 increased (Fig. 4B, C), whereas after reducing JAML expression in HCT116 and LOVO cells, the expression of P-PI3K, P-AKTSER473, and P-mTORSer2448Ser2448 decreased (Fig. 4D, E). Next, we treated JAML-overexpressing DLD-1 and SW480 cells with the mTOR inhibitor rapamycin (Fig. 4F). The results of the Transwell migration and EdU proliferation assays showed that the migration and proliferation ability of DLD-1 and SW480 cells increased after JAML was overexpressed, while the increase in migration and proliferation caused by the overexpression of JAML was partially inhibited by the addition of the mTOR inhibitor (Fig. 4G-K). These results suggested that JAML promoted the migration and proliferation of CRC cells by activating the PI3K-AKT-mTOR signalling pathway in vitro.
Overexpression of JAML promoted the proliferation of CRC by activating the PI3K-AKT-mTOR signalling pathway in vivo
LOVO cells transfected with the JAML knockdown virus or control cells were subcutaneously inoculated into BALB/c nude mice, after which tumours were subcutaneously implanted and cultured. The tumour volumes of the mice were recorded regularly. The two groups of mice were sacrificed on the 40th day, after which the tumour tissue was weighed and embedded. After plotting the tumour growth curve, we found that the subcutaneous implanted tumours in the LOVOshJAML group grew significantly more slowly than those in the control group did (Fig. 5A–D). We obtained tumour tissue sections from the mice and then performed IHC staining experiments. The results showed that in tumour tissues with low JAML expression, the expression of P-PI3K, P-AKT, and P-mTOR decreased significantly, while the expression of PI3K, AKT, and mTOR was similar in two groups (Fig. 5E–K). These results suggested that JAML promoted the proliferation of CRC by increasing the phosphorylation of PI3K-AKT-mTOR signalling pathway in vivo.
T-lymphocytes infiltration was reduced in tumour tissues with JAML high expression by decreasing chemokines
To verify the relationship between CRC-related JAML and tumour infiltrating lymphocytes (TILs) in the tumour immune microenvironment in vivo, we used MC38 cells to establish a CRC transplantation model in C57BL/6 mice. We also used lentivirus transfection to knockdown the expression of JAML in MC38 cells. MC38shJAML cells and control cells were subcutaneously implanted into C57BL/6 mice after we obtained the corresponding MC38shJAML cell line. We found that tumour growth in the MC38shJAML group was significantly slower than that in the control group (Fig. 6A-D). We detected T-lymphocyte subsets in the tumour, by flow cytometry. As shown in Fig. 6E–G, downregulation of JAML in tumour cells significantly increased the proportion of CD8+T cells in the tumour (p < 0.05). Immunohistochemical staining revealed that CD8+T cells infiltration within tumour tissues increased significantly in MC38shJAML group (Fig. 6H-K).
To clarify the mechanism by which JAML regulates the infiltration of T cells, we used transcriptome analysis, and we found that cytokine secretion was enriched in LOVOshJAML cells (Fig. 6L). Subsequently, we used RQ-PCR to verify the relationship between JAML and the expression of various cytokines in CRC cells. The results showed that the expression of cytokines such as CCL2, CCL16, CCL20, CCL22, CXCL9, CXCL10 and CXCL11 increased when JAML decreased in LOVO cells (Fig. 6M). Similar results were observed for MC38 cells by RQ-PCR (Fig. 6N). After treatment with the mTOR inhibitor rapamycin, the expression of CCL20 and CXCL9/10/11 in LOVO and MC38 cells was upregulated significantly (Fig. 6O-R).