Syncytin 1, CD9, and CD47 regulating cell fusion to form PGCCs associated with cAMP/PKA and JNK signaling pathway

Abstract

Background: We have previously reported the formation of polyploid giant cancer cells (PGCCs) through endoreduplication or cell fusion after cobalt chloride (CoCl₂ ) induction. Cell fusion plays an important role in development and disease. However, the underlying molecular mechanism concerning cell fusion in PGCCs formation and clinicopathological significances remains unclear.

Methods: We treat HCT116 and LoVo cell with CoCl₂ and observed the cell fusion via fluorescent markers of different colors. Western blot and immunocytochemical staining were used to compare the expression and subcellular location of the fusion-related proteins syncytin 1, CD9, and CD47 along with PKA RIα, JNK1, and c-Jun between PGCCs and control cells from the HCT116 and LoVo cell lines. Moreover, 173 cases of colorectal tumor tissue samples were analyzed, including 47 cases of well-differentiated primary colorectal cancer (group I) and 5 cases of corresponding metastatic tumors (group II), 38 cases of moderately differentiated primary colorectal cancer (group III) and 14 cases of corresponding metastatic tumors (group IV), and 42 cases of poorly differentiated primary colorectal cancer (group V) and 27 cases of corresponding metastatic tumors (group VI).

Results: The expression of syncytin 1, CD9, and CD47 is higher in PGCCs than in control cells and they are located in the cytoplasm. The expression of PKA RIα and JNK1 decreased, and that of c-Jun increased in PGCCs. The syncytin 1 expression was significantly different between groups I and II (P = 0.000), groups III and IV (P = 0.000), groups V and VI (P = 0.029), groups I and III (P = 0.001), groups III and V (P = 0.000), and groups I, III, and V (P = 0.000).

Conclusions: These data indicate that the cell fusion-related proteins syncytin 1, CD9, and CD47 may be involved in PGCC formation, and that cAMP/PKA and JNK signaling is likely to promote PGCC formation via the regulation of cell fusion processes.

Keywords: cell fusion; colorectal cancer; polyploid giant cancer cells; syncytin 1.

Figure 1

PGCCs with budding daughter cells. A, HCT116 PGCCs and control HCT116 cells. (a) Control HCT116 cells (100×). (b). HCT116 PGCCs induced by 375 μM CoCl₂ treatment for 60 h (100×). (The large black arrow heads indicate the PGCCs; the small black arrow heads indicate the budding daughter cells; 200×). (c) The PGCCs generated daughter cells via budding. The black arrow heads indicate the budded daughter cells; 100×). (d) Generation of daughter cells by budding contributes to the reproduction of the PGCCs (100×). B, LoVo PGCCs and control LoVo cells. (a) Control LoVo cells (200×). (b) HCT116 PGCCs induced by 375 μM CoCl₂ treatment for 30 h (100×). (The large black arrow heads indicate the PGCCs; the small black arrow heads indicate the budding daughter cells; 100×). (c) The PGCCs generated daughter cells via budding. The black arrow heads indicate the budded daughter cells; 100×). (d) Generation of daughter cells by budding contributes to the reproduction of the PGCCs (100×). C, Fluorescent markers of different colors were used to detect the cell fusion in HCT116 and LoVo before CoCl₂ treatment (100×). (a) HCT116 cells with red fluorescence before CoCl₂ treatment. (b) HCT116 cells with green fluorescence before CoCl₂ treatment. (c) Merge image of (a) and (b). (d) LoVo cells with red fluorescence before CoCl₂ treatment. (e) LoVo cells with green fluorescence before CoCl₂ treatment. (f) Merge image of (d) and (e). D, Fluorescent markers of different colors were used to detect the cell fusion in HCT116 and LoVo after CoCl₂ treatment (100×). (a) HCT116 cells with red fluorescence after CoCl₂ treatment and white arrow points the PGCC. (b) HCT116 cells with green fluorescence after CoCl₂ treatment and white arrow points the same PGCC of (a). (c) Merge image of (a) and (b) and white arrow points the PGCC with yellow. (d) LoVo cells with red fluorescence after CoCl₂ treatment and white arrow points the PGCC. (e) LoVo cells with green fluorescence after CoCl₂ treatment and white arrow points the same PGCC of (d). (f) Merge image of (d) and (e) and white arrow points the PGCC with yellow. E, H&E staining of the HCT116 and LoVo cells before and after CoCl₂ treatment (100×). (a) H&E staining of the control HCT116 cells. (b) Many PGCCs appeared in HCT116 cells after CoCl₂ treatment. (c) H&E staining of the control LoVo cells. (d) Many PGCCs appeared in LoVo cells after CoCl₂ treatment. F, Quantitative results of the percentage of PGCCs of control cells and PGCCs of HCT116 and LoVo cells

Figure 2

Syncytin 1, CD9, and CD47 expression in HCT116 and LoVo PGCCs with budding and HCT116 and LoVo control cells. A, Western blotting was used to assess differences in syncytin 1, CD9, and CD47 expression in HCT116 and LoVo cells before and after CoCl₂ treatment. (a) Total expression of syncytin 1, CD9, and CD47 in the HCT116 and LoVo control HCT116 and LoVo cells and HCT116 and LoVo PGCCs. (b) Cytoplasmic expression of syncytin 1 and CD9 in the control HCT116 and LoVo cells and HCT116 and LoVo PGCCs. B, ICC staining was used to detect the subcellular location of syncytin 1, CD9, and CD47 in HCT116 and LoVo PGCCs with budding and control HCT116 and LoVo cells. (a) The subcellular location of syncytin 1 in control HCT116 and LoVo cells and HCT116 and LoVo PGCCs. (b) The subcellular location of CD9 in control HCT116 and LoVo cells and HCT116 and LoVo PGCCs in. (c) The subcellular location of CD47 in control HCT116 and LoVo cells and HCT116 and LoVo PGCCs. C, Quantitative results of total and cytoplasmic protein expression differences are shown as histograms. (a) The histogram of cytoplasmic syncytin 1 expression in HCT116 and LoVo. (b) The histogram of cytoplasmic CD9 expression in HCT116 and LoVo. (c) The histogram of total syncytin 1 expression in HCT116 and LoVo. (d) The histogram of total CD9 expression in HCT116 and LoVo. (e) The histogram of total CD47 expression in HCT116 and LoVo. The corresponding densitometric analyses of each protein band were performed using image‐J software; the signals of each protein band were normalized to the β‐actin signal

Figure 3

PKA RIα, JNK1, and c‐Jun expression in HCT116 and LoVo PGCCs with budding and control cells. A, Western blotting was used to test differences in the total expression of PKA RIα, JNK1, and c‐Jun in HCT116 and LoVo cells before and after CoCl₂ treatment. B, Cytoplasmic and nuclear expression of PKA RIα, JNK1, and c‐Jun in control HCT116 and LoVo cells and HCT116 and LoVo PGCCs. C, ICC staining was used to assess the subcellular location of PKA RIα, JNK1, and c‐Jun in HCT116 and LoVo PGCCs with budding and control cells (100×). (a) PKA RIα ICC staining in HCT116 control cells. (b) PKA RIα ICC staining in HCT116 PGCCs. (c) PKA RIα ICC staining in LoVo control cells. (d) PKA RIα ICC staining in LoVo PGCCs. (e) JNK1 ICC staining in HCT116 control cells. (f) JNK1 ICC staining in HCT116 PGCCs. (g) JNK1 ICC staining in LoVo control cells. (h) JNK1 ICC staining in LoVo PGCCs. (i) c‐Jun ICC staining in HCT116 control cells. (j) c‐Jun ICC staining in HCT116 PGCCs. (k) c‐Jun ICC staining in LoVo control cells. (l) c‐Jun ICC staining in LoVo PGCCs. D, Quantitative results of total, cytoplasmic, and nuclear protein expression differences are shown as histograms. The corresponding densitometric analyses of each protein band were performed using image‐J software; the signals of each protein band were normalized to the β‐actin signal. (a) The histogram of total PKA RIα expression in HCT116 and LoVo. (b) The histogram of total JNK1 expression in HCT116 and LoVo. (c) The histogram of total c‐Jun expression in HCT116 and LoVo. (d) The histogram of cytoplasmic PKA RIα expression in HCT116 and LoVo. (e) The histogram of cytoplasmic JNK1 expression in HCT116 and LoVo. (f) The histogram of cytoplasmic c‐Jun expression in HCT116 and LoVo. (g) The histogram of nuclear PKA RIα expression in HCT116 and LoVo. (h) The histogram of nuclear JNK1 expression in HCT116 and LoVo. (i) The histogram of nuclear c‐Jun expression in HCT116 and LoVo

Figure 4

The expression of syncytin 1 in human colorectal tumor tissues. A, Syncytin 1 expression in (a) well‐differentiated primary colorectal cancer (group I), (b) moderately differentiated primary colorectal cancer (group II), and (c) poorly differentiated primary colorectal cancer (group III) (200×). B, Corresponding metastases. (d) Corresponding metastasis of the well‐differentiated primary colorectal cancer (group IV), (e) Corresponding metastasis of the moderately differentiated primary colorectal cancer (group V), and (f) Corresponding metastasis of the poorly differentiated primary colorectal cancer (group VI) (200×)