Correlations of the expression of Cx43, SCFFBXW7, p-cyclin E1 (Ser73), p-cyclin E1 (Thr77) and p-cyclin E1 (Thr395) in colon cancer tissues

Abstract

BACKGROUND

Previous cellular studies have demonstrated that elevated expression of Cx43 promotes the degradation of cyclin E1 and inhibits cell proliferation through ubiquitination. Conversely, reduced expression results in a loss of this capacity to facilitate cyclin E degradation. The ubiquitination and degradation of cyclin E1 may be associated with phosphorylation at specific sites on the protein, with Cx43 potentially enhancing this process by facilitating the phosphorylation of these critical residues.

AIM

To investigate the correlation between expression of Cx43, SKP1/Cullin1/F-box (SCF)^FBXW7, p-cyclin E1 (ser73, thr77, thr395) and clinicopathological indexes in colon cancer.

METHODS

Expression levels of Cx43, SCF^FBXW7, p-cyclin E1 (ser73, thr77, thr395) in 38 clinical colon cancer samples were detected by immunohistochemistry and were analyzed by statistical methods to discuss their correlations.

RESULTS

Positive rate of Cx43, SCF^FBXW7, p-cyclin E1(Ser73), p-cyclin E1 (Thr77) and p-cyclin E1 (Thr395) in detected samples were 76.32%, 76.32%, 65.79%, 5.26% and 55.26% respectively. Positive expressions of these proteins were not related to the tissue type, degree of tissue differentiation or lymph node metastasis. Cx43 and SCF^FBXW7(r = 0.749), p-cyclin E1 (Ser73) (r = 0.667) and p-cyclin E1 (Thr395) (r = 0.457), SCF^FBXW7 and p-cyclin E1 (Ser73) (r = 0.703) and p-cyclin E1 (Thr395) (0.415) were correlated in colon cancer (P < 0.05), and expressions of the above proteins were positively correlated in colon cancer.

CONCLUSION

Cx43 may facilitate the phosphorylation of cyclin E1 at the Ser73 and Thr195 sites through its interaction with SCF^FBXW7, thereby influencing the ubiquitination and degradation of cyclin E1.

Key Words: Colon cancer; Cx43; SCF^FBXW7; Phosphorylation of cyclin E1; Sites of cyclin E1; Correlation analysis

Core Tip: In this study, we found that SKP1/Cullin1/F-box (SCF)^FBXW7 exhibited a positive correlation with cyclin E1 expression at positions ser73 and thr395 in colon cancer tissues, suggesting that SCF^FBXW7 may facilitate phosphorylation-dependent degradation by interacting with cyclin E1 at these specific sites. Furthermore, the expressions of Cx43 and SCF^FBXW7 were positively correlated, as well as their association with the levels at positions ser73 and thr395. This indicates that Cx43 may enhance the phosphorylation of these two sites through its interaction with SCF^FBXW7, thereby promoting the ubiquitination and degradation of cyclin E1.

INTRODUCTION

Colon cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related mortality[1]. Despite significant advancements in recent years with chemotherapeutic agents targeting colon cancer, severe side effects, toxicity, and drug resistance remain major clinical challenges[2]. Therefore, it is imperative to identify additional therapeutic targets for colon cancer treatment to mitigate these adverse effects.

Cx43 is a crucial component of cell gap junctions, regulating cell proliferation and differentiation through intercellular communication via gap junctions[3]. Cx43 plays a significant role in modulating the cell cycle of tumor cells, and its abnormal expression is often associated with tumorigenesis[3,4-6]. Our previous studies have demonstrated that Cx43 expression is reduced in various cancer tissues, including lung and esophageal cancers[7-9].

Our previous study has indicated that AKAP95 and Cx43, functioning as a pair of molecular switches, influence the cell cycle by regulating the expression of cyclin Ds and cyclin Es, as well as the expression and activity of their associated cyclin-dependent kinases[10,11]. In lung cancer, Cx43 down-regulates cyclin E1 expression, thereby inhibiting Rb phosphorylation and E2F activation, which hinders cell cycle progression through competitive bindings to cyclin E1 with AKAP95[10]. Furthermore, Cx43 can negate the protective effect of AKAP95 on cyclin Es[10].

Ubiquitination is a crucial and widespread mechanism of protein degradation within cells. This pathway is catalyzed by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3)[12-14]. Among the family of E3 Ligases, SCF has garnered significant attention in recent years. SCF^FBXW7 has been identified as a specific ubiquitin ligase for cyclin E1, mediating its ubiquitination and subsequent degradation[15-17]. The SCF protein complex contains phosphate-binding domains that recognize and bind to phosphorylated sites on substrates, thereby facilitating substrate reactions[18]. We hypothesize that phosphorylation of cyclin E1 is a prerequisite for its ubiquitination and degradation; thus, we examined the expression levels of Cx43, SCF^FBXW7, and the phosphorylation status at various sites of cyclin E1 in 38 clinical colon cancer tissues while analyzing correlations among these proteins.

MATERIALS AND METHODS

Sample sources

Thirty-eight colon cancer tissue samples were all surgical specimens from colon cancer patients in Air Force Hospital of Eastern Theater during 2015-2020. The age range of the patients was 30-94, with an average of 67.3 years. The pathological diagnosis of patient samples was clear, including 1 case of T1N0M0, 1 case of T1N1aM1a, 5 cases of T2N0M0, 1 case of T2N1aM0, 2 cases of T3N0M0, 16 cases of T4aN0M0, 1 case of T4aN0M1a, 3 cases of T4aN1bM0, 1 case of T4aN1bM1a, 3 cases of T4aN1cM0, 2 cases of T4aN1cM1a, 1 case of T4aN2bM0 and 1 case of TisN0M0 (TNM typing).

Reagents and antibodies

General two-step immunohistochemical kit (PV-9000) and DAB chromogenic solution were purchased from Fuzhou Maxim Biotechnology Development Co., Ltd; Hematoxylin dye was purchased from Solarbio Co., Ltd; Anti-rabbit Cx43 (AF0137), FBXW7(DF12400), p-cyclin E1 (Ser73) (AF4413), and p-cyclin E1 (Thr395) (AF3235) were purchased from Affinity Biosciences (Ohio, United States). Anti-rabbit p-cyclin E1 (Thr77) (ET1612-31) was purchased from Hangzhou HuaAn Biotechnoloy Co., Ltd (Hangzhou, China).

Immunohistochemical assay

The specimens were fixed in 10% neutral formaldehyde and embedded in paraffin, and then 4 µm serial sections were cut. After hydration with xylene and gradient ethanol, the sections were placed in citric acid/ sodium citrate buffer for thermal repair. After blocking endogenous peroxidase, the primary antibody was incubated overnight (8 hours) and then the reaction enhancer and horseradish peroxidase labeled secondary antibody were incubated successively. After incubating DAB reagent, slides were put into hematoxylin staining solution to stain the nucleus, and dehydrate the slides after cleaning.

Positive criteria

Brownish yellow in cancer tissues was considered as protein positive expression under the light microscope. Each tissue slide was randomly observed in 10 different visual fields, and 200 tumor cells were counted in each visual field. The ratio of the number of positive cells to the total number of counted cells and the positive intensity were taken as the judgment criteria. Under the light microscope, no brownish yellow in the field of vision or proportion of positive cells was less than 10% would be record as ‘-’; proportion of positive cells was ≥ 10% and < 30% would be recorded as ‘+’; proportion of positive cells was ≥ 30% and < 50% would be recorded as ‘++’; proportion of positive cells was ≥ 50% would be recorded as ‘+++. ‘-’ was regarded as negative results while ‘+’, ‘++’ and ‘+++’ were regarded as positive results. The slides were read by different experimenters for 3 times, and the average results were taken for statistical analysis.

Statistical analysis

IBM SPSS statistical was used for statistical analysis. χ² test was used for rate comparison, Spearman rank correlation analysis was used for correlation analysis, and test level α = 0.05.

RESULTS

Expression of Cx43, SCF^FBXW7 and p-cyclin E1(Ser73, Thr77, Thr395) in colon cancer tissues

In 38 cases of colon cancer, positive expression of Cx43 and SCF^FBXW7 were 29 and 29 cases respectively, and their positive rates were 76.32% and 76.32%; positive expression of phosphorylation site Ser73, Thr77 and Thr395 of cyclin E1 were 25, 2 and 21 cases, and positive rates were 65.79%, 5.26% and 55.26% respectively (Table 1).

Table 1 Expressions of proteins in colon cancer tissues (n = 38).

	Cx43	SCFFBW7	p-cyclin E1 (Ser73)	p-cyclin E1 (Thr77)	p-cyclin E1 (Thr395)
Positive	29	29	25	2	21
Negative	9	9	13	36	17
Positive rate (%)	76.32	76.32	65.79	5.26	55.26

Positive rates of expression of Cx43, SCF^FBXW7, p-cyclin E1 (Ser73) and p-cyclin E1 (Thr395) were significantly higher than p-cyclin E1 (Thr77). Cx43, SCF^FBXW7, p-cyclin E1 (Ser73) and p-cyclin E1 (Thr395) were expressed in both nucleus and cytoplasm (Figure 1).

Figure 1 Expression of Cx43, Skp1-Cullin1-F-box (SCF)^FBXW7, Phosphorylation of cyclin E1 (p-cyclin E1) (Ser73, Thr77, Thr395) in 38 colon cancer tissue cases. A: High expression of Cx43 in normal tissues; B: Low expression of Cx43 in colon cancer tissues; C: Medium expression of Cx43 in colon cancer tissues; D: High expression of Cx43 in colon cancer tissues; E: High expression of SCF^FBXW7 in normal tissues; F: Low expression of SCF^FBXW7 in colon cancer tissues; G: Medium expression of SCF^FBXW7in colon cancer tissues; H: High expression of SCF^FBXW7in colon cancer tissues; I: High expression of p-cyclin E1 (Ser73) in normal tissues; J: Low expression of p-cyclin E1(Ser73) in colon cancer tissues; K: High expression of p-cyclin E1 (Ser73) in colon cancer tissues; L: Negative expression of p-cyclin E1 (Thr77) in normal tissues; M: Negative expression of p-cyclin E1 (Thr77) in colon cancer tissues; N: High expression of p-cyclin E1(Thr395) in normal tissues; O: Medium expression of p-cyclin E1(Thr395) in colon cancer tissues; P: High expression of p-cyclin E1 (Thr395) in colon cancer tissues.

Correlation analysis of SCF^FBXW7, p-cyclin E1(Ser73, Thr77, Thr395)

Tables 2, 3, and 4 showed analysis results of correlation between expression of SCF^FBXW7 and p-cyclin E1 (Ser73), SCF^FBXW7 and p-cyclin E1 (Thr77), and SCF^FBXW7 and p-cyclin E1 (Thr395) in 38 colon cancer tissue cases. The expression of SCF^FBXW7 and p-cyclin E1 (ser73), SCF^FBXW7 and p-cyclin E1 (Thr395) were statistically significant (P < 0.05) and their spearman correlation coefficient were 0.703 and 0.415 respectively. The results showed that there were positive correlations between the expression of SCF^FBXW7 and p-cyclin E1 (ser73), and SCF^FBXW7 and p-cyclin E1 (thr395) in colon cancer. No correlation between SCF^FBXW7 and p-cyclin E1 (Thr77) was detected this time.

Table 2 Correlation analysis between FBXW7 and p-cyclin E1 (Ser73) in colon cancer tissues.

FBXW7	Ser73(-)	Ser73(+)	Ser73(++)	Ser73(+++)	r	P value
-	7	2	0	0	0.703	0.001
+	5	7	2	2
++	1	1	4	2
+++	0	0	2	3

Table 3 Correlation analysis between FBXW7 and p-cyclin E1 (Thr77) in colon cancer tissues.

FBXW7	Thr77(-)	Thr77(+)	Thr77(++)	Thr77(+++)	r	P value
-	9	0	0	0	0.079	0.638
+	15	0	0	0
++	9	0	0	0
+++	5	0	0	0

Table 4 Correlation analysis between FBXW7 and p-cyclin E1 (Thr395) in colon cancer tissues.

FBXW7	Thr395(-)	Thr395(+)	Thr395(++)	Thr395(+++)	r	P value
-	5	3	0	0	0.415	0.010
+	8	5	2	1
++	4	2	3	0
+++	0	1	2	2

Correlation analysis of Cx43, SCF^FBXW7, p-cyclin E1(Ser73), p-cyclin E1(Thr77) and p-cyclin E1(Thr395)

Tables 5, 6, 7, and 8 showed analysis results of correlation between expression of Cx43 and SCF^FBXW7, Cx43 and p-cyclin E1 (Ser73), Cx43 and p-cyclin E1 (Thr77), and Cx43 and p-cyclin E1 (Thr395) in 38 colon cancer tissue cases. The expression of Cx43 and SCF^FBXW7, Cx43 and p-cyclin E1 (ser73), Cx43 and p-cyclin E1 (Thr395) were statistically significant (P < 0.05) and their spearman correlation coefficient were 0.749, 0.667 and 0.457 respectively. The results showed that there were positive correlations between the expression of Cx43 and SCF^FBXW7, Cx43 and p-cyclin E1 (ser73), and Cx43 and p-cyclin E1 (Thr395) in colon cancer while there was no correlation between Cx43 and p-cyclin E1 (Thr77).

Table 5 Correlation analysis between Cx43 and FBXW7 in colon cancer tissues.

Cx43	FBXW7(-)	FBXW7(+)	FBXW7(++)	FBXW7(+++)	r	P value
-	6	3	0	0	0.749	0.001
+	3	6	3	0
++	0	6	4	1
+++	0	0	2	5

Table 6 Correlation analysis between Cx43 and p-cyclin E1 (Ser73) in colon cancer tissues.

Cx43	Ser73(-)	Ser73(+)	Ser73(++)	Ser73(+++)	r	P value
-	8	1	0	0	0.667	0.001
+	2	7	2	1
++	3	2	3	3
+++	0	0	3	3

Table 7 Correlation analysis between Cx43 and p-cyclin E1 (Thr77) in colon cancer tissues.

Cx43	Thr77(-)	Thr77(+)	Thr77(++)	Thr77(+++)	r	P value
-	9	0	0	0	0.262	0.112
+	12	0	0	0
++	11	0	0	0
+++	6	0	0	0

Table 8 Correlation analysis between Cx43 and p-cyclin E1(Thr395) in colon cancer tissues.

Cx43	Thr395(-)	Thr395(+)	Thr395(++)	Thr395(+++)	r	P value
-	5	3	0	1	0.457	0.004
+	8	3	1	0
++	4	4	3	0
+++	0	1	3	2

DISCUSSION

Our long-term studies have showed that AKAP95 and Cx43 are a pair of "molecular switches", regulating the progress of cell cycle by affecting the expression of cyclin Es[7-11,19]. AKAP95 and Cx43 could competitively bind to cyclin Ds and cyclin Es, regulating degradation of them: AKAP95 could bind to cyclin Ds and cyclin Es, preventing them from degradation, on the one hand, and inhibiting Cx43 promoting degradation of cyclin Ds and cyclin Es by binding to Cx43; Cx43, on the contrary to AKAP95, could accelerate the degradation of cyclin Ds and cyclin Es[10].

Ubiquitination is an important way of protein hydrolysis in cells. To discuss whether Cx43 affects the degradation of cyclin E1 and its expression level by regulating cyclin E1’s ubiquitination, we further detected the expression of Cx43 and SCF^FBXW7 and their correlation in clinical colon cancer tissues this time. Our data showed that there was a positive correlation between expression of Cx43 and SCF^FBXW7, which was in line with the relevant report of ‘Cx43 promote the degradation of cyclin E1/2 and hinder the progress of cell cycle’[15-17].

Protein ubiquitination and degradation mediated by SCFs are related to the phosphorylation of substrate molecules[18]. For instance, phosphorylation of Thr286 site of cyclin D1 has been reported to be a necessary condition for cyclin D1’s ubiquitination and degradation[20]. However, there is still a lack of researches on the relationship between ubiquitination and phosphorylation of related site and of cyclin E1. We detected correlation between ubiquitination and three phosphorylation sites of cyclin E1 this time, and our data showed that phosphorylation of sites Ser73 and Thr395 of cyclin E1 was positively correlated with SCF^FBXW7 and Cx43 respectively, and Cx43 and SCF^FBXW7 were also positively correlated as well, suggesting that ubiquitination and degradation of cyclin E1 might be related to the phosphorylation of sites Ser73 and Thr395 of cyclin E1, and Cx43 might promote the ubiquitination and degradation of cyclin E1 by promoting the phosphorylation of these two sites.

Low positive rate of phosphorylation level of sites Thr77 of cyclin E1 was detected and no correlation between expression levels of Cx43 or SCF^FBXW7 showed this time, suggesting that the phosphorylation of site Thr77 might not be related to ubiquitination and degradation of cyclin E1, and Cx43 did not affect its phosphorylation.

In general, our results of 38 clinical colon cancer samples preliminarily showed that ubiquitination and degradation of cyclin E1 were related to phosphorylation of site Ser73 and Thr395, but further experimental verification was still needed. In addition, Cx43 might affect degradation of cyclin E1 by affecting its phosphorylation and the expression of SCF^FBXW7.

CONCLUSION

It is postulated that Cx43 might facilitate the phosphorylation of cyclin E1 at the Ser73 and Thr395 sites via its interaction with SCF^FBXW7, thereby exerting an impact on the ubiquitination and degradation of cyclin E1.