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. 2023 Dec 30;44(12):809-823.
doi: 10.1093/carcin/bgad070.

KCNN1 promotes proliferation and metastasis of breast cancer via ERLIN2-mediated stabilization and K63-dependent ubiquitination of Cyclin B1

Bin Xiao  1 , Qin Xiang  1 , Zihua Deng  2 , Daxiang Chen  1 , Shunhong Wu  1 , Yanxia Zhang  1 , Yaru Liang  1 , Shi Wei  3 , Guoqing Luo  2 , Linhai Li  1
Affiliations

KCNN1 promotes proliferation and metastasis of breast cancer via ERLIN2-mediated stabilization and K63-dependent ubiquitination of Cyclin B1

Bin Xiao et al. Carcinogenesis. .

Abstract

Potassium Calcium-Activated Channel Subfamily N1 (KCNN1), an integral membrane protein, is thought to regulate neuronal excitability by contributing to the slow component of synaptic after hyperpolarization. However, the role of KCNN1 in tumorigenesis has been rarely reported, and the underlying molecular mechanism remains unclear. Here, we report that KCNN1 functions as an oncogene in promoting breast cancer cell proliferation and metastasis. KCNN1 was overexpressed in breast cancer tissues and cells. The pro-proliferative and pro-metastatic effects of KCNN1 were demonstrated by CCK8, clone formation, Edu assay, wound healing assay and transwell experiments. Transcriptomic analysis using KCNN1 overexpressing cells revealed that KCNN1 could regulate key signaling pathways affecting the survival of breast cancer cells. KCNN1 interacts with ERLIN2 and enhances the effect of ERLIN2 on Cyclin B1 stability. Overexpression of KCNN1 promoted the protein expression of Cyclin B1, enhanced its stability and promoted its K63 dependent ubiquitination, while knockdown of KCNN1 had the opposite effects on Cyclin B1. Knockdown (or overexpression) ERLNI2 partially restored Cyclin B1 stability and K63 dependent ubiquitination induced by overexpression (or knockdown) of KCNN1. Knockdown (or overexpression) ERLIN2 also partially neutralizes the effects of overexpression (or knockdown) KCNN1-induced breast cancer cell proliferation, migration and invasion. In paired breast cancer clinical samples, we found a positive expression correlations between KCNN1 and ERLIN2, KCNN1 and Cyclin B1, as well as ERLIN2 and Cyclin B1. In conclusion, this study reveals, for the first time, the role of KCNN1 in tumorigenesis and emphasizes the importance of KCNN1/ERLIN2/Cyclin B1 axis in the development and metastasis of breast cancer.

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Conflict of interest statement

None of the authors has any conflict of interest.

Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
The expression of KCNN1 in breast cancer. (A) Heat map showing the mRNA expression of KCNN family proteins, including KCNN1, KCNN2 and KCNN3, in different molecular subtypes of breast cancer samples. The mean expressions of these genes in each subtype were converted by log2 transformation. The original gene expression data comes from TCGA database. (B) The mRNA level of KCNN1 in 1109 breast cancer samples and 113 normal samples. The original gene expression data comes from TCGA database. (C) The protein expression of KCNN1 in 140 breast cancer samples and 90 paracancer tissues were evaluated by IHC. The H-score represented the relative KCNN1 staining intensity. (D) The mRNA expression of KCNN1 in representative breast cancer cells with different molecular subtypes was determined by RT-qPCR. (E) Western blot showing the protein expression of KCNN1 in the same breast cancer cell lines as indicated in (D). **P < 0.01 by One-way ANOVA. ns, No significant.
Figure 2.
Figure 2.
The effect of KCNN1 on breast cancer cell proliferation and metastasis. (A) Western blot identified the KCNN1 overexpression and KCNN1 knockdown cells using lentiviral expression system and siRNA in MCF7 and MDA-MB-231. (B and C) CCK8 assay showing the effect of KCNN1 overexpression (B) and KCNN1 knockdown (C) on the proliferation of MCF7 and MDA-MB-231, respectively. (D) Clone formation assay measuring the clone number of MCF7 with KCNN1 overexpression and MDA-MB-231 with KCNN1 knockdown. (E and F) Edu assay showing the number of Edu staining cells in MCF7 with KCNN1 overexpression (F) and MDA-MB-231 with KCNN1 knockdown (G). (G and H) Cell migration ability affected by KCNN1 overexpression (G) and KCNN1 knockdown (H) was detected by wound healing assay. (I and J) The effect of KCNN1 overexpression (I) and KCNN1 knockdown (J) on breast cancer cell migration and invasion was measured by transwell assay. *P < 0.05, **P < 0.01 and ***P < 0.001 by One-way ANOVA.
Figure 3.
Figure 3.
Identification of the key gene expression and signaling pathway regulated by KCNN1. (A) Volcano Plot of the DEGs in KCNN1 overexpressing cells compared with the control cells. (B) GO analysis of the DEGs. (C) KEGG analysis of the DEGs. (D) Identification of 32 DEGs by qPCR and the comparison of qPCR result with the original RNA sequencing result for each DEG.
Figure 4.
Figure 4.
Validation of the interaction between KCNN1 and ERLIN2, as well as KCNN1 and Cyclin B1. (A) Co-immunoprecipitation (co-IP) showing the interaction between KCNN1 and ERLIN2. ERLIN2 immunoprecipitates were analyzed with HA tag antibody recognizing ERLIN2 and Flag tag antibody recognizing KCNN1. (B) co-IP showing the interaction between KCNN1 and ERLIN2. ERLIN2 immunoprecipitates were analyzed with HA tag antibody recognizing ERLIN2 and Flag tag antibody recognizing KCNN1. (C) The colocalization of KCNN1 and ERLIN2 in MCF7 cells. Scan bar = 50 μM. (D) The interaction between KCNN1 and Cyclin B1 was identified by co-IP. KCNN1 immunoprecipitates were analyzed with Flag tag antibody recognizing KCNN1 and HA tag antibody recognizing Cyclin B1. (E) The interaction between KCNN1 and Cyclin B1 was identified by co-IP. Cyclin B1 immunoprecipitates were analyzed with HA tag antibody recognizing Cyclin B1 and Flag tag antibody recognizing KCNN1.
Figure 5.
Figure 5.
The effect of KCNN1 on the mRNA expression, protein stability and ubiquitination of Cyclin B1. (A) The mRNA expression of ERLIN2 and Cyclin B1 was detected by qPCR in KCNN1 overexpression and the control cells. (B) The mRNA expression of ERLIN2 and Cyclin B1 was detected by qPCR in MDA-MB-231 cells transfected with siNC or siKCNN1. (C) The protein expression of ERLIN2 and Cyclin B1 in KCNN1 overexpression, KCNN1 knockdown and the control cells was quantified by western blot. (D) MCF7 cells in response to KCNN1 overexpression were treated with MG132 or cycloheximide at different time points. The expression of Cyclin B1 was quantified by western blot. (E) MCF7 cells were transfected with HA-Ub, Flag-Cyclin B1 or GFP-KCNN1 as indicated grouping. After transfection for 36 h, cells were treated with 20 μM MG132 for 6 h. The cells were immunoprecipitated with a Flag antibody and polyubiquitination of Cyclin B1 was analyzed by western blot. (F) MCF7 cells were transfected with HA-Ub (WT), HA-Ub (K63R), Flag-Cyclin B1 or GFP-KCNN1 as indicated grouping. After transfection for 36 h, cells were treated with 20 μM MG132 for 6 h. The cells were immunoprecipitated with a Flag antibody and polyubiquitination of Cyclin B1 was analyzed by western blot. (G) MCF7 cells with KCNN1 overexpression were transfected with siNC or siERLIN2. The protein expression of KCNN1, ERLIN2 and Cyclin B1 was measured by western blot. (H) MDA-MB-231 cells were transfected with siNC, siKCNN1, vector or Flag-ERLIN2. The protein expression of KCNN1, ERLIN2 and Cyclin B1 was measured by western blot. (I) HA-Ub, GFP-KCNN1, Flag-Cyclin B1 or siERLIN2 were transfected into MCF7 cells for 36 h. The cells were treated with 20 μM MG132 for 6 h. The polyubiquitination of Cyclin B1 was detected in the immunoprecipitates by HA antibody. ns, No significant; WT, wild type.
Figure 6.
Figure 6.
The effect of ERLIN2 in KCNN1 mediated breast cancer cell proliferation, migration and invasion. (A) CCK8 assay measuring the effect of ERLIN2 silencing on the proliferation of MCF7 cells with KCNN1 overexpression. (B) MDA-MB-231 cells were transfected with siNC, siKCNN1, vector or Flag-ERLIN2. The proliferation activity was detected by CCK8 assay. (C[1 and 2]) The colony formation assay showed the effect of ERLIN2 overexpression or knockdown on the cell survival of MCF7 cells with KCNN1 overexpression and MDA-MB-231 cells with KCNN1 knockdown. (D[1 and 2]) The wound healing assay evaluated the migration of KCNN1 overexpressing cells and KCNN1 knockdown cells transfected with siERLIN2 and Flag-ERLIN2 respectively. (E) The transwell migration (up) and invasion (down) assay evaluated the effect of ERLIN2 silencing or ERLIN2 overexpression in MCF7 cells with KCNN1 overexpression and MDA-MB-231 cells with KCNN1 knockdown. *P < 0.05, **P < 0.01 and ***P < 0.001 by One-way ANOVA.
Figure 7.
Figure 7.
Correlation of KCNN1, ERLIN2 and Cyclin B1 in breast cancer tissues. (A) The mRNA level of ERLIN2 in 1109 breast cancer samples and 113 normal samples. The original gene expression data comes from TCGA database. (B) The mRNA level of Cyclin B1 in 1109 breast cancer samples and 113 normal samples. The original gene expression data comes from TCGA database. (C) Representative IHC pictures showing the expression and locolization of KCNN1, ERLIN2 and Cyclin B1 in 30 breast cancer tissues and 30 paired adjacent tissues. The expressions of KCNN1, ERLIN2 and Cyclin B1 in each samples were represented by H-score. (D–F) The expression correlation of KCNN1 and ERLIN2 (D), KCNN1 and Cyclin B1 (E), as well as ERLIN2 and Cyclin B1 (F). (G and H) Kaplan–Meier plot depicting the overall survival of breast cancer patients with high or low expression of ERLIN2 (G) and Cyclin B1 (H) was analyzed using KM-plotter.
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