KIFC1 depends on TRIM37-mediated ubiquitination of PLK4 to promote centrosome amplification in endometrial cancer

Abstract

Endometrial cancer (EC), as one of the most common cancers, severely threatens female reproductive health. Our previous study has shown that Kinesin family member C1 (KIFC1) played crucial roles in the progression of EC. In addition, abnormal centrosome amplification, which was reported to be partially regulated by KIFC1, usually occurred in different cancers. However, whether KIFC1 promoted EC through centrosome amplification and the potential mechanism remain to be revealed. The present study demonstrated that overexpressed KIFC1, which exhibited a worse prognosis, had a positive correlation with an increased number of centrosomes in human EC samples. In addition, KIFC1 overexpression in EC cells prompted centrosome amplification, chromosomal instability, and cell cycle progression. Moreover, we demonstrated that KIFC1 inhibited E3 ubiquitin-protein ligase TRIM37 to maintain the stability of PLK4 by reducing its ubiquitination degradation, and finally promoting centrosome amplification and EC progression in vitro. Finally, the contributing role of KIFC1 and the inhibitory effect of TRIM37 on EC development and metastasis was verified in a nude mouse xenograft model. Our study elucidated that KIFC1 depends on TRIM37-mediated reduced ubiquitination degradation of PLK4 to promote centrosome amplification and EC progression, thus providing a potential prognostic marker and promising therapeutic target for EC in the future.

Fig. 1. Higher expression of KIFC1 and more centrosome amplification were observed in EC.

A Gene expression of KIFC1 in normal (n = 35) and EC samples (n = 546) based on the TCGA database. B Survival curves of KIFC1 expression in EC patients from Kaplan–Meier plotter. C The protein expressions of KIFC1 and γ-tubulin in EC specimens (T) and adjacent normal tissues (N). D The correlation analysis between KIFC1 and γ-tubulin protein expressions based on EC specimens and normal tissues. E The H & E staining and immunofluorescence analysis with KIFC1 and γ-tubulin antibodies in EC specimens (left) and adjacent normal tissues (right) (scale bar: 200 μm). **P < 0.01.

Fig. 2. KIFC1 promoted centrosome amplification.

A The immunofluorescence analysis with γ-tubulin and α-tubulin antibodies in KIFC1-overexpressed EC cells (scale bar: 10 μm). B The protein expressions in KIFC1-overexpressed EC cells. *P < 0.05, **P < 0.01.

Fig. 3. KIFC1 promoted centrosome amplification dependent on PLK4.

A The mRNA expression of PLK4 in EC cell lines. B The protein expression of PLK4 in EC cell lines. C The mRNA expression of PLK4 in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. D The protein expression of PLK4 in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. E The mRNA expressions of PLK4 and KIFC1 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). F The protein expressions of PLK4 and KIFC1 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). G The immunofluorescence analysis with γ-tubulin and α-tubulin antibodies in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa) (scale bar: 10 μm). *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01.

Fig. 4. PLK4 promoted the EC progression.

A CCK8 assay in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). B Colony formation assay in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Transwell assay and quantitative analysis of migratory and invasive abilities of PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). **P < 0.01, ##P < 0.01.

Fig. 5. KIFC1 controlled the ubiquitination of PLK4.

A CHX chase analysis of PLK4 protein half-life after KIFC1 overexpression and knockdown in EC cell lines (HEC-1A and Ishikawa). B The protein expression of KIFC1 and PLK4 in HEC-1A and Ishikawa cells treated with MG132 or CQ after KIFC1 knockdown. C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 overexpression detected by ubiquitination assay. **P < 0.01, ##P < 0.01, ^^P < 0.01.

Fig. 6. KIFC1 regulated the EC progression by controlling TRIM37 expression.

A The mRNA and protein expressions of TRIM37 in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. B The mRNA and protein expressions of TRIM37 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 or TRIM37 overexpression detected by ubiquitination assay. D The mRNA expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). E CCK8 assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). F Colony formation assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). G Transwell assay and quantitative analysis of migratory and invasive abilities of TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). H The protein expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01.

Fig. 7. KIFC1 promoted tumor metastasis dependent on TRIM37 in vivo.

A General images of mice and tumors in the subcutaneous xenograft of HEC-1A cells after TRIM37 and KIFC1 overexpression. B The tumor volume and weight. C Representative H & E staining and IHC staining of Ki-67, KIFC1, TRIM37, and PLK4 in tumors (scale bar: 200 μm for left; 100 μm for right). D Representative TUNEL images of tumors (scale bar: 200 μm for left; 100 μm for right). E Representative H & E staining images of lung tissues (scale bar: 200 μm for left; 100 μm for right); F Fluorescence images (left) and quantitative analysis (right) of mouse lung metastasis. *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01.