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Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Published on: December 5, 2017

E2F1 promotes cell cycle progression by stabilizing spindle fiber in colorectal cancer cells

Zejun Fang^1,2,3, Min Lin¹, Shenghui Chen^3,4

¹Central Laboratory, Sanmen People's Hospital of Zhejiang Province, Sanmen, 317100, China.

Cellular & Molecular Biology Letters

|October 11, 2022

Abstract

Background:

E2F1 is a transcription factor that regulates cell cycle progression. It is highly expressed in most cancer cells and activates transcription of cell cycle-related kinases. Stathmin1 and transforming acidic coiled-coil-containing protein 3 (TACC3) are factors that enhance the stability of spindle fiber.

Methods:

The E2F1-mediated transcription of transforming acidic coiled-coil-containing protein 3 (TACC3) and stathmin1 was examined using the Cancer Genome Atlas (TCGA) analysis, quantitative polymerase chain reaction (qPCR), immunoblotting, chromatin immunoprecipitation (ChIP), and luciferase reporter. Protein-protein interaction was studied using co-IP. The spindle structure was shown by immunofluorescence. Phenotype experiments were performed through MTS assay, flow cytometry, and tumor xenografts. Clinical colorectal cancer (CRC) specimens were analyzed based on immunohistochemistry.

Results:

The present study showed that E2F1 expression correlates positively with the expression levels of stathmin1 and TACC3 in colorectal cancer (CRC) tissues, and that E2F1 transactivates stathmin1 and TACC3 in CRC cells. Furthermore, protein kinase A (PKA)-mediated phosphorylation of stathmin1 at Ser16 is essential to the phosphorylation of TACC3 at Ser558, facilitating the assembly of TACC3/clathrin/α-tubulin complexes during spindle formation. Overexpression of Ser16-mutated stathmin1, as well as knockdown of stathmin1 or TACC3, lead to ectopic spindle poles including disorganized and multipolar spindles. Overexpression of wild-type but not Ser16-mutated stathmin1 promotes cell proliferation in vitro and tumor growth in vivo. Consistently, a high level of E2F1, stathmin1, or TACC3 not only associates with tumor size, lymph node metastasis, TNM stage, and distant metastasis, but predicts poor survival in CRC patients.

Conclusions:

E2F1 drives the cell cycle of CRC by promoting spindle assembly, in which E2F1-induced stathmin1 and TACC3 enhance the stability of spindle fiber.

Keywords:

Cell cycle Colorectal carcinoma E2F1 Spindle fiber Stathmin1 TACC3

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