Depletion of SUMO ligase hMMS21 impairs G1 to S transition in MCF-7 breast cancer cells

• Published in: Biochimica et biophysica acta Vol. 1820; no. 12; pp. 1893 - 1900
• Main Authors: Ni, Huey-Juin, Chang, Ying-Nang, Kao, Pu-Hong, Chai, Shin-Pei, Hsieh, Ya-Hsin, Wang, Duo-Hsiang, Fong, Jim C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2012
• Subjects: Blotting, Western; breast neoplasms; Breast Neoplasms - genetics; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; cell cycle; cell growth; Cyclin E - genetics; Cyclin E - metabolism; Cyclin E/CDK2; Cyclin-Dependent Kinase 2 - genetics; Cyclin-Dependent Kinase 2 - metabolism; Cyclin-Dependent Kinase Inhibitor p21 - genetics; Cyclin-Dependent Kinase Inhibitor p21 - metabolism; Cyclin-Dependent Kinase Inhibitor p27 - genetics; Cyclin-Dependent Kinase Inhibitor p27 - metabolism; cyclins; DNA repair; E2F1; E2F1 Transcription Factor - genetics; E2F1 Transcription Factor - metabolism; Female; flow cytometry; Fluorescent Antibody Technique; G1 Phase - physiology; G1–S transition; Humans; Immunoprecipitation; Ligases - antagonists & inhibitors; Ligases - genetics; Ligases - metabolism; MCF-7; messenger RNA; MMS21; mutants; neoplasm cells; Oncogene Proteins - genetics; Oncogene Proteins - metabolism; p21; Phosphorylation; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction; Retinoblastoma Protein - genetics; Retinoblastoma Protein - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA interference; RNA, Messenger - genetics; RNA, Small Interfering - genetics; S Phase - physiology; Signal Transduction; Tumor Cells, Cultured; uterine cervical neoplasms; Western blotting; E2F1; G1–S transition; MMS21; Cyclin E/CDK2; MCF-7; p21
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2012.08.002

hMMS21 is a human SUMO ligase required for DNA damage repair and mitotic progression in HeLa cervical cancer cells. Owing to the diversity of cancer, we further investigated the effect of hMMS21-depletion on MCF-7 breast cancer cells. hMMS21-depletion was achieved by RNA interference. Cellular hMMS21 and E2F1 mRNA levels were estimated by RT-PCR and real-time PCR. Cell cycle profile was assessed by flow cytometry. Western blot and co-immunoprecipitation were used to determine the protein levels of various factors involved in G1–S transition and CDK2- or CDK4-associated p21 and p27. Kinase activity of cyclin E/CDK2 was measured in anti-cyclin E immunoprecipitate. hMMS21-depletion induced slower cell growth and G1–S transition. While it had no effect on cyclin D1 or phospho-Rb (S807/811) levels, hMMS21-depletion provoked lower E2F1 levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity correlated with increased cellular p21CIP1 levels and CDK2–p21 association. Moreover, ectopic expression of Flag-hMMS21 but not its ligase-inactive mutant rescued the decreased growth rates of hMMS21-depletd cells. Thus, depletion of hMMS21 seems to impair G1–S transition due to lowered E2F1 protein levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity is probably attributable to its greater association with p21 as a result of increased p21 levels. In addition, hMMS21-mediated sumoylation appears to be involved. This study demonstrates that hMMS21 is required for G1–S transition in breast cancer cells and implies that manipulation of hMMS21-mediated sumoylation may alter the growth rates of breast cancer cells. ► hMMS21-depletion slows MCF-7 breast cancer cell growth. ► hMMS21-depletion impairs G1–S progression. ► hMMS21-depletion lowers E2F1 expression and cyclin E/CDK2 activity. ► hMMS21-depletion increases p21 expression and CDK2-p21 association. ► SUMO ligase activity of hMMS21 is involved.