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The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-?5 in Breast Cancer Cells.

ABSTRACT: BACKGROUND:MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS:We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (?-galactosidase analysis). RESULTS:In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-?5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-?5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-?5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P=4.3 × 10-4 and P=7.1 × 10-4, respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-?5 both lead to increased mRNA levels of the endogenous MDM2-FL (P=.039 and P=.070, respectively) but also the proapoptotic gene PUMA (P=.010 and P=.033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION:We found P2-MDM2-10 and MDM2-?5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE:Alternative splicing may influence the oncogenic effects of the MDM2 gene.

SUBMITTER: Huun J

PROVIDER: S-EPMC5576977 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells.

Huun Johanna J Gansmo Liv B LB Mannsåker Bård B Iversen Gjertrud T GT Sommerfelt-Pettersen Jan J Øvrebø Jan Inge JI Lønning Per E PE Knappskog Stian S

Translational oncology 20170829 5

<h4>Background</h4>MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood.<h4>Methods</h4>We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apopto ...[more]

PMID: 28844019

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Project description:IntroductionMany human breast cancers overexpress the E3 ubiquitin ligase MDM2 and its homolog MDMX. Expression of MDM2 and MDMX occurs in estrogen receptor α-positive (ERα+) breast cancer and triple-negative breast cancer (TNBC). There are p53-independent influences of MDM2 and MDMX, and 80% of TNBC express mutant p53 (mtp53). MDM2 drives TNBC circulating tumor cells (CTCs) in mice, but the context-dependent influences of MDM2 and MDMX on different subtypes of breast cancers expressing mtp53 have not been determined.MethodsTo assess the context-dependent roles, we carried out MDM2 and MDMX knockdown in orthotopic tumors of TNBC MDA-MB-231 cells expressing mtp53 R280K and MDM2 knockdown in ERα+ T47D cells expressing mtp53 L194F. The corresponding cell proliferation was scored in vitro by growth curves and in vivo by orthotopic tumor volumes. Cell migration was assessed in vitro by wound-healing assays and cell intravasation in vivo by sorting GFP-positive CTCs by flow cytometry. The metastasis gene targets were determined by an RT-PCR array card screen and verified by qRT-PCR and Western blot analysis.ResultsKnocking down MDMX or MDM2 in MDA-MB-231 cells reduced cell migration and CTC detection, but only MDMX knockdown reduced tumor volumes at early time points. This is the first report of MDMX overexpression in TNBC enhancing the CTC phenotype with correlated upregulation of CXCR4. Experiments were carried out to compare MDM2-knockdown outcomes in nonmetastatic ERα+ T47D cells. The knockdown of MDM2 in ERα+ T47D orthotopic tumors decreased primary tumor volumes, supporting our previous finding that estrogen-activated MDM2 increases cell proliferation.ConclusionsThis is the first report showing that the expression of MDM2 in ERα+ breast cancer and TNBC can result in different tumor-promoting outcomes. Both MDMX and MDM2 overexpression in TNBC MDA-MB-231 cells enhanced the CTC phenotype. These data indicate that both MDM2 and MDMX can promote TNBC metastasis and that it is important to consider the context-dependent roles of MDM2 family members in different subtypes of breast cancer.

Dataset Information

The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-?5 in Breast Cancer Cells.

Publications

The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells.

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