High MDMX (MDM4) in Mutant p53 Expressing Breast Cancer Cells Activates the CXCL12-CXCR4 Metastasis Signaling Axis

Author

Rusia H. Lee, The Graduate Center, City University of New YorkFollow

Date of Degree

6-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Jill Bargonetti

Committee Members

Frida Kleiman

David Foster

Anjana Saxena

Zhaohui Feng

Subject Categories

Cancer Biology | Cell Biology | Developmental Biology

Keywords

TNBC, Metastasis, CTC, MDM2, MDMX, CXCR4

Abstract

The metastasis-promoting G-protein coupled receptor (GPCR) CXCR4 is activated by the cytokine CXCL12 (also known as SDF-1) to promote metastasis through CXCR4 upregulation. High CXCR4-expressing cancer cells utilize CXCL12, the exclusive endogenous ligand of CXCR4, to intravasate into the blood as circulating tumor cells (CTCs) which then extravasate to distant metastatic sites. We previously determined the oncogenes, Mouse Double Minute 2 and 4 (MDM2 and MDM4, also known as MDMX), drive TNBC metastasis and positively correlate with high expression of CXCR4. Interestingly, this upregulation of CXCR4 was only observed in primary tumor cells grown in the tumor microenvironment (TME) of xenograft mice, but not in cancer cells grown in tissue culture, suggesting a cell non-autonomous CXCL12 ligand to the CXCR4 receptor-mediated pathway.

To assess if the MDMX-dependent upregulation of CXCR4 in a TME context could be recapitulated by addition of chemokine CXCL12 in a tissue culture system. We used a variety of assays (e.g., immunoblotting, qRT-PCR, migration/chemotaxis, and affinity purification assays) with MDM2 or MDM4 expressing- or depleted- MDA-MB-231 cell lines, Consistent with the literature to date, addition of CXCL12 in a cell culture system increased CXCR4 protein and activated the PI3K/AKT signaling pathway, as measured by phospho-AKT S473. Surprisingly, we observed CXCL12-mediated increase in MDM2 and MDMX protein levels in a manner dependent only on MDMX, suggesting a positive feedforward activation loop between the CXCL12-CXCR4 and MDMX. We began investigating if CXCL12-mediated increase in MDMX protein may be a result of increased protein stability, although future studies may help further address this question.

Consistent with this model, the xenograft mice CTC cell lines derived from the MDM2- and MDMX-knockdowns exhibited significant reduced capacity for cell migration. However, MDMX nor MDM2 knockdown CTC cells did not have a significant decrease in CXCL12-CXCR4/AKT signaling, unlike in the non-CTC MDA-MB-231 knockdown cells. This suggests that these CTC cells extravasated into the bloodstream independently of MDMX. Together these results demonstrate the potential for MDMX to impact the primary TME for metastatic signaling through the activation of the CXCL12-CXCR4/AKT signaling pathway. Our findings highlight a novel regulatory loop between MDMX protein and CXCL12-CXCR4/AKT signaling and demonstrate differences in MDM2 and MDMX pathways towards metastasis.

Recommended Citation

Lee, Rusia H., "High MDMX (MDM4) in Mutant p53 Expressing Breast Cancer Cells Activates the CXCL12-CXCR4 Metastasis Signaling Axis" (2024). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/5790