Trim24 Promotes Mammary Tumor Development By Upregulating Metabolic Reducing Power

Author

Shucheng Miao, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical SciencesFollow

Author ORCID Identifier

https://orcid.org/0000-0002-7306-0966

Date of Graduation

8-2021

Document Type

Thesis (MS)

Program Affiliation

Genetics and Epigenetics

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Michelle Barton and Guillermina Lozano

Committee Member

Powel Brown

Committee Member

Went Li

Committee Member

Wenliang Li

Committee Member

Sendurai Mani

Abstract

Metabolic reprograming is an emerging hallmark of cancer cells. Changes in cellular metabolism can contribute to cancer cell survival and tumor progression. Tripartite motif-containing protein 24 (TRIM24) is an E3 ligase for p53, a nuclear receptor co-regulator, and a histone reader. Over expression of TRIM24 correlates with poor overall survival of breast cancer patients. Previously, our lab created a mouse model that conditionally over-expresses (COE) TRIM24 protein in mammary epithelia (Trim24^COE) and develops carcinosarcoma or metaplastic mammary tumors (70% of all Trim24^COE tumors), a rare and aggressive triple-negative subtype called metaplastic TNBC in humans (MpBC). RNA-sequencing revealed that Trim24^COE mouse mammary tumors have upregulated glycolysis and epithelial-to-mesenchymal transition (EMT). Trim24^COE carcinosarcoma cell-derived spheroids showed irregular, poorly differentiated structures that were more invasive compared to spontaneous, control MMTV-cre mammary tumor-derived spheroids. Using ¹³C₆-glucose/glutamine tracing, we found Trim24^COE spheroids recapitulated some of the metabolic features of Trim24^COE carcinosarcomas like upregulated glycolysis and exhibited aberrantly upregulated antioxidant defenses. We found Trim24^COE spheroids exhibited low intracellular and mitochondrial ROS, an increased NADP(H) pool, and were more resistant to oxidative stress. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and immunoblotting validated that enzymes of one-carbon metabolism and de novo glutathione synthesis pathways were upregulated in Trim24^COE spheroids and that upregulation was depended on TRIM24 over expression. NRF2 is a key regulator of cellular antioxidant response and often found over expressed in many different types of cancers. Nrf2 mRNA level and NRF2 protein expression were both upregulated in the Trim24^COE spheroids and knocking down TRIM24 significantly decreased Nrf2 expression as well as NRF2-regulated gene expression. Metabolic inhibitors that target enzymes in one-carbon metabolism and de novo glutathione synthesis effectively decreased Trim24^COE spheroids viability. The effect of PHGDH inhibitor CBR5884, GLS inhibitors CB839, and GCLC inhibitor was enhanced with TRIM24 knock-down, indicating that TRIM24 plays a critical role in oxidation-reduction (REDOX)-related cell survival.

Here, with metabolomic studies, we uncovered the potential metabolic vulnerabilities in Trim24^COE MpBC spheroids. TRIM24 over expression led to upregulated production of reducing equivalents which further reduce oxidative stress and increase antioxidant defense in Trim24^COE spheroids. Targeting TRIM24 and the corresponding metabolic enzymes in combination could shed light on potential strategies for inhibiting tumor progression of TRIM24-overexpressing MpBC. The work described here supports further investigations of these pathways in MpBC, for which no targeted therapies exist.

Keywords

metaplastic breast cancer, metabolism, ROS, redox, NADPH