Targeting ketone body metabolism in mitigating gemcitabine resistance
Krizia Rohena-Rivera, Sungyong You, Minhyung Kim, Sandrine Billet, Johanna ten Hoeve, Gabrielle Gonzales, Chengqun Huang, Ashley Heard, Keith Syson Chan, Neil A. Bhowmick
Krizia Rohena-Rivera, Sungyong You, Minhyung Kim, Sandrine Billet, Johanna ten Hoeve, Gabrielle Gonzales, Chengqun Huang, Ashley Heard, Keith Syson Chan, Neil A. Bhowmick
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Research Article Oncology

Targeting ketone body metabolism in mitigating gemcitabine resistance

Abstract

Chemotherapy is often combined with surgery for muscle invasive and nonmuscle invasive bladder cancer (BCa). However, 70% of the patients recur within 5 years. Metabolic reprogramming is an emerging hallmark in cancer chemoresistance. Here, we report a gemcitabine resistance mechanism that promotes cancer reprogramming via the metabolic enzyme OXCT1. This mitochondrial enzyme, responsible for the rate-limiting step in β-hydroxybutyrate (βHB) catabolism, was elevated in muscle invasive disease and in patients with chemoresistant BCa. Resistant orthotopic tumors presented an OXCT1-dependent rise in mitochondrial oxygen consumption rate, ATP, and nucleotide biosynthesis. In resistant BCa, knocking out OXCT1 restored gemcitabine sensitivity, and administering the nonmetabolizable βHB enantiomer (S-βHB) only partially restored gemcitabine sensitivity. Suggesting an extrametabolic role for OXCT1, multi-omics analysis of gemcitabine sensitive and resistant cells revealed an OXCT1-dependent signature with the transcriptional repressor OVOL1 as a master regulator of epithelial differentiation. The elevation of OVOL1 target genes was associated with its cytoplasmic translocation and poor prognosis in a cohort of patients with BCa who have been treated with chemotherapy. The KO of OXCT1 restored OVOL1 transcriptional repressive activity by its nuclear translocation. Orthotopic mouse models of BCa supported OXCT1 as a mediator of gemcitabine sensitivity through ketone metabolism and regulating cancer stem cell differentiation.

Authors

Krizia Rohena-Rivera, Sungyong You, Minhyung Kim, Sandrine Billet, Johanna ten Hoeve, Gabrielle Gonzales, Chengqun Huang, Ashley Heard, Keith Syson Chan, Neil A. Bhowmick

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Figure 5

Genomic analysis of isogenic bladder cancer lines revealed OVOL1 as a master regulator.

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Genomic analysis of isogenic bladder cancer lines revealed OVOL1 as a ma...
(A) Master regulator analysis highlighting the significant transcription factors SOX15, OVOL1, and TBX2. (B) Survival curve associating OVOL1 targets in pretreatment and gemcitabine/cisplatin-treated samples from the Peter C. Black dataset (HR = 2.35, P = 0.0119) (24). (C) Venn diagram highlighting the proportion of OVOL1 and OVOL2 targets in the differentially expressed genes. More details in Supplemental Tables 5 and 6. (D) qPCR confirmation of the top OVOL1 and OVOL2 targets — 5637: P (parental), GR (gemcitabine resistant), KO (GR-OXCT1 KO); T24: PC (parental, untreated), PG (parental, gemcitabine-treated), EC (gemcitabine-exposed, untreated), EG (gemcitabine-exposed, gemcitabine treated); and UM-UC3: PC (parental, untreated) PG (parental, gemcitabine-treated) KC (OXCT1-KO, untreated), KG (OXCT1-KO, gemcitabine treated) — all normalized to their respective control. Resistant or exposed cells show a higher expression of OVOL1 targets suggesting that OVOL1 is not repressing its targets. Heatmap represents fold change to each respective control. (E) Representative Western blot images for OXCT1, OVOL1, and OVOL2 using β-actin, lamin B1, and Tom20 as cytoplasmic, nuclear, and mitochondrial markers, respectively. Representative densitometry values were normalized to their respective loading control and relative to the parental vehicle sample. Western blots were repeated 4 times. (F) Cell surface CD44 and CD36 were measured by flow cytometry of indicated lines following 72 hours gemcitabine treatment at IC50 doses. (G) Schematic representation of the roles of OXCT1 and OVOL1 in gemcitabine sensitive and gemcitabine resistant cells. Under lower OXCT1, OVOL1 is free to translocate to the nucleus and repress target genes including PPARG, promoting a more differentiated state and reduced FAO. Under gemcitabine-resistant conditions, there is higher OXCT1, and cytoplasmic OVOL1 promotes PPARG, FAO, and a more dedifferentiated state.