14 th International VHL Medical/Research Symposium Oct 29-31, 2020 UQCRH Downregulation Promotes Warburg Effect in Renal Cell Carcinoma Cells Xin Lu, Ph.D. Boler Assistant Professor Department of Biological Sciences Boler-Parseghian Center for Rare and Neglected Diseases Harper Cancer Research Institute University of Notre Dame Full member, Indiana University Simon Cancer Center
Lu Laboratory at University of Notre Dame Genito- Myeloid urinary MDSC, neutrophil, ICB Prostate cancer, penile cancer cells and cancers Wang*, Lu* et al. Can Disc. 2016 Lu et al. G&D. 2017 immuno- Lu et al. Nature. 2017 and Lu et al. Can Res. 2018 therapy Feng…Lu, PNAS. 2018 Huang…Lu, Nat Comm. 2020 biology Liu…Lu, Int J Mol Sci. 2020 Rahmy…Lu, BBRC. 2020 Wen…Lu, J Pathol Clin Res. 2020 Zhao…Lu, Pharmacol Ther. 2020 VHL disease Synonymous yet Gene function in pathogenic VHL mutation familial and sporadic VHL disease See poster by Dr. Fang Liu • UQCRH in ccRCC Yanting Luo Louise Mouse modeling for Medina Bengtsson VHL hemangioblastoma and pheochromocytoma Undergrads at Notre Dame
Prominence of aerobic glycolysis in human ccRCC Wettersten, H. I. et al. (2017) Nat. Rev. Nephrol. Clear cell renal cell carcinoma (ccRCC) is among the most aggressive tumor types for patients with VHL syndrome. ccRCC is considered a “disease of cell metabolism” (Linehan et al., 2010). Warburg effect in ccRCC is supported by results from TCGA, CPTAC, metabolomics and human cancer [U- 13 C]glucose-tracing experiments. HIF-1 negatively regulates mitochondrial biogenesis and O 2 consumption in VHL-deficient ccRCC (Zhang et al., 2007).
UQCRH: methylated and downregulated in ccRCC Ubiquinol-cytochrome c reductase hinge protein (UQCRH), also known as cytochrome b-c1 complex subunit 6, is an integral subunit of complex III. UQCRH functions as a regulatory protein for the electron transfer reaction between cytochrome c1 and c. In TCGA study of ccRCC, UQCRH is the top-ranked gene with promoter hypermethylation and inversed correlation at the mRNA level. The role of UQCRH in cancer is poorly understood. We hypothesize that UQCRH modulates the metabolic activity and proliferative potential of ccRCC cells.
UQCRH downregulation associated with poor prognosis TCGA P = 5.1 X 10 -50 Protein level (Z-value) UQCRH protein level in Human Protein Atlas Normal ccRCC ccRCC High Medium Low Not detected Not detected High Low Normal Tumor Normal Tumor (n=84) (n=110) (n=3) (n=12) Human Protein Atlas CPTAC
UQCRH-like is unlikely to compensate for low UQCRH Align UQCRH and UQCRHL Identities Positives Gaps 88/91(97%) 89/91(97%) 0/91(0%) UQCRH MGLEDEQKMLTESGDPEEEEEEEEELVDPLT UQCRHL MGLEDEQKMLTESGDPEEEEEEEEELVDPLT UQCRH TVREQCEQLEKCVKARERLEL C DE R VSSRSH UQCRHL TVREQCEQLEKCVKARERLEL Y DE H VSSRSH UQCRH TEEDCTEELFDFLHA R DHCVAHKLFNNLK UQCRHL TEEDCTEELFDFLHA K DHCVAHKLFNNLK TCGA It is unlikely for UQCRHL to functionally compensate for UQCRH downregulation in human ccRCC development.
Ectopic overexpression and CRISPR/cas9 knockout of UQCRH in ccRCC cell lines UQCRH DNA methylation (RRBS) 10 mRNA (RNAseq) 1.1 786O KMRC2 1.0 UQCRHL 5 UQCRH 0.9 786O 0.8 β -actin KMRC2 0 0.7 0.0 0.5 1.0 0.0 0.2 0.4 0.6 0.8 1.0 DNA methylation (RRBS) UQCRH KMRC2 786O Decitabine ( μ M) 0 1 3 10 UQCRH UQCRH β -actin β -actin KMRC2
Restored UQCRH expression in KMRC2 repolarized mitochondrial membrane potential JC-1 dye KMRC2-pcDNA KMRC2-UQCRH MFI=2.38 X 10 4 MFI=1.84 X 10 4 JC-1 aggregate (red) ΔΨ m Green low Green high Green low Green high 72.4% 24.9% 94.8% 3.99% ΔΨ m JC-1 monomer (green)
UQCRH overexpression in KMRC2 partially overturns Warburg effect
UQCRH knockout in 786O has litter effect metabolically 786O cells already had very high glycolytic activity and severely weakened mitochondrial function, due to hyperactivated HIF signaling as a result of VHL mutation (Zhang et al. Cancer Cell 11, 407-420, 2017). Therefore, it is conceivable that UQCRH knockout would not further strengthen the Warburg-like metabolic activity in this cell line.
UQCRH overexpression slows down KMRC2 growth in vitro and in vivo • UQCRH knockout did not affect 786O proliferation in vitro or as orthotopic tumors in vivo
Summary: UQCRH downregulation constrains OXPHOS and reinforces Warburg effect in ccRCC Luo Y*, Medina Bengtsson L*, Wang X*, … Lu X. Sci Rep. 2020. PMID: 32929120
Acknowledgements Lu Lab Yanting “Raven” Luo (undergrad student) Louise Medina Bengtsson (undergrad student) Xuechun Wang (PhD student) Collaborators Kasturi Haldar Zach Schafer Curtis Pettaway (MD Anderson) Magaly Martinez (U. Puerto Rico) Liang Cheng (IUSM) Jun Wan (IUSM) Boler Family Foundation I save human lives!
https://biology.nd.edu/labs/lu-lab/
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