UQCRH Downregulation Promotes Warburg Effect in Renal Cell - - PowerPoint PPT Presentation

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 14th International VHL Medical/Research Symposium Oct 29-31, 2020

Lu Laboratory at University of Notre Dame

Genito- urinary cancers and biology

VHL disease

Myeloid cells and immuno- therapy

MDSC, neutrophil, ICB Wang*, Lu* et al. Can Disc. 2016 Lu et al. Nature. 2017 Feng…Lu, PNAS. 2018 Liu…Lu, Int J Mol Sci. 2020 Wen…Lu, J Pathol Clin Res. 2020 Zhao…Lu, Pharmacol Ther. 2020 Prostate cancer, penile cancer Lu et al. G&D. 2017 Lu et al. Can Res. 2018 Huang…Lu, Nat Comm. 2020 Rahmy…Lu, BBRC. 2020

See poster by Dr. Fang Liu

Synonymous yet pathogenic VHL mutation Mouse modeling for VHL hemangioblastoma and pheochromocytoma Gene function in familial and sporadic VHL disease

• UQCRH in ccRCC

Yanting Luo Louise Medina Bengtsson

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-13C]glucose-tracing experiments.  HIF-1 negatively regulates mitochondrial biogenesis and O2 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

Normal (n=84) Tumor (n=110) Protein level (Z-value)

P = 5.1 X 10-50 Normal (n=3) Tumor (n=12) High Medium Low Not detected

UQCRH protein level in Human Protein Atlas Normal ccRCC

Low High

ccRCC

Not detected

TCGA CPTAC Human Protein Atlas

UQCRH-like is unlikely to compensate for low UQCRH

UQCRH MGLEDEQKMLTESGDPEEEEEEEEELVDPLT UQCRHL MGLEDEQKMLTESGDPEEEEEEEEELVDPLT UQCRH TVREQCEQLEKCVKARERLELCDERVSSRSH UQCRHL TVREQCEQLEKCVKARERLELYDEHVSSRSH UQCRH TEEDCTEELFDFLHARDHCVAHKLFNNLK UQCRHL TEEDCTEELFDFLHAKDHCVAHKLFNNLK

Identities Positives Gaps 88/91(97%) 89/91(97%) 0/91(0%) Align UQCRH and UQCRHL

 It is unlikely for UQCRHL to functionally compensate for UQCRH downregulation in human ccRCC development.

TCGA

Ectopic overexpression and CRISPR/cas9 knockout of UQCRH in ccRCC cell lines

0.0 0.5 1.0 5 10

UQCRH DNA methylation (RRBS) mRNA (RNAseq)

KMRC2 786O 0.0 0.2 0.4 0.6 0.8 1.0 0.7 0.8 0.9 1.0 1.1

DNA methylation (RRBS) UQCRH UQCRHL

KMRC2 786O

β-actin UQCRH

β-actin UQCRH 0 1 3 10 Decitabine (μM) KMRC2

β-actin UQCRH KMRC2 786O

Restored UQCRH expression in KMRC2 repolarized mitochondrial membrane potential

JC-1 dye ΔΨm JC-1 aggregate (red) JC-1 monomer (green) Greenlow 72.4% Greenhigh 24.9% Greenlow 94.8% Greenhigh 3.99% KMRC2-pcDNA KMRC2-UQCRH ΔΨm

MFI=2.38 X 104 MFI=1.84 X 104

UQCRH overexpression in KMRC2 partially

• verturns 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
• rthotopic 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

Collaborators

Kasturi Haldar Zach Schafer Curtis Pettaway (MD Anderson) Magaly Martinez (U. Puerto Rico) Liang Cheng (IUSM) Jun Wan (IUSM)

Acknowledgements

Lu Lab

Yanting “Raven” Luo (undergrad student) Louise Medina Bengtsson (undergrad student) Xuechun Wang (PhD student)

I save human lives!

Boler Family Foundation

https://biology.nd.edu/labs/lu-lab/