Brautigan, David L.
F. Palmer Weber Medical Research Professor, Emeritus, Microbiology, Immunology, and Cancer Biology
- BA, Chemistry, Kalamazoo College, Kalamazoo, MI
- MS, Chemistry, Northwestern University, Evanston, IL
- PhD, Biochemistry, Northwestern University, Evanston, IL
- Postdoc, Biochemistry, University of Washington School of Medicine, Seattle, WA
Biochemistry, Biotechnology, Cancer Biology, Molecular Biology, Structural Biology
Cell signaling in Cancer and Ciliopathies.
Research: Cell Signaling in Cancer and Ciliopathies
As a Professor Emeritus I no longer operate my own research laboratory or supervise graduate students, but am active as a collaborator and advisor for projects listed below. We are investigating the role of protein post-translational modifications, namely ubiquitination and phosphorylation, in intracellular signaling pathways that govern cell proliferation and survival. We have a project to develop drugs to inhibit deubiquitinases (DUBs) to reduce hormone receptor levels in prostate and breast cancers. Another project uses a novel natural agent, caffeic acid, to locally and selectively poison tumors in the liver. Lastly, we are studying how protein phosphorylation involving an extremely specific kinase called CILK1 controls the structure and function of the primary cilium, the cellular antenna for sensing hormonal stimuli and transducing the signals.
Targeting DUBs for Androgen Receptor (AR) ablation in prostate cancer.
(A collaboration with James Larner, M.D., Chairman of UVA Department of Radiation Oncology, in association with the Chemical Genomics Center of NCATS, NIH)
Prostate cancer is a major health challenge that afflicts about one in six American men. Hormone deprivation is a primary approach to therapy, depriving the androgen receptor (AR) of its activating ligand to limit tumor growth. However, tumor cells still survive because of mutations in the AR, or by AR-dependent signaling in the absence of ligand. Jim Larner and I are interested in promoting the degradation of the AR protein in cancer cells, which would potentially arrest tumor growth. UVA investigator George Amorino, who died from cancer at a young age, found that the metabolite 2-methoxyestradiol (2-ME) a compound tested as an anti-tumor agent itself, caused a synergistic enhancement of radiation-induced tumor regression in xenograft mouse models (Cancer Res. 2007; see Figure). The 2-ME alone (solid squares) had no effect on tumor growth compared to untreated controls (open squares), and radiation alone (open circles) gave some delayed growth inhibition, compared to the combination of 2-ME plus radiation that stopped the tumors from growing (solid circles).
We studied this effect of 2-ME and found by FACS that it involved cell cycle arrest of the cells at G2/M. There was a loss of the AR from the cells and we went on to find 2-ME induces this time-dependent loss of AR from human prostate cancer cells via ubiquitin-mediated proteolysis, which is inhibited by adding the drug MG-132 (see Figure). (Oncogene, 2014 PMID 23246967).
Using RNAi to knockdown different proteins we showed that degradation of AR was dependent on hsp70 and its partner CHIP, an E3 ligase that conjugates ubiquitin to AR. More recent data indicates this involves CHIP activation in a phosphorylation-dependent mechanism that involves Aurora A kinase phosphorylation of CHIP (Molec Cancer Res. 2017). The polyubiquitination of AR is opposed by deubiquitinases (DUBs), so inhibiting these proteases would in effect enhance AR polyubiquitination and promote AR protein degradation. Indeed, we have found that siRNA knockdown of certain DUBs does reduce AR levels in human prostate cancer cell lines. We narrowed down candidate DUBs, produced these highly active recombinant enzymes and adapted an extremely sensitive assay for DUB activity. In a collaboration with the National Center for Advancing Translational Science (NCATS) Chemical Genomics Center (NCGC) this DUB assay is being applied in high throughput screening of chemical libraries to discover compounds that act as DUB inhibitors. That is our first step down the path of drug discovery, and we anticipate doing hit-to-lead development in the near future.
Application of Caffeic Acid as A Novel Therapy for Liver Cancer
A collaboration with Luke R. Wilkins, MD from the section of Vascular and Interventional Radiology in the UVA Department of Radiology and Medical Imaging
Patients with hepatocellular carcinoma (HCC) tumors are treated clinically by interventional radiology, involving embolization of the hepatic arteries via catheterization (called Trans Arterial Embolization, TAE). This treatment blocks blood flow to create a hypoxic tumor microenvironment that limits growth of the tumors. Malignant foci with low intratumor O2 levels use glucose for anaerobic glycolysis that generates significant levels of lactate (see Figure 6, from review article). However, high intracellular lactate levels are deleterious. The low pH caused by lactate accumulation reduces glycolysis via inhibition of the rate-limiting enzyme phosphofructokinase. Cells have multiple mechanisms to regulate intracellular pH (see Figure). One mechanism reduces intracellular lactate by efflux of lactate out of cells via transmembrane proteins known as monocarboxylate transporters (MCTs), especially MCT4.
Caffeic or ferulic acids are derivatives of cinnamic acid that inhibit monocarboxylate transporters. Our hypothesis is that under hypoxia the HCC cells shift to anaerobic glycolysis and survive by exporting the excess lactate produced via MCT4, which is induced by the hypoxia-induced transcription factor HIF-1a. We showed in vitro that caffeic and ferulic acid blocked MCT4 and reduced extracellular acidification of N1S1 cells in response to glucose. This work was published recently in Cardiovasc. Intervent. Radiol. 2017, PMID 27872984). We continued to characterize the pharmacological effects of CA on the metabolism and survival of HCC cells and published the results in 2018 (Brautigan et al., BBRC 505: 612-617 PMID 30278886). We have devised a proprietary slow-release version of CA in plastic minibeads used for TAE procedures. We were able to show in a pharmacokinetic study that essentially none of the CA leaks into the circulation or urine for hours after arterial placement of the beads in vivo, showing restricted locoregioanl delivery of CA as an anti-tumor agent. Continuing work is going to compare the efficacy of CA TACE vs TAE and TACE in a pre-clinical animal model. Our work was supported by a Thelma R. Swortzel collaborative research award from the UVA School of Medicine and an American Cancer Society Institutional Research Grant (ACS-IRG), and now by DOD and ACS.
Ciliopathies and the Primary Cilium
(A collaboration with Zheng (John) Fu, Ph.D., UVA Department of Pharmacology)
On the surface of essentially every animal cell is a diminutive (2-5 um) protruberance called the primary cilium. This microtubule-based villus emanates from the basal body of the centriole and functions as a cellular antenna that senses and transduces environmental and hormonal stimuli to regulate intracellular signaling events. The primary cilium is dynamic, and is resorbed before centrosome duplication in each cell cycle, then reassembled following cell division. The primary cilium is enriched in receptors and multiple signaling pathways including Hedgehog (Hh), Wnt, Notch, Hippo, GPCR, RTK, mTOR, and TGF?. The formation of tissues during embryonic development and the homeostasis and function of adult organs depends on signaling in the primary cilium. At least 35 different human diseases are collectively called ciliopathies because they are associated with mutations in proteins localized in the primary cilium that affect cilium assembly and signaling. These include Bardet-Biedl syndrome, Joubert syndrome, Polycystic Kidney Disease, endocrine-cerebro-osteodysplasia (ECO) syndrome, and forms of juvenile epilepsy.
We have been studying CILK1 (ciliogenesis associated kinase 1), an essential gene for human development that encodes a serine/threonine protein kinase in the MAPK superfamily. Unlike classic MAPKs, CILK1 is not acutely activated by growth factors through the canonical dual-specificity MEKs. Instead, CILK1 is activated by phosphorylation of Thr157 in the TDY motif by CDK20 (cyclin-dependent kinase 20), also known as CCRK (cell cycle-related kinase). Mutated homologs of vertebrate CILK1 and CDK20 in Chlamydomonas and C.elegans cause elongated flagella and cilia. In mice, both Cilk1 knock-in and Cilk1 knockout mutations have recapitulated a human ciliopathy phenotype with defective cilia morphology and function in multiple organ systems including lung, brain, and skeleton. Our research seeks to understand how cAMP and other upstream signals from receptors activate CILK1 and what are the substrates of CILK1 critical for its functions in control of cilia formation and length.
We recently published a review article on the subject: Fu, Z., Gailey, C. D., Wang, E. J. and D.L. Brautigan (2019) Ciliogenesis associated Kinase 1 (CILK-1) targets and functions in various organ systems. FEBS Lett. 593: 2990-3002. PMID 31506943