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Scott Bultman, Department of Genetics, Lineberger Comprehensive Cancer Center, UNC-Chapel Hill
Bridging nutrition, metabolism, the microbiome, and cancer susceptibility
Nutrition
Immune/Inflammation
Metabolism Genetic background
Cancer susceptibility
Microbiome
Lumen Intestinal Epithelium Lamina Propria
Gut microbiota
B cell T cell
Macrophage Dendritic cell Immune cells
Fiber
+ Dietary/digestive components Metabolites that influence cancer =
Polyphenols
Meat Bile acids Oncometabolites: 2° bile acids (DCA), hydrogen sulfide Tumor-suppressive metabolites: SCFAs (butyrate), equol, urolithins
Diet & Microbiota Influence Cancer Risk and Progression
Fiber-Microbiota-Butyrate Axis
Fiber
Bacterial fermentation
Butyrate Mitochondria: Energetics
Nucleus: Epigenetics
SCFAs FAO
HDACi Ligand for GPCRs
Evidence for Butyrate & Cancer Prevention Tumor-derived cell lines: Microbiome studies:
Butyrate Proliferation Apoptosis Differentiation
Butyrate producers: CRC cases < controls
A Limitation of Metagenomic Studies
Microbiota composition: Cases vs controls
Association study: ∆microbiome = cause or consequence?
Gnotobiotic Facility
GEMMs
Metagenomic Sequencing
Next Phase of Microbiome Research Bacterial Culture
from cataloging to function
Does Dietary Fiber Protect Against Colorectal Cancer (CRC)?
Complex polysaccharides
Butyrate
Maintains digestive movement
(insoluble)
(soluble)
Two proposed general mechanisms:
But …
#2
#1
…Lack of Universal Consensus: Conflicting Results from Human Epidemiology Studies
Not Protective Protective
Fuchs et. al. 1999
Lanza et. al. 2002
Park et. al. 2005
Lanza et. al. 2007
Ferguson et. al. 2003
Bingham et. al. 2003
Magalhães et. al. 2011
Janike et. al. 2011
grain/fiber
Ecologic and migration supportive; prospective cohort mixed:
Possible Reasons for Conflicting Results
Various fiber sources
Differences in microbiota
Other dietary factors
Compliance
Hypothesis: Dietary fiber protects against CRC in a microbiota- and butyrate-dependent manner.
Genetic heterogeneity
AOM Tumors Resemble Human CRC
mouse
human CRC T S M A ED LD adult
normal human
4.00
0.25
1.00
Kaiser et al. (2007) Genome Biol.
Similar mutation spectrum: Similar transcriptome profiles:
Apc Kras Braf β Catenin p53
“Vogelgram”
Experimental Approach- Part 2
Control Control Control Experimental
AOM AOM
Low Fiber Diet Low Fiber Diet High Fiber Diet High Fiber Diet
Butyrate
Hypothesis: tumor burden
Combination of High Fiber & B. fib Confer Tumor Suppressive Effect
High Dose (5 AOM)
Experimental: Diminished incidence & multiplicity
Tum
ors/
mou
se
*
Combination of High Fiber & B. fib Also Inhibits Tumor Growth & Progression
Experimental: Diminished size
*
Combination of High Fiber & B. fib Also Inhibits Tumor Growth & Progression
Experimental: Less progressed
High Grade Low Grade
A Mutant Strain of B. fibrisolvens to Confirm Butyrate Mechanism
Asanuma et al. (2005) Current Microbiology
Butyryl-CoA synthesis operon:
Mutant
Wild type
0.8-kb deletion
Butyrate is a Causal Factor
High Dose (5 AOM)
Tum
ors/
mou
se
Attenuated protective effect
Stronger protective effect
* *
+
# # # #
What is Butyrate Doing Mechanistically?
#1 It accumulates in tumors…
LC-MS:
Tumor
Control Groups
HFD WT B. fib
TB (-) B. fib
Buty
rate
(pm
ols/
µg o
f pro
tein
)
**
*
* *
Tumor Normal
What is Butyrate Doing Mechanistically?
#2 …which increases histone acetylation due to HDAC inhibition and…
Tumor
H3
H3ac
HFD LFD TB HFD LFD
(-) B. fib WT B. fib
IHC: Western Blot:
What is Butyrate Doing Mechanistically? #3 … cell proliferation and apoptosis
Accumulation HDAC inhibitor Epigenetic gene regulation Cell proliferation Apoptosis (Ki-67) (cleaved caspase 3)
Q: Why does butyrate accumulate in tumors in first place?
( p21)
Fewer/smaller tumors
( Fas)
A: Warburg Effect- Part 1 of 2
LC-MS:
*
Normal Tumor
*
LDHA IHC:
Tumor
Tumors undergo Warburg effect …
A: Warburg Effect- Part 2 of 2
Butyrate Proliferation +Warburg
-Warburg
… butyrate dependent on Warburg effect in cell culture model
(siLDHA, glutamine>glucose)
Current Working Model
Fiber
Cell proliferation Apoptosis
Butyrate
Treg cells & anti-inflammatory effects, barrier function, GPCR signaling
Caveats: • Approach is reductionistic for diet and microbiome. One genetic bkg. • Model is simplistic. Focused on cancer cell autonomous effects.
Nevertheless …
… Human Cancer Relevance
LC-MS: H3ac WBA:
*
H3ac
H3
Normal Colon Adenocarcinoma 1 2 1 2
Normal Colon Adenocarcinoma (n = 11) (n = 11)
Advantages of Chemoprevention Strategy
Endogenous HDAC inhibitor: Synthetic HDAC inhibitors:
Probiotics & prebiotics Anticancer chemotherapeutics Minimal off-target/adverse effects Clinical trials & FDA approval
Tumor Proliferation
Apoptosis
Future: Combinatorial Human Studies
Prospective-cohort fiber study
+ GWAS or exome sequencing + Gut microbiome
Hypothesis: Responders and non-responders should have microbiota and/or genetic differences. Resolve previous conflicting results.
Precision medicine
Integrating Precision Medicine into Cancer Prevention
Population-based studies
Biomarkers: Dietary compliance or disease state
Mechanism: Etiology and disease progression
Translational potential
Genetic background/GWAS
Microbiome
Metabolome
Emerging Evidence for Precision Medicine & Interactome
Nutrition (fiber)
Immune/Inflammation (Treg cells)
Metabolism (butyrate)
Genetic background
Cancer susceptibility
Microbiome (Clostridium)
Milk Bifidobacteria SNPs
Diet Choline production/ 1C metabolism
SNPs Tumor Tumor Normal Normal
Bultman lab Dallas Donohoe Darcy Holley Sarah Renner Kaitlin Curry Alicia Greenwalt
Funding:
Acknowledgments Texas A&M David Threadgill Colorado State University Elizabeth Ryan
UNC Maureen Bower Leonard Collins Jim Swenberg Virginia Godfrey Stephanie Montgomery
Ongoing Experiments
Control Diet High Fiber Diet
Butyrate
Hypothesis: Butyrate mediates tumor suppression, in part, through a cancer cell non-autonomous manner via diminished inflammation
Wild type RAG2-deficient IL10-deficient
Tumor burden Inflammation
ChIP seq: Colon, tumor, immune cells H3K9ac, H3K27ac, HDACs
Other dietary factors? Omega-3 fatty acids (PUFAs) Synergize with butyrate in vitro
±
Lumen Intestinal Epithelium Lamina Propria
Commensal bacteria
B cell T cell
Macrophage
Dendritic cell
Immune cells
Fiber
+ Dietary/digestive components Metabolites (oncometabolites, tumour-suppressive metabolites)
=
Polyphenols
Meat Bile acids 2° bile acids (DCA), hydrogen sulfide SCFAs (butyrate), equol, urolithins
Cancer incidence
Cancer treatment
Beyond Butyrate: Gut Microbiota & Cancer
CpG oligo, platinum chemotherapy Anti-CTLA4, anti-PD-L1 immunotherapy β-glucuronidase & chemotherapy (CPT)
Systemic effects via circulation
Irinotecan
SN-38-glucuronide
HUMAN ESTERASE
SN-38
HUMAN UGT
HUMAN TOPO I
efficacy
SN-38-glucuronide
SN-38
BACTERIAL GUS
epithelial cell death Dose-Limiting
Diarrhea
Large GI
Prodrug
Active
Active
Inactive
Inactive
Liver
Drugs Targeting Bacterial Enzymes to Prevent Adverse Effects of Chemotherapeutics in GI Tract
Matt Redinbo, UNC Aadra Bhatt, UNC
Gnotobiotic Facility
GEMMs
Metagenomic Sequencing
Next Phase of Microbiome Research Bacterial Culture
from cataloging to function
Organoid co-culture system -Higher throughput -Less $ -Not Caco-2 cells -Not exisiting miniguts
3D Crypts-on-a-Chip
-Lgr5+ stem cells & each differentiated cell type on ECM scaffold in media with Wnt gradient -Anatomically & physiologic normal crypt -Customize genetic background & disease state -Assay barrier function, transport, metabolism, etc. -Amenable to microbial co-culture -Can be interconverted to 2D culture system for high-throughput screening
Nancy Allbritton, UNC Scott Magness, UNC
The Warburg Effect Cancer cells shift from oxidative metabolism to aerobic glycolysis:
Glycolysis
Pyruvate
2-4 ATP TCA cycle
36 ATP
Glucose
cytosol
mitochondria
+ O2
Lactate
Otto Warburg 1931 Nobel Prize Recently “rediscovered”
OXPHOS
Cancer Cells Take Up Glucose at High Levels
Warburg effect is basis of tumor imaging in the clinic:
FDG
Butyrate Accumulation & Histone Acetylation
IC50 = 300
13C-Butyrate (mM)
Reported IC50
Warburg Effect No Warburg Effect
LC-MS/MS: WBA:
H3ac
H3
Butyrate (mM)
Hypothesis: Butyrate can also increase histone acetylation by a mechanism other than HDAC inhibition.
Butyrate Regulates Histone Acetylation via ACL-Dependent Mechanism
#1
#2
Butyrate
Butyrate
Butyrate
ACL
TCA cycle
Acetyl CoA
Citrate
β ox
idat
ion
mitochondria
Citrate Acetyl CoA
nucleus
Ac
nucleus HDACs
HATs
Lipid biosynthesis
AKT
Glucose
X
C13
C13
TSA Control
- + - + siACL
Vehicle TSA
H3ac
β actin
ACL
#1
#2
Butyrate
Butyrate
Butyrate
ACL
TCA cycle
Acetyl CoA
Citrate
β ox
idat
ion
mitochondria
Citrate Acetyl CoA
nucleus
Ac
nucleus HDACs
HATs
Lipid biosynthesis
AKT
Glucose
X
ACL-Dependent Mechanism Predominates in Normal Colonocytes
#1
#2
Butyrate
Butyrate
Butyrate
ACL
TCA cycle
Acetyl CoA
Citrate
β ox
idat
ion
mitochondria
Citrate Acetyl CoA
nucleus
Ac
nucleus HDACs
HATs
Lipid biosynthesis
AKT
Glucose
X
Cancerous Colonocyte
Warburg Effect No Warburg Effect
Reg
ulat
ion
of H
isto
ne
Acet
ylat
ion
and
Tran
scrip
tion
0.5 1.0 2.0
Acetyl-CoA Donor/HAT Mechanism
HDAC Inhibitor Mechanism
Butyrate (mM)
A
Butyrate
Lys
Mitochondria
Histone
Ac
HDACs HATs
Butyrate Butyrate Glucose B C
D
Butyrate Glucose
Nucleus
Histone
Ac
HATs HDACs
Histone
Ac
HATs HDACs
CO2
Normal Colonocyte
Proliferation
5.0
Butyrate Increases Histone Acetylation by Stimulating HATs & Inhibiting HDACs
Histone Histone Lys Lys Ac
Ac Coenzyme A
HATs
Coenzyme A
Ac
HDACs
Normal cells; lower doses; proliferation genes
Cancer cells; higher doses; pro-apoptotic genes (Fas)
Biological material (buccal swabs, stool samples, tissue biopsies, etc.): -Can be obtained from disease cases and controls
Principal Component Analysis to identify differences between samples such as disease cases and controls Identification of microbes significantly enriched in different body sites or at one site in cases vs controls
Prepare genomic DNA
Computational assembly and analysis of microbial sequence reads to quantify abundance of microbiota
Next-generation DNA sequencing: -16S rRNA -whole-genome shotgun (discard human sequence reads)
Microbiome Studies
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