The Environment’s Influence on Colon Cancer: A Disease Example that

Generates Questions to Answer for the Near Future

October 31, 2019

John M. Carethers, M.D., M.A.C.PC. Richard Boland Distinguished University Professor

John G. Searle Professor of Internal MedicineUniversity of Michigan

Disclosures• NONE (except NIH and University of Michigan research

funding)

Adenoma (polyp)-to-Carcinoma (cancer)

Adenoma Adenoma Adenoma

Adenoma Cancer Cancer

Familial Colorectal Cancer Risk

Sporadic (~65%) Familial (~35%)

Lynch (3-5%)

FAP (<1%)

Rare syndromes (~4%)

Colorectal Cancer145,600 cases annually in the US

Genetics of Adenomatous Polyposis Syndromes(dysplastic tissue)

AdenomatousSyndrome

Chromosomal Location(s)

Mutated Gene(s) Inheritance Pattern

Familial Adenomatous PolyposisGardner’s variantTurcot’s variant

5q21 APC Autosomal Dominant

MYH-Associated Polyposis 1p32-34 MYH (bi-allelic) Autosomal Recessive

POLE/POLD1 Polyposis 12q24.319q13.3

POLE (L424V)POLD1 (S478N)

AutosomalDominant

Lynch SyndromeMuir-Torre variantTurcot’s variant

2p16, 3p21, 2q32, 7p22, 2p16, 3p22

MSH2, MLH1, PMS2, MSH6, EPCAM (TACSTD1)

Autosomal Dominant

MSH3-Associated Polyposis(CMMRD)

5q14.1 MSH3 (bi-allelic) Autosomal Recessive

Familial CRC Type X 8q12 + 1q22 RPS20 + SEMA4A + HNRNPA0 + WIF1 + others

Autosomal Dominant

1991

2003

1993-1997

2005, 2014-

2013

2016

Genetics of Hamartomatous Polyposis Syndromes(disorganized but mature tissue or cells indigenous to the site of origin)

Hamartomatous Syndrome Chromosomal Location

Mutated Gene Frequency in Germline

PTEN Hamartoma SyndromeCowden’s Disease

Lhermitte-Duclos variantBannayan-Riley-Ruvalcaba Syndrome

10q22-23

10q22-23

PTEN/MMAC1/TEP1

PTEN/MMAC1/TEP1

>80%

~60%

Juvenile Polyposis Syndrome(with HHT overlap)

18q21.110q22-239q34

SMAD4BMPR1A/ALK3ENG

~20%~25%?

Peutz-Jeghers Syndrome 19p13.3 STK11/LKB1 70-90%

Hereditary Mixed Polyposis Syndrome

15q13-q14 duplication

GREM1 overexpression ?

Hyperplastic (Serrated) Polyposis Syndrome

17q22?1p

RNF43 (extremely rare)May not be familial

?

1997

1998, 2001, 2005

1997

2003/*2012

1996-*Nature Genetics 2012;44:699-703

Environmental and Heritable Factors for Cancer

Lichtenstein P et al. N Eng J Med 2000;343:78-85

• 44,788 pairs of twins• 10,803 persons among

9512 pairs of twins with cancer

• Overall, twin of a person with cancer had increased risk for that same cancer

• Only 3 tumors showed statistical heritablecomponent (prostate, colorectum, breast) among 28 sites

• Environment has overall principal role in causing sporadic cancer

Examples of Gene – Environmental Interactions for Cancer Development

VirusesHPVHBV

UV Exposure

Tobacco

GeneticallySusceptibleIndividual

Exposure Types of Cancer

High Fat Diet

Inflammation

Cervical, Anal, Head & NeckLiver

Skin, Melanoma

Lung, others

Colon, Breast

Colon, Pancreas, Stomach

DNA Damage Occurs Constantly• Block DNA

replication, transcription

• Genomic instability• Impair gene

expression• Mutagenic: human

disease• Stem cell

cytosol

nucleus

environment

UVOxidativeDamage

Carcinogens Cytosine DeaminationTo Uracil

DNAReplication

Errors

MUTATIONor

CELL DEMISE(without any repair)

Patient Risk Factors for CRC Development

Carethers JM. Dig Dis Sci 2015;60:711-721.

Comprehensive Mutation

Frequencies in 224 Primary

CRCs

Cancer Genome Atlas NetworkNature 2012;487:330-337

Hypermutated: MLH1 and POLε deficiencyNon-hypermutated: APC, TP53, KRAS, TTN, PIK3CA

~15% ~85%

Diet and CRC Risk in African Americans

O’Keefe SJD at al. Nat Comm 2015;6:6342

• 2 week food exchanges; mucosal biopsy• AA: fed high fiber, low-fat African-style diet• Rural Africans: fed high fat, low-fiber Western-style diet• Observed reciprocal changes in mucosal biomarkers

Butyrate(short chain fatty acid)

Deoxycholic acid (DCA)(secondary bile acid)

• During Dietary Intervention (DI)• AA: increased butyrate and decreased DCA• Rural Africans: decreased butyrate and increased DCA

ED: endoscopyHE: home environmentDI: dietary intervention

Aspirin Use Reduces CRC Death with Mutant PIK3CA

NEJM 2012;367:1596-1606

• Aspirin inhibits COX-2, down-regulates WT PI3K

• Mutant PI3K is predictor for aspirin effectiveness

Lynch Normal Accelerated (1-3 years)

80% Risk

FAP Normal 100% Risk

Normal Colonocyte

Tumor Initiation

Tumor Progression Carcinoma

Sporadic Normal(10-20 years)

5% Risk

Mean Age ~ 40 yrs

Mean Age ~ 40 yrs

Mean Age ~ 68 yrsNormal

(30-50 years)

Accelerated(5-20 years)

Normal colon Tubular adenoma High grade dysplasia CancerGrady W & Carethers JM. Gastroenterol 2008;135:1079-1099.

• Microbiome?• Inflammation Inflammation?• Microbiome?

• InflammationMetastasis

Type, Density and Location of Immune Cells Better Predictor Than Stage for Colorectal Cancer

• Immune cells– Can release mediators with pro-

angiogenic and pro-metastatic effects

• Tumor-infiltrating lymphocytes (CD3+)– Higher densities in center (CT)

and at invasive margin (IM) of tumor associated with improved patient survival

• CD8+ and granzyme B+ cytotoxic T lymphocytes (TH1 via IFNγ)

• CD45RO memory T cells

Galon et. al. Science 2006;313:1960-1964

Genetics Changing the Environment

Some Common Features of Cancer with Defective DNA MMR

• 15% of all sporadic CRCs• Tissues display Microsatellite Instability (frameshifts)

• Lost of DNA MMR protein expression

• CRCs more common in proximal (Right) colon• CRCs often show poor differentiation and mucin• Tumors are hypermutated, containing 102-103 somatic mutations• Tumors are mostly diploid; lack p53 mutations• CRCs attract T cells and form sub-epithelial lymphoid nodules

(neo-antigens from novel frameshifted peptides)• More susceptible to PD-1 immune checkpoint blockade

• CRCs are resistant to 5-fluorouracil treatment• Patients with MSI CRCs show longer survival, earlier stage

• Caveat: EMASTCarethers and Jung. Gastroenterology 2015;149:1177-1190.

Normal AdenomaHypermutable

PhenotypeCancer

Wnt:APC/β-catenin

Detection of Microsatellite

instability-High

Immunogenic neo-peptides fromACVR2, TGFBR2,BAX, MMR genes,

IGF2R, E2F4, MBD4, etc.frameshifts

Genomic/Epigenomicinstability

-Lynch (germline MMR mutation)-Lynch-like (bi-allelic somatic mutation)-Sporadic (hMLH1 hypermethylation)

B-RAFMutation

(non-Lynch)

Loss of DNA Mismatch Repair

somatic frameshift mutations

MSI Pathway and CRC Progression

Carethers JM. Curr Colorectal Cancer Rep 2017;13:73-80

PD-1

Induction of “protective” T cells

Schema for MSI Neo-peptide Inflammation

Induction of T Cell “Protective” Inflammation

Alteration of DNA MMR(somatic/germline MLH1, MSH2, MSH6, PMS2, EPCAM)

Target Gene Mutations (mononucleotide; flanking sequences)TGFBR2, ACVR2, etc.

Biological consequences(reduced metastasis)

Estimate of patients anti-PD-1 benefits

Le DT et al. Science 2017;357:409-413

Environment Changing the Genetics

Some Common Features of EMAST CRCs

• Observed in 50% of all sporadic CRCs (plus other cancers)• Tissues display EMAST (elevated microsatellite alterations at

selected tetranucleotide repeats)• Heterogeneous (reduced) MSH3 nuclear expression

• CRCs appear distributed throughout the colon• Tumors are non-hypermutated (excluding complete MMR defects)• CRCs associated intimately with inflammatory cells

• Could be a driver for EMAST

• CRCs are sensitive to 5-fluorouracil treatment• Patients with EMAST CRCs show shorter survival, more

metastases, poor prognosis, advanced staged disease• Higher frequency among African American cancers

Carethers and Jung. Gastroenterology 2015;149:1177-1190.

IL-6 Changes MSH3 Subcellular Location and Induces EMAST

Tseng-Rogenski SS et al. Gastroenterology 148:579-589, 2015.

MYCL1

Schema for Inflammatory-Associated Microsatellite Alterations AKA EMAST

Inflammation (IL-6 driven)

Initiation of Neoplasia and tumor advancement

Alteration of DNA MMR(MSH3 “loss of function”)

Potential Target GeneMutations (di, tri, tetranucleotide),

Double strand breaks

Biological consequences(increased metastasis)

Carethers JM et al. Genes 6:185-205, 2015

Microbiome?

50% of CRCs

Microbes and CRC Progression

Brennan CA, Garrett WS. Annu Rev Microbiol 2016;70:395-411.

CRC-Associated Microbes

Brennan CA, Garrett WS. Annu Rev Microbiol 2016;70:395-411.

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Earlyneoplasia

Lateneoplasia

pks+E. Coli and ETBF in FAP Patients and Mutant APC mice

Dejea CM et al. Science 2018;359:592-597.

FAP Patients

Mutant APC mice

Combination of pks+E. coli and ETBF• increased inflammation• faster tumor onset • poor survival• IL17-dependent

Colonized with both pks+E. coli and ETBF and assessed at 15 weeks

Colon inflammation score

Biofilm • Found in majority of Rt. Side

CRCs and associated normal mucosa

• Biofilm-negative CRCs are associated with normal mucosa that does NOT possess a biofilm

• Risk of proximal CRC development is 5-fold higher with biofilm

• Rt. Side: MSI, hypermutated, hypermethylation of MLH1, BRAFV600E

Right-sided Location of Biofilm in Sporadic CRCs

**

Dejea CM et al. PNAS 2014;111:18321-18326.

Biofilms Trigger Immune Response and Proliferation

Dejea CM et al. PNAS 2014;111:18321-18326.

Fusobacterium Persistence in Primary CRC and Liver Metastasis

Bullman S et al. Science 2017;258:1443-1448.

Primary CRC

Paired Liver Mets

Primary CRC

Paired Liver Mets

RNA ISH

Serial PDX Passage

Antibiotics Affect Fusobacterium Infected Tumor Growth

Bullman S et al. Science 2017;258:1443-1448.

Fusobacterium ROS

8-oxoGCpG islands

Translocation of MSH3 from nucleus to

cytoplasm

Recruitment of MSH2/MSH6

DNMTsPRC4

Replication errors at microsatellite

with di- or tetranucleotide

repeats

Hypermethylation of promoter CpG

islands

Hypomethylation of CpG at non-

promoter region

CIMP

Hypermethylation of MLH1 MSI-H

MSI-LEMAST

IL6PGE2

DNA damage

Proposed Pathways of Genetic/Epigenetic Alterationsin CRC Induced by F. nucleatum Infection

Koi et al. JARC 2018;2:37-46

Diet and Fusobacterium nucleatum status in CRC

Liu L et al. Clin Gastroenterol and Hepatol 2018;16:1622-1631.

EDIP: empiric dietary inflammatory pattern score (weighted intake of 18 foods constructed to predict plasma levels of IL6, CRP, TNFRSF1B, or TNF alpha-receptor 2)

CRC Trends by Birth Year or Year Diagnosis

Wolf AMD et al. CA Cancer J Clin 2018;68:250-281 and Murphy CC et al. Gastroenterol 2018;155:1716-1719

45-49 yrs

40-44 yrs

35-39 yrs30-34 yrs

Triclosan Increases Tumorigenesis (AOM/DSS)

Yang H et al. Science Translational Medicine 2018;10:1-10

Summary• The environment plays a major role in cancer

formation, progression, and metastasis– Colorectal cancer is a great example

• Understanding role and pathways involved affords intervention strategies– Diet intervention– Aspirin optimal usage– Microbiome intervention– Inflammation prevention or modification

Future Addressable Questions• How does the environment influence different

genetic susceptibilities?– e.g. aspirin and PIK3CA mutations in colorectal

cancer• How does the environment contribute to disease

formation?– e.g. microbiome and adenomas in colorectal cancer

• How does the environment generate changes in cellular pathways (that could be targeted)?– e.g. IL-6 inflammation and loss of DNA repair in CRC

Future Addressable Questions• How do we identify the genetically susceptible?

– Prior to disease formation (e.g. germline)– With disease (e.g. somatic mutation analysis)

• What in the environment leads to changes in the genome for disease formation?– e.g. what is increasing young-onset CRC

Thank you!