1. Linking Adipokines and Obesity
with Colorectal Cancer Risk
Jenifer I. Fenton, PhD, MPH
Michigan State University
East Lansing, MI
IASO/WCRF April, 2013

2. Diet and cancer risk: Is it body
composition?
The Shape of Things to Come. The Economist; Dec 11, 2003.
(http://www.economist.com/displaystory.cfm?story_id=2282754)

3. Obesity-induced inflammatory changes in
adipose tissue

4.  Serum leptin levels elevated in endometrial,
breast, prostate and colon cancer patients
 Leptin levels associated with greater risk of
being diagnosed with larger and riskier tumors
 Increased proliferation of breast, esophagus,
colon and prostate cancer cells
 stimulated the growth and proliferation of colon
cancer cells (HT-29 and CACO-2)
 promotes invasiveness of kidney and colonic
epithelial cells
Leptin and cancer

5. Copyright ©2005 American Association for Cancer Research
Otake, S. et al. Clin Cancer Res 2005;11:3642-3646
Adenoma risk

6. Evolution of a lethal cancer
Jones S et al. PNAS 2008;105:4283-4288
©2008 by National Academy of Sciences

7. Hallmarks of cancer paradigm
• The authors suggest that most if not all cancers have
acquired the same set of functional capabilities during
their development, albeit through various mechanistic
strategies.
Cell. 2000; Cell Press. 2011

8. Model system

9. Cell characteristics
 Two cell lines: “Normal” (Apc+/+; YAMC) and
 “Preneoplastic” (ApcMin/+; IMCE)
 Neither will grow in soft agar or form tumors in athymic
mice
 Cells are grown in presence of IFN-gamma at 33º C
(growth permissive); experiments done in IFN-free,
serum-free media at 39ºC (non-permissive for growth).
 Under non-permissive conditions: cells form tight
junctions, polarize, differentiate and undergo apoptosis
over 5 to 8 days.
These cells serve as an excellent model to study early events in colon carcinogenesis
Fenton et al, Carcinogenesis, 2005.

10. Colon epithelial cells
Normal MetastaticNon-tumorigenic Tumorigenic
YAMC
IMCE
SMAD3
-/-
YAMC-Ras IMCE-Ras
MC38
Modeling colon epithelial cell
carcinogenesis

11. Cell culture methodology
Cells grow to 70%
confluence in flasks
in growth media.
33ºC
Cells are transferred
to 39ºC, media is
aspirated, and
serum-free, IFN free
media is placed on
cells.
39ºC 24hr
• Collect cells
in lysis buffer
for Western
blot
• Angiogenesis
• Cell signaling
• Cell
proliferation
Treat cells

12. Leptin induces IMCE cell proliferation
YAMC IMCE
Ob-R
Proliferation results verified by flow cytometric analysis

13. Microarray analysis
Up in IMCE
IMCE Time-independent
217 genes
IMCE Time-dependent
2-fold 707 genes
Down in YAMC
YAMC Time-independent
588 genes
YAMC Time dependent 2-
fold 229 genes
Fenton et al, Molecular Carcinogenesis, 2007

14. Gene Category
Number of
genes
Genes
Proliferation Up 42 Btg2, Bub1, Bub1b, Ccna2, Ccnf, Cd68, Cdc25b,
Cdc25c, Cdkn2c, Cdkn2d, Crip1, Gsg2, Il18, Incenp,
Lxn, Mxd4, Mdm2, Mki67, Gadd45b, Nmyc1, Pdgfa,
Pdgfra, Plk1, Dusp1, Kif20a, Rb1, Aurkb, Aurka, Tacc3,
Tgfb2, Gadd45g, Pttg1, Cks2, 1190002H23Rik, Cdca1,
Kntc2, Rprm, Ube2c, Rhou, Sesn3, Pard6g, Cdc20
Down 14 Cdkn2a, Cpeb1, Ddit3, Dst, Ereg, Igfbp4, Cxcl12, Plk2,
Vegfa, Zfr, Ets2, Pdgfc, Atf5, Sesn2
Cell cycle Up 34 Bub1, Bub1b, Ccna2, Ccnf, Cdc25b, Cdc25c, Cdkn2c,
Cdkn2d, Gsg2, Incenp, Mdm2, Mki67, Gadd45b,
Nmyc1, Pdgfa, Plk1, Dusp1, Rb1, Aurkb, Aurka, Tacc3,
Tgfb2, Gadd45g, Pttg1, Cks2, 1190002H23Rik, Cdca1,
Kntc2, Rprm, Ube2c, Rhou, Sesn3, Pard6g, Cdc20
Down 11 Cdkn2a, Cpeb1, Ddit3, Dst, Ereg, Plk2, Vegfa, Zfr,
Ets2, Atf5, Sesn2
MAPK signaling Up 10 Cd14, Cdc25b, Hspb1, Gadd45b, Pdgfa, Pdgfra,
Dusp1, Tgfb2, Gadd45g, Dusp14
Down 9 Atf4, Casp11, Ddit3, Fgfr1, Mknk1, Pdgfrb, Fgf21,
Pla2g12a, Rasa1
IMCE (Apc-/+): EASE Analysis

15. Leptin induces angiogenic potential
Colon epithelial cells
Leptin
↑ VEGF
Chemoattact endothelial cells to site
↑ SAPK/MAPK in endothelial cell
↑ NFkB activation
Endothelial cell proliferation Adhesion protein upregulation
↑ Capillary formation/Angiogenesis
End result=↑ blood supply to Apc (min/+) cells
Carcinogenesis. 2009 Apr;30(4):690-7

17. Leptin induces 3-4 Hallmarks of Cancer
in IMCE cells

18. Adiponectin
 Inhibits leptin-induced IMCE cell proliferation
 Via reduced IL-6 production by the IMCE cells
 Increases sgp130 to inhibit trans-IL-6 signaling
 Blocks NFkB nuclear translocation and DNA binding
Fenton and Birmingham. Mol Carcinog. 2010

19. Adipokines and Colon Epithelial Cell Homeostasis
LEPTIN IL-6 INSULIN
IL-6 Homeostatic
Apoptosis
Caspase Activation
Proliferation
Proliferation
NFκB activation/binding
MAPK
Leptin induced sIL-6R
Leptin-induced IL-6
VEGF production
capillary formation
Promotion of Transformed
Cells
YAMC (Apc +/+) Normal IMCE (Apc Min/+) Transformed
Supports Normal Phenotype
Adenocarcinoma (MC38)
•No leptin receptor
•No response to leptin treatment
•IL-6 induces proliferation
•Insulin key proliferative signal
•VEGF ?
Rapid Expansion of Tumor
Adiponectin
Overarching
inhibitor

20. The Question
 How is obesity and its associated factors related
to colorectal polyp formation and severity?
 Hypothesis:
 Obesity and its related factors would be associated
with increased #’s of polyps as well as increased
polyp severity

21. Zauber 2012 NEJM
Removal of Colorectal Polyps Decreases
Mortality from Colorectal Cancer

22. The Study Population
 Caucasian men (n=126)
 48 to 65 yrs old
 Asymptomatic
 BMI distribution:
1WHO Global Database on BMI
0
25
50
lean
(n=28)
overweight
(n=46)
obese
(n=52)
%ofPopulation
study population U.S. males
1
0
25
50
lean
(n=28)
overweight
(n=46)
obese
(n=52)
%ofStudyPopulation

23. Methods
 Blood sample, weight, height, waist
circumference, clinical metadata, colonoscopy reports
 Categorical variables were constructed using either
natural biological cut off points (BMI) or tertiles within the
data (waist
circumference, leptin, adiponectin, HMW, MMW, LMW, IL
-6, TNF-alpha, IP-10)
 Odds Ratios were determined using polytomous logistic
regression
 Test for trend was carried out across categories.
 p ≤ 0.05 indicates significance

24. Conclusions
 In vitro, leptin induces 3-4 hallmarks of cancer.
 In men 50-65 years of age BMI, waist
circumference and leptin were associated with
tubular adenoma risk.
 If these observations are confirmed in larger
populations, the recommended age for first
colonoscopy in individuals with a BMI over 30
and elevated adipokines should be revisited.

25. Acknowledgements
 Kari Hortos
 Sarah Comstock
 Sarah McCaskey
 Dorothy Pathak
 Funding from:
 NCI-CPF Program
 NCI 1R03CA142000
 MSU CTSI
 Steve Hursting
 Julia Busik
 JP Steibel
 Norman Hord
 Sarah Comstock
 Bruce Kovan
 Tri-County
Gastroenterology
 Macomb, MI

26. Question???