Download Novel Colon Cancer Tumor Suppressor Gene, Defensin-β1

Novel Colon Cancer Tumor Suppressor Gene, Defensin-β1, Predicts Recurrence in Patients with Stage II and III Colon Cancer.
Melissa J. LaBonte1/2, Pierre O. Bohanes2, Dongyun Yang2, Fotios Loupakis2, Peter M. Wilson2, Armin Gerger2, Yan Ning2, Takeru Wakatsuki2, Wu Zhang2,
Leonor Benhaim2, Robert D. Ladner2, Rita El-Khoueiry2, Syma Iqbal2,Anthony El-Khoueiry2, and Heinz-Josef Lenz2
 Genomic DNA was extracted from 234 patients with stage III and high-risk stage II
colon cancer patients.
Pre-Clinical Results
DU145
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Figure 1: DEFB1 mRNA expression in various colorectal cancer cell lines. The bar graph
illustrates the relative amount of DEFB1 mRNA expression fold change in various cancer cell
lines relative to HT29. The value of DEFB1 mRNA expression depicted here is low when
compared to normal prostate and colon cell expression. Error bars represent standard
deviation of two independent experiments in duplicate.
HCT116
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Figure 2: Validation of DEFB1 overexpression from pCMV6-XL4-DEFB1 in colon cancer
cell lines. Validation of pCMV6-XL4-DEFB1 overexpression by mRNA in 4 colon cancer cell
lines relative to their normal expression.
Estimated Recurrence-Free Probability
CaCo2
HT29
HCT116
1.0
Estimated Recurrence-Free Probability
0.6
0.5
0.4
0.3
0.2
48 H
0.8
0.6
Log-rank test
= 0.008
C/Cp-value
(n=74)
0.7
0.5or G/G (n=36)
C/G
0.6
0.5
0.4
0.4
0.3
0.2
0.3
0.1
0.2
0
2
4
0.1
B
6
8
10
Years Since Diagnosis of Stage III Colon Cancer
0.0
0
2
4
6
12
14
8
test p-value
= 0.008
Time toLog-rank
Recurrence:
DEFB1
rs1799946
Time-to-Tumor Recurrence
1.0
0.9
Median TTR, yrs (95% CI)
DEFB1 N
rs1799946
C/C (n=74)45
G/G
5.9 (4.8, 12.2+)
HR (95% CI)
1 (Reference)
Log-rank test p-value = 0.017
G/A
A/AG/G51
C/Gor or
(n=36)16.8 (9.4, 16.8+)
0.38 (0.16, 0.87)
G/G (n=45)
0.8
Log-rank test p-value = 0.017
G/A or A/A (n=51)
0.7
0.6
0.5
0.4
0.3
0.2
0.0
0.0
0
10
2
12
4
6
8
10
0.1
Years Since Diagnosis of Stage III Colon Cancer
0.0
14
0
2
4
6
8
Years Since Diagnosis of Stage III Colon Cancer
12
10
14
12
14
16
18
Years Since Diagnosis of Stage II Colon Cancer
Figure 5. Defensin polymorphisms and time to recurrence (TTR) in patients with stage II or III colon cancer, univariate analysis. (A) DEFB1 rs1800972 (DEFB1 44) in Stage III colon
cancer (B) DEFB1 rs1799946 (DEFB1 52). Abbreviations: CI, confidence interval; HR, hazard ratio; N, number; yrs, years. P-value based on log-rank test using the dominant genetic model.
24 H
48 H
Summary of Results
Figure 3: Images demonstrating the effects of overexpression of hBD-1 in various CRC cell lines at 24 and 48
hours. DU145 is a prostate cell line that was used as a control for the Caco2, HCT116 and HT29 CRC cell lines.
Magnification at x100.
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1.E. Zhuravel1*, T. Shestakova1, O. Efanova1, Yu. Yusefovich1, D. Lytvin2, M. Soldatkina1, P. Pogrebnoy1 1R.E.Human beta-defensin-2 controls cell cycle in
malignant epithelial cells: in vitro study. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv,Ukraine 2Institute of Food
Biotechnology and Genomics,NAS of Ukraine, Kyiv, Ukrain
2.Ouellette, A.J., and J.C. Lualdi. 1990. A novel mouse gene family coding for cationic, cysteine-rich peptides. Regulation in small intestine and cells of myeloid
origin. J. Biol. Chem. 265:9831-9837
3.Hoover DM, Rajashankar KR, Blumenthal R, et al. The structure of human beta-defensin-2 shows evidence of higher order oligomerization. Biol Chem. 2000;
275:32911-8
4.C. Sun, R. Arnold, et al. Human B-Defensin-1, a potential chromosome 8p tumor suppressor: control of transcription and induction of apoptosis in renal cell
carcinoma. American Association for Cancer Research Journal. 2006; 66:17
5.Takanishi DM, Kim SY, et al. Chromosome 8 loses in colorectal carcinoma: localization and correlation with invasive disease. Mol Diagn. 1997 Mar 2 (1):3-10
6.American Cancer Society. Cancer Facts and Figures, 2010. www.cancer.org/
7.Origene. http://www.origene.com/
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CaCo2
Time-to-Tumor Recurrence
0.8
N rs1800972
DEFB1
Median TTR, yrs
(95% CI)
HR (95% CI)
C/C (n=74)74
C/C
2.7 (1.8, 6.6)
1 (Reference)
0.7
Log-rank
0.008
C/G
G/GG/G36(n=36)
11.3+ (5.2,
11.3+) test p-value
0.41=(0.20,
0.82)
C/Gor or
0.9
0.7
pCMV6DEFB1
ND
48 H
DU145
HCT116
 β-Defensin variants predict a stage specific
survival in patients with early colon cancer
 Validation of DEFB1 overexpression in colon
cancer cell lines by mRNA
 DEFB1 has detectable expression in 5 of the 8
colon cancer cell lines tested
 Following DEFB1 overexpression:
o Cell growth inhibited
o Increase in apoptosis
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Abbreviations: 5-FU, 5-fluorouracil; LV, leucovorin.
CaCo2
pCMV6- pCMV6- pCMV6- pCMV6- pCMV6EV
EV
DEFB1
EV
DEFB1
ND = Not Detected
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DU145
Region
Genotyping
5’UTR
DS
5’UTR
DS
3’UTR
DS
Time-to-Tumor
Recurrence
5’UTR
DS
DEFB1 rs1800972
Abbreviations: UTR, translated region; DS, direct DNA sequencing.
Time toLog-rank
Recurrence:
DEFB1
rs1800972
test p-value
= 0.008
A
24 H
0.1
pCMV6-pCMV6pCMV6- pCMV6pCMV6- pCMV6pCMV6- pCMV6- pCMV6- pCMV6- pCMV6- pCMV6EV
DEFB1
DEFB1
EV
DEFB1
EV
EV DEFB1
DEFB1
EV
DEFB1
EV
 Flow cytometry (FACS): Cells were collected and stained with propidium iodine
according to standard protocols. Cells were harvested at 24h, fixed, stained, and
analyzed using an EPICS ELITE flow cytometer with cell populations quantified using
Expo32 software (Beckman Coulter) in collaboration with USC. Cells with DNA content
>1, were considered apoptotic.
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References
was
Base Exchange
G/C
G/A
G/A
G/A
0.9
0.9
 qRT-PCR: Total RNA was isolated from cell lines according to TRIzol protocol
(Invitrogen). cDNA synthesis was cond. TaqMan primers were obtained from Applied
Biosystems.

Transfection: pCMV6-XL4-DEFB1 was obtained from Origene.
used according to manufactures protocol (Denville).
Time-to-Tumor Recurrence
DEFB1 rs1800972
1.0
 Log-rank test and stratified Cox regression, with adjustment for treatment, were used
for associations. Genotypes were parameterized as the number of dominant alleles. No pvalue adjustments were made for multiple comparisons.
ExpressfectTM
rs-number
rs11362
rs1047031
rs1799946
rs1800972
1.0
DEFB1
 Four DEFB1 SNPs were evaluated using PCR-based and direct DNA sequencing
protocols.
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This study tested:
1. Whether 4 germline single nucleotide polymorphisms
(SNPs) in DEFB1 could predict time to tumor recurrence
(TTR) in stage II and III colon cancer (CC) patients.
2. Evaluate if hBD-1 had tumor suppressor functions in CC
models.
Gene
All patients treated with 5-FU-based adjuvant chemotherapy at the Norris Comprehensive
Cancer center/ University of Southern California or the Los Angeles Count/USC-Medical
Center.
Colorectal cancer (CRC) was the third leading cause of cancer related
incidence and second deadliest malignancy in the United States with an
estimated 143,000 new cases and 51,000 deaths in 2010 (6).
Objective
Table 2. Single Nucleotide Polymorphisms (SNPs) Analyzed
Table 1. Patient Characteristics
pCMV6EV
Human β-Defensin 1 (hBD-1) is encoded by the DEFB1 gene and is found on
chromosome 8p23 (4). Previous experiments have revealed that the deletion of
chromosome 8p is the most common genetic alteration in prostate (82%) and
renal cell carcinoma (90%). In addition, 40-50% of colorectal cancers
demonstrate a deletion of the short arm of chromosome 8, indicating the
presence of several tumor suppressor genes (5). Other experiments have
shown that hBD-1 exhibits tumor suppressor functions and its loss contributes
to tumorigenesis in renal and urothelial cancer cells.
Clinical Results
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B
The defensin molecules biophysical properties allow them to function as
antimicrobial agents by forming dimers and octamers in solution (3). This
quaternary structure either creates pores in target membranes or disrupts the
membrane integrity that results in cell lysis and death of bacterial and
potentially tumor cells (3).
Patients and Method
Estimated Recurrence-Free Probability
Introduction
Human β-defensins (hBDs) are part of a family of antimicrobial peptides made
by neutrophils that are an important component of both the innate and adaptive
immune defense responses (1). To date, six hBDs have been identified
primarily found in human epithelial tissues via reverse transcriptase-PCR (2).
Abstract ID: 3622
Pacific University, Azusa, CA; 2USC/Norris Comprehensive Cancer Center, Los Angeles, CA
Estimated Recurrence-Free Probability
1Azusa
Conclusion
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Figure 4: Overexpression of hBD-1 induces apoptosis (% Sub-G1) in CRC cell lines at 24 hours. Flow cytometry
was used to determined the % of cells in Sub-G1 as represented by the mean ± SD from 2 independent experiments.
The results demonstrate for the first time evidence of hBD-1’s potential influence on the
microenvironment and its role as a tumor suppressor in colon cancer. Further studies need to be
conducted to better understand the role of hBD-1 in colon cancer development and progression
and evaluate the potential utility of hBD-1 as a therapeutic strategy.