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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 ! "$! ! # ! "! $! # ! "! ! $# ! "! ! ! # ND %&' ( )*# + , + )$# - . / ! # - %01# - %0$$. # - 0*2# , ( 3( # 45 . *! # 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 :;$2 "34$ <;$( 2 =>:?!@:AB%CD$ ! ####" ! ###" ! ##" ! #" HT29 !" $" %" ! "! #$% $" %" &! ' ( ( ) % $" %" &' $*% $" %" +, ) $- % Figure 2: Validation of DEFB1 overexpression from pCMV6-XL4-DEFB1 in colon cancer cell lines. Validation of pCMV6-XL4-DEFB1 overexpression by mRNA in 4 colon cancer cell lines relative to their normal expression. Estimated Recurrence-Free Probability CaCo2 HT29 HCT116 1.0 Estimated Recurrence-Free Probability 0.6 0.5 0.4 0.3 0.2 48 H 0.8 0.6 Log-rank test = 0.008 C/Cp-value (n=74) 0.7 0.5or G/G (n=36) C/G 0.6 0.5 0.4 0.4 0.3 0.2 0.3 0.1 0.2 0 2 4 0.1 B 6 8 10 Years Since Diagnosis of Stage III Colon Cancer 0.0 0 2 4 6 12 14 8 test p-value = 0.008 Time toLog-rank Recurrence: DEFB1 rs1799946 Time-to-Tumor Recurrence 1.0 0.9 Median TTR, yrs (95% CI) DEFB1 N rs1799946 C/C (n=74)45 G/G 5.9 (4.8, 12.2+) HR (95% CI) 1 (Reference) Log-rank test p-value = 0.017 G/A A/AG/G51 C/Gor or (n=36)16.8 (9.4, 16.8+) 0.38 (0.16, 0.87) G/G (n=45) 0.8 Log-rank test p-value = 0.017 G/A or A/A (n=51) 0.7 0.6 0.5 0.4 0.3 0.2 0.0 0.0 0 10 2 12 4 6 8 10 0.1 Years Since Diagnosis of Stage III Colon Cancer 0.0 14 0 2 4 6 8 Years Since Diagnosis of Stage III Colon Cancer 12 10 14 12 14 16 18 Years Since Diagnosis of Stage II Colon Cancer Figure 5. Defensin polymorphisms and time to recurrence (TTR) in patients with stage II or III colon cancer, univariate analysis. (A) DEFB1 rs1800972 (DEFB1 44) in Stage III colon cancer (B) DEFB1 rs1799946 (DEFB1 52). Abbreviations: CI, confidence interval; HR, hazard ratio; N, number; yrs, years. P-value based on log-rank test using the dominant genetic model. 24 H 48 H Summary of Results Figure 3: Images demonstrating the effects of overexpression of hBD-1 in various CRC cell lines at 24 and 48 hours. DU145 is a prostate cell line that was used as a control for the Caco2, HCT116 and HT29 CRC cell lines. Magnification at x100. pCMV6EV !"#$%"&' $( ) *+#, $ -. , &*/ 0, $1"$2 "34$56*+78, 31, ' 9$ 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/ ! #####" 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 ( ) *+', - . /01') 2', 3, ', 1**4'5/'! "#$%&'67'8 '9) 4:'; <( =&' > ?1@1AB@1445) /' %#" ! " #$%&'( ) *+', - . / 01' ND ! "! ! ! # pCMV6DEFB1 $"! ! ! # 0.8 Abbreviations: 5-FU, 5-fluorouracil; LV, leucovorin. CaCo2 pCMV6- pCMV6- pCMV6- pCMV6- pCMV6EV EV DEFB1 EV DEFB1 ND = Not Detected $! "! ! ! # pCMV6DEFB1 ! "#$%&'( ) *&$+ , - . /$- " '0$12( 3#&$ %&'( ) *&$4" $5 6789$ $! ! "! ! ! # 24 H 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. . , &*/ 0, $AB%CD$E . F G$H0, 6, I J 6, 77KK"+$*1$@L$M$ J ( 2 =>:?!@:AB%CD$ 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. 1"'":&; 4( '$1( 3; &:$1&''$!<3&=$ %&'( ) *&$+ , - . /$> %? @$, AB:&==<"3$ 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 %! " $#" HT29 $! " #" !" $" HCT116 %" HT29 pCMV6DEFB1 B The defensin molecules biophysical properties allow them to function as antimicrobial agents by forming dimers and octamers in solution (3). This quaternary structure either creates pores in target membranes or disrupts the membrane integrity that results in cell lysis and death of bacterial and potentially tumor cells (3). Patients and Method Estimated Recurrence-Free Probability Introduction Human β-defensins (hBDs) are part of a family of antimicrobial peptides made by neutrophils that are an important component of both the innate and adaptive immune defense responses (1). To date, six hBDs have been identified primarily found in human epithelial tissues via reverse transcriptase-PCR (2). Abstract ID: 3622 Pacific University, Azusa, CA; 2USC/Norris Comprehensive Cancer Center, Los Angeles, CA Estimated Recurrence-Free Probability 1Azusa Conclusion ' ( )*" +, ' - . /012/3456$" &" SW620 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.