Dina Kazanov

CD24 knockout prevents colorectal cancer in chemically induced colon carcinogenesis and in APCMin/CD24 double knockout transgenic mice

Increased expression of CD24 is seen in a large variety of solid tumors, including up to 90% of gastrointestinal (GI) tumors. Stable derivatives of SW480 colorectal cancer (CRC) cells that overexpress CD24 proliferate faster, and increase cell motility, saturation density, plating efficiency, and growth in soft agar. They also produce larger tumors in nude mice as compared to the parental SW480 cells. Most significantly, even depletion of one copy of the CD24 allele in the APCMin/+ mice of a transgenic mouse model led to a dramatic reduction in tumor burden in all sections of the small intestine. Homozygous deletion of both CD24 alleles resulted in complete abolishment of tumor formation. Moreover, CD24 knockout mice exhibited resistance to chemically induced inflammation‐associated CRC. Finally, a new signal transduction pathway is suggested: namely, CD24 expression downstream to COX2 and PGE2 synthesis, which is directly regulated by β‐catenin. CD24 is shown in vitro and in vivo as being an important oncogene in the gut, and one that plays a critical role in the initiation and progression of carcinogenesis.

Role of CD24 polymorphisms in the susceptibility to inflammatory bowel disease

Background Inflammatory bowel disease (IBD) results from an inappropriate inflammatory response in which genetic, immune, and environmental factors all play important roles. Recently, single nucleotide polymorphisms (SNPs) in the CD24 gene have been associated with the development of several autoimmune diseases. Aim To evaluate whether CD24 SNPs, are associated with risk of ulcerative colitis (UC) and Crohns disease (CD). Methods The CD24 polymorphisms C170T (rs8734), TG1527del (rs3838646), A1626G (rs1058881), and A1056G (rs1058818) were assessed in a case-control study of an Israeli cohort comprising 117 IBD patients and 105 age and gender-matched healthy controls. Restriction fragment length polymorphism (RFLP) analysis was performed using BstX1, Bsr1, Mfe1, and BstU1 restriction enzymes. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression models. Results Carriers of the C170T SNP were at increased risk of IBD (OR=3.022, 95% CI: 1.748–5.223, p=0.001), UC (OR=3.002, 95% CI: 1.661–5.427, p=0.001) and CD (OR=3.077, 95% CI: 1.334–7.095, p=0.008). Carrying the A1626G and A1056G SNPs was found to be a risk factor for IBD (OR=2.460, 95% CI: 1.420–4.259, p=0.001 and OR=1.856, 95% CI: 1.011–3.405, p=0.01), UC (OR=2.218, 95% CI: 1.207–4.075, p=0.01 and OR=1.944, 95% CI: 0.995–3.798, p=0.01) but not for CD (p=0.086 and p=0.299). The A1626G and TG1527del were found to be associated with younger age of IBD onset (p=0.022 and p=0.027, respectively). Conclusions The CD24 C170T polymorphism is associated with IBD risk. The A1626G and A1056G SNPs might be associated only with UC risk. These findings suggest CD24 as a new genetic susceptibility factor, with clinical implications in the prediction of IBD prognosis and therapy.

Risk of colorectal neoplasia associated with the adenomatous polyposis coli E1317Q variant

BACKGROUND Reports of the risk of colorectal neoplasia associated with a variant of the adenomatous polyposis coli (APC E1317Q) gene are conflicting. Using a case-control design, we investigated this relationship within a clinic-based cohort followed through the Integrated Cancer Prevention Center and the Tel-Aviv Sourasky Medical Center. MATERIALS AND METHODS All study subjects were tested for the APC E1317Q variant at enrollment. Subjects underwent colonoscopic evaluation (+/-biopsy and/or polypectomy) and had cancer history and colorectal neoplasia risk factors assessed. The crude and adjusted risks of neoplasia associated with the E1317Q variant were calculated. RESULTS The prevalence of the E1317Q variant was 1.4% in the entire study sample and 3.2% in Sephardic Jews. E1317Q was more prevalent among cases: 15 of 458 (3.3%) cases were carriers compared with 11 of 1431 (0.8%) controls [odds ratio (OR) 4.4, 95% CI 2.0-9.6]. When stratified by neoplasia type, adenoma risk was significantly elevated in carriers (OR 4.1, 95% CI 1.8-9.4) but colorectal cancer risk was not (OR 2.1, 95% CI 0.8-5.3). After adjustment, the E1317Q variant remained a significant predictor of colorectal adenoma (OR 4.6, 95% CI 2.0-10.8). CONCLUSIONS The APC E1317Q variant is associated with colorectal neoplasia, particularly colorectal adenomas, but further studies are still needed. Variant prevalence is elevated in Sephardic Jews.

Chemopreventive effects of Coltect, a novel dietary supplement, alone and in combination with 5-aminosalicylic acid in 1,2-dimethylhydrazine-induced colon cancer in rats

Objectives: Coltect is a novel dietary supplement containing curcumin, green tea and selenomethionine. Previous reports have suggested that these agents can prevent colorectal cancer (CRC). The present study examined the chemopreventive effect of Coltect alone or combined with 5-aminosalicylic acid (5-ASA) using the 1,2-dimethylhydrazine (DMH) model in rats. Methods: The effect of Coltect was examined on HT-29 CRC cells by growth inhibition assay. Apoptosis was determined by annexin V-FITC/PI staining. Male rats were injected with DMH in vivo and treated with Coltect 150 mg/kg, 5-ASA 50 mg/kg or their combination, by oral gavage. Aberrant crypt foci (ACF) were identified by methylene blue staining. Results: HT-29 cells exhibited a dose-dependent response to Coltect. Part of the growth inhibition can be explained by the induction of mild-moderate apoptosis in cancer cells (28%) compared with the untreated cells (10%). In the in vivo model, the average number of ACF was divided into small (1-3 crypts) or large (≥4 crypts). The Coltect compound reduced the number of small and large ACF similarly to 5-ASA (40% reduction). This reduction was amplified by combining the two agents (70% reduction). Conclusion: Coltect inhibits the growth of colon cancer cells, induces apoptosis and inhibits ACF development. Furthermore, it augments the growth inhibitory effect of 5-ASA in vivo. This may be clinically important since this safe dietary supplement-drug combination can be administered as a chemopreventive regimen for the treatment of CRC.

Increased expression of CD24 in nonmelanoma skin cancer

Background Skin cancer detection is based on the macroscopic and microscopic appearance of the lesions and the experience of the surgeon. The final diagnosis is done by pathological analysis, based on established criteria. Currently, there is no serum marker that can be used for the diagnosis of skin cancer. CD24, a mucin-like glycoprotein, is overexpressed in a variety of cancers including skin malignancies. Objective Evaluate the potential utility of CD24 expression in peripheral blood leukocytes (PBLs) for the detection of nonmelanoma skin cancers (NMSC). Methods Twenty-nine consented individuals attending Tel Aviv Sourasky Medical Center for excision of suspected skin lesions, and 21 age- and gender-matched subjects were prospectively recruited. The resected lesions were examined by an expert dermatopathologist. PBLs were isolated from blood samples and protein extracts were subjected to sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The study was double blinded. Results CD24 expression in PBLs distinguishes between NMSC and healthy subjects, with high sensitivity (81%) and specificity (67%) for basal cell carcinoma, and 100% and 71%, respectively, for squamous cell carcinoma. Conclusion The CD24 test can successfully distinguish NMSC from healthy subjects. CD24 may serve as a new potential and promising diagnostic biomarker for the detection and surveillance of NMSC.

Delayed Wound Healing in Heat Stable Antigen (HSA/CD24)-Deficient Mice

Background Healthy individuals rarely have problems with wound healing. Most skin lesions heal rapidly and efficiently within one to two weeks. However, many medical and surgical complications can be attributed to deficiencies in wound repair. Open wounds have lost the barrier that protects tissues from bacterial invasion and allows the escape of vital fluids. Without expeditious healing, infections become more frequent. The CD24 gene encodes a heavily-glycosylated cell surface protein anchored to the membrane by phosphatidylinositol. CD24 plays an important role in the adaptive immune response and controls an important genetic checkpoint for homeostasis and autoimmune diseases in both mice and humans. We have previously shown that overexpression of CD24 results in increased proliferation and migration rates. Aim To examine the role of CD24 in the wound healing process. Methods An excisional model of wound healing was used and delayed wound healing was studied in genetically modified heat stable antigen (HSA/CD24)-deficient mice (HSA -/-) compared to wild-type (WT) mice. Results Large full-thickness skin wounds, excised on the back of mice, exhibited a significant delay in the formation of granulation tissue, and in wound closure when compared to their WTHSA +/+ littermates. Wounds were histologically analyzed and scored, based on the degree of cellular invasion, granulation tissue formation, vascularity, and re-epithelialization. Additionally, in stitched wounds, the HSA -/- mice failed to maintain their stitches; they did not hold and fell already 24 hours, revealing erythematous wound fields. Re-expression of HSA, delivered by lentivirus, restored the normal healing phenotype, within 24 hours post-injury, and even improved the healing in WT, and in BalbC mice. Conclusions Delayed wound-healing in the absence of HSA/CD24 suggests that CD24 plays an important role in this process. Increased expression of CD24, even in the normal state, may be used to enhance wound repair.

Of mice and men: a novel dietary supplement for the treatment of ulcerative colitis:

Background: Curcumin, green tea polyphenols and selenium possess anti-inflammatory and anti-oxidant properties. Individually they have demonstrated some efficacy in animal models and human subjects with inflammatory bowel disease (IBD). To evaluate the efficacy and safety of Coltect [Curcumin (500 mg), green tea (250 mg) and selenium (100 µg)] in vivo and in patients with ulcerative colitis (UC). Methods: Each component was compared to placebo in a DSS mice colitis model. The efficacy was validated in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) rat colitis model. Twenty patients with mild-to-moderate UC received two Coltect tablets twice daily for 8 weeks. Enrollees underwent sigmoidoscopy at study entrance and closure, and physical and laboratory evaluation at baseline, 4 and 8 weeks. Results: Coltect showed a synergistic therapeutic effect in the DSS and TNBS models. Disease activity was significantly higher in the placebo versus the treated group (p < 0.05). Selenium was the more active component. The contribution of green tea was minor. In the TNBS model, the Wallace scores for macroscopic lesions were 4.8 ± 1.5 (treatment) and 8.2 ± 0.5 (placebo) (p = 0.01). In humans, Coltect was well tolerated and effective. Fourteen subjects (70%) improved: nine (45%) went into complete remission, four (20%) experienced marked improvement and one (5%) experienced moderate improvement at the end of the trial. Clinical activity index decreased significantly at 4 and 8 weeks (p < 0.001). Two patients had no change in their symptoms, and one withdrew after 4 weeks. Flare-up in four subjects caused three to withdraw from the study after less than 4 weeks. Endoscopic improvement was observed in 11 (69%) patients, and four patients (25%) achieved complete remission. Conclusions: Coltect may serve as a first-line or add-on therapy in patients with mild-to-moderate UC.

CD24 and APC Genetic Polymorphisms in Pancreatic Cancers as Potential Biomarkers for Clinical Outcome

Background There are no validated biomarkers that correlate with the prognosis of pancreatic ductal adenocarcinoma (PDA). The CD24 and adenomatous polyposis coli (APC) genes are important in the malignant transformation of gastrointestinal cells. This study examined APC and CD24 genetic polymorphisms and their possible impact on survival of patients with PDA. Methods Clinical and pathological data as well as blood samples for extracting DNA were obtained for 73 patients with PDA. Real-time PCR assessed genetic variants of APC (I1307K and E1317Q), and four different single nucleotide polymorphisms (SNPs) in the CD24 gene: C170T (rs52812045), TG1527del (rs3838646), A1626G (rs1058881) and A1056G (rs1058818). Results The median age at diagnosis was 64 (41–90) years. Thirty-one patients (42.5%) were operable, 16 (22%) had locally advanced disease and 26 (35.5%) had disseminated metastatic cancer. The malignancy-related mortality rate was 84%. Median survival was 14 months (11.25–16.74). Survival was similar for wild-type (WT), heterozygous and homozygous variants of the APC or CD24 genes. The three most frequent CD24 SNP combinations were: heterozygote for A1626G and WT for the rest of the alleles (14% of patients), heterozygote for C170T, A1626G, A1056G and WT for the rest (14% of patients), and heterozygote for C170T, A1056G and WT for the rest (10% of patients). All patients were APC WT. The first two groups were significantly younger at diagnosis than the third group. Conclusions Specific polymorphisms in the APC and CD24 genes may play a role in pancreatic cancer development. Correlation with survival requires a larger cohort.

Abstract 3645: CD24 polymorphisms and susceptibility to inflammatory bowel disease and colorectal cancer risk

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohns disease (CD), are chronic inflammatory disorders of the gastrointestinal tract probably resulting from an aberrant immune response to luminal microbial antigens in a genetically predisposed host. Despite significant progress in the understanding and treatment of IBD, patients have an increased risk of colorectal cancer. We have previously shown that CD24 plays an important role in the multistep process of colorectal carcinogenesis and that it may be a target for chemoprevention and antitumor therapy (Sagiv, Gastroenterol, 2006; Sagiv, Can Res, 2008; Shapira, Gastroenterol 2011). However, the role of CD24 in mediating colitis has not been elucidated. Recently, single nucleotide polymorphisms (SNPs) in the CD24 gene have been associated with disease risk and progression in autoimmune diseases and may impact breast cancer prognosis. Aim: Evaluate whether CD24 SNPs are associated with a risk for IBD. Methods: The CD24 polymorphisms: C170T (rs8734), TG1527del (rs3838646), A1626G (rs1058881) and A1056G (rs1058818) were assessed in a case-control study of an Israeli cohort comprising 138 IBD patients and 105 age- and gender-matched healthy controls. Restriction fragment length polymorphism (RFLP) analysis was performed using BstX1, Bsr1, Mfe1, and BstU1 restriction enzymes, respectively. Odds ratio (OR) and 95% confidence interval (CI) were estimated by logistic regression models. Results: C170T carriers had more IBD (OR=3.022, 95% CI: 1.748-5.223, p=0.001): UC (OR=3.002, 95% CI: 1.661-5.427, p=0.001) and CD (OR=3.077, 95% CI: 1.334-7.095 p=0.008). Carrying the A1626G and A1056G SNPs was a risk factor for IBD: OR=2.460, 95% CI: 1.420-4.259, p=0.001 and OR=1.856, 95%: 1.011-3.405, p=0.01, respectively, specifically UC: OR=2.218, 95% CI: 1.207-4.075, p=0.01 and OR=1.944, 95% CI: 0.995-3.798, p=0.01, respectively, but not for CD (p=0.086, p=0.299). A1626G and TG1527del were associated with a younger age of IBD onset (p=0.022, p=0.027, respectively). Conclusions: 1. The CD24 C170T polymorphism is associated with IBD risk. 2. CD24 A1626G and A1056G SNPs might be specifically associated with UC risk. 3. CD24 SNPs associated with an earlier onset of IBD (A1626G and TG1527del) may have a protective role. 4. CD24 may be a new IBD genetic susceptibility factor, with clinical implications in the prediction of IBD phenotype, the course of the disease, and colitis-associated cancer. Citation Format: Victoria Lisiansky, Sarah R. Kraus, Inna Naumov, Dina Kazanov, Ilana Nabiochtchikov, Ohad Toledano, Moshe Leshno, Doran Avivi, Menachem Moshkowitz, Iris Dotan, Nadir Arber. CD24 polymorphisms and susceptibility to inflammatory bowel disease and colorectal cancer risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3645. doi:10.1158/1538-7445.AM2014-3645

Abstract 1004: Impact of genetic polymorphisms on colorectal adenoma recurrence and toxicity in a COX-2 inhibitor trial (Celecoxib): Results from a pilot study

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Chemoprevention trials have shown that Celecoxib, a selective COX-2 inhibitor can reduce adenoma recurrence but can also increase risk of cardiac toxicity. Therefore, in this pilot study, we evaluated the associations between genetic variation in several candidate pathways (e.g. prostaglandin, arachidonic acid and leukotriene synthesis), the incidence of adenoma recurrence as well as toxicity (any, gastrointestinal, cardiac, and non-gastrointestinal/non-cardiac toxicities). Methods: This analysis includes 119 Israeli patients with colorectal adenoma who participated in the Prevention of Colorectal Sporadic Adenomatous Polyps (PreSAP) trial. Reassessment followed after ∼3 years on Celecoxib, followed by 2 years off drug. Incidence of colorectal adenomas was measured by colonoscopy at years 1, 3, and 5. The presence of any, gastrointestinal, cardiac and non-gastrointestinal/non-cardiac toxicities was assessed by investigators for the on-treatment period and collected by patient self-report after treatment. A linkage-disequilibrium-based tagSNP selection algorithm (r2≥0.90, MAF≥4%) identified tagSNPs in candidate pathways. Genotyping was performed using IlluminaTM GoldenGate bead-based genotyping technology. Results: Multiple variants were associated with adenoma recurrence and toxicities. Variability, particularly in COX-1 (rs10306164, rs1236913, rs1330344, rs3119773), COX- 2 (rs4648268), and ALOX12 (rs2073438, rs2292350) played a major role in adenoma recurrence risk among patients on placebo compared to patients on Celecoxib. The highest risk was observed for the COX-1 rs3119773 variant (1890 G>A intron 2) (HR=4.65, 95%CI 1.71-12.64, p=0.0026). Variants in COX2 (rs2206593, rs2745557), EGFR (rs1558544, rs17336919), ALOX15 (rs2619112, rs2619118, rs4796535), SRC (rs6018027, rs7269342), SEPP1 (rs230819, rs230820), and WNT6 (rs6747776, rs6754599) genes were associated with any toxicity. In contrast to gastrointestinal toxicity, a larger number of gene variants (especially variants in PGES, CRP, SRC and GPX3) were associated with increased risk of cardiac toxicity. The increased risk of cardiac toxicity associated with the SRC gene variants (rs6017996, rs6018256, rs6018257) ranged from 7- to 11-fold. Conclusion: Genetic polymorphisms in multiple inflammation-related genes appear to mediate the effect of Celecoxib on adenoma recurrence and the resultant toxicity, particularly cardiac toxicity. Identification of these genetic variants can potentially help in the provision of tailored and optimum care for colorectal adenoma patients. Larger studies validating these pharmacogenetic relationships are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1004. doi:1538-7445.AM2012-1004