Inna Naumov

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.

Aberrant methylation and reduced expression of RASSF1A in Ewing sarcoma.

Ewing sarcoma (ES) is the second most common solid bone and soft tissue malignancy in children and young adults with low cure rates indicating the need to identify further prognostic markers. The importance of methylation in the inactivation of key tumor suppressor genes including RASSF1A has begun to be appreciated in context of cancer development, prognosis and therapy. However there is lack of similar broad based studies in ES. The objective of this study was to analyze RASSF1A methylation and assess its clinical significance in ES.

Malignant transformation of normal enterocytes following downregulation of Bak expression.

Bak is a pro-apoptotic gene, which plays an important role in the multi-step process of gastrointestinal tumorigenesis. We hypothesized that downregulation of Bakexpression in normal enterocytes will result in a transformed phenotype. The nontumorigenic intestinal epithelial cell line (IEC18) was transfected with the vector pMV12-AS-bak (encoding anti-sense bak). Three clones, with Bakprotein levels similar to those seen in colon cancer cell lines and significantly lower than those found in the parental cells, were further evaluated. The three clones proliferated faster, demonstrated anchorage-independent growth in soft agar and a higher saturation density and plating efficiency. Furthermore, when injected into nude mice, these cells generated tumors after approximately 2–3 weeks. The cells were more resistant to the induction of apoptosis by sulindac sulfide and sulindac sulfone but more sensitive to COX 2 inhibitors (celecoxib and nimesulide). The levels of p16, cyclin D1 and COX 2 were higher in the three transformed clones. In summary,downregulation of Bak expression in normal enterocytes contributes to abnormal growth and tumorigenesis. COX 2 inhibitors may serve as important agents in the prevention and treatment of CRC as they only inhibit the growth of malignant cells.

One stop screening for multiple cancers: The experience of an integrated cancer prevention center

BACKGROUND Cancer is a leading cause of mortality worldwide. Screening is a key strategy for reducing cancer morbidity and mortality. METHODS We aimed to describe the experience of an integrated cancer prevention center in screening an asymptomatic population for the presence of neoplasia. One-thousand consecutive asymptomatic, apparently healthy adults, aged 20-80 years, were screened for early detection of 11 common cancers that account for 70-80% of cancer mortality. RESULTS Malignant and benign lesions were found in 2.4% and 7.1% of the screenees, respectively. The most common malignant lesions were in the gastrointestinal tract and breast followed by gynecological and skin. The compliance rate for the different screening procedures was considerably higher than the actual screening rate in the general Israeli population - 78% compared to 60% for mammography (p<0.001) and 39% compared to 16% for colonoscopy (p<0.001). Advanced age, family history of cancer and certain lifestyle parameters were associated with increased risk. Moreover, polymorphisms in the APC and CD24 genes indicated high cancer risk. When two of the polymorphisms existed in an individual, the risk for a neoplastic lesion was extremely high (OR 2.3 [95% CI 0.94-5.9]). CONCLUSIONS One stop shop screening for 11 common cancers in the setting of a multidisciplinary outpatient clinic is feasible and can detect cancer at an early stage.

Gene Expression following Exposure to Celecoxib in Humans: Pathways of Inflammation and Carcinogenesis Are Activated in Tumors but Not Normal Tissues

Background: The Cox-2 inhibitor, celecoxib (Pfizer Inc., N.Y., USA), is a promising chemopreventive agent [Arber et al.: N Engl J Med 2006;355:885–895; Bertagnolli et al.: N Engl J Med 2006;355:873–884]. This study aims to explore its mechanism by defining changes in gene expression between neoplastic and normal tissue samples before and after treatment. Methods: Patients with documented colorectal neoplasia in screening colonoscopy, destined to undergo surgical colectomy, were randomized for treatment with celecoxib (n = 11; 400 mg/day) or placebo (n = 3) for 30 days. Tissue samples were taken from the tumor and from normal adjacent mucosa during both colonoscopy and surgery. RNA was extracted and analyzed using Affymetrix Genechip®. Results: 687 genes differentiated tumor samples before and after treatment, among which 310 genes did not show the same differential expression in the placebo group or normal samples. These genes were significantly related to pathways of cell cycle regulation and inflammation, and of note was the TGF-β pathway, which held a strong association with the list of genes formerly found to be associated with the colorectal cancer expression profile in microarray analyses, as summarized in a meta-analysis by Cardoso et al. [Biochim Biophys Acta 2007;1775:103–137]. Conclusions: Celecoxib selectively affects genes and pathways involved in inflammation and malignant transformation in tumor but not normal tissues, this may assist in the development of safer and more effective chemopreventive agents.

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.

Predictive Levels of CD24 in Peripheral Blood Leukocytes for the Early Detection of Colorectal Adenomas and Adenocarcinomas.

CD24 is expressed in 90% of colorectal adenomas and adenocarcinomas. Colorectal cancer (CRC) can be mostly prevented but average risk population screening by stool testing or colonoscopy faces many hurdles. Blood testing is clinically needed. We aimed to evaluate the utility of CD24 expression in peripheral blood leukocytes (PBLs). Two independent case studies were conducted in eligible individuals undergoing colonoscopy. Protein extracted from PBLs was subjected to immunoblotting using anti-CD24 monoclonal antibodies. CD24 sensitivity and specificity were determined using receiver operating characteristic (ROC) analysis. Initially, 150 subjects were examined: 63 had CRC, 19 had adenomas, and 68 had normal colonoscopies. The sensitivity and specificity of CD24 for distinguishing CRC from normal subjects were 70.5% (95% CI, 54.8–83.2%) and 83.8% (95% CI, 74.6–92.7%) and for adenomas 84.2% (95% CI, 60.4–96.4%) and 73.5% (95% CI, 61.4–83.5%), respectively. In the second trial (n = 149), a similar specificity but higher sensitivity was achieved: 80.0% (95% CI, 63.1–91.6%) for CRC and 89.2% (95% CI, 74.6–97%) for adenomas. A simple noninvasive blood test evaluating CD24 levels has high sensitivity and specificity for detecting colorectal adenomas and cancer in patients undergoing colonoscopy at an urban medical center. Larger multicenter studies are warranted to establish the potential of this promising test.

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 1338: Knockdown of CD24 prevents colorectal polyp formation in APCmin/CD24 double knockout transgenic mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Previous studies in our lab have shown that CD24 is a potential oncogene in the colon. CD24, a mucin-like glycoprotein, is overexpressed in 90% of colorectal cancer (CRC) tumors at a fairly early stage in the multistep process of CR carcinogenesis (Sagiv et al., Gastroenterology, 2006). We have also shown that CD24 downregulation by shRNA interference (Sagiv et al., Can Res, 2008) or anti-CD24 mAb targeting (Shapira et al., Gastroenterology, 2011) retarded tumorigenicity of human CRC cell lines and/or reduced tumor volume in nude mice. The APCmin/+ is a popular animal model for studies of human colon carcinogenesis and the molecular changes associated with neoplasia in this system have been partially characterized. These mice have a germline nonsense mutation at codon 850 of the APC gene and spontaneously develop hundreds of polyps, mostly in the small intestine at the age of 10-12 weeks. Aim: To validate the importance of CD24 in intestinal carcinogenesis using a transgenic mouse model with a double knockout (KO) of CD24−/+ /CD24+/− and APCMin/+ alleles. Methods: We generated the CD24−/+/APCMin/+ double KO by crossing the mice with each of the single gene knockouts, in order to obtain both CD24+/−/APCMin/+ and CD24−/+ APCMin/+ genotypes. The double KO mice were followed up thrice a week and sacrificed at 12 weeks of age. Tumors from the entire small and large intestine were counted and verified by histological analysis Results: There was almost a complete absence of polyps in the small intestine in APCMin/+/CD24+/− double heterozygotes compared to that of the parental APCMin/+, CD24+/+ animals: 273±55.4 vs. 6±1.7 polyps, respectively (P=0.0001). APCMin/+and CD24−/+ homozygotes showed a striking lack of polyps (P< 0.0001). This reduction occurred in all sections of the small intestine. Histophatological analysis confirmed the absence of malignant lesions in the double KO mice as compared to the parental mice. Conclusions: 1. A deficiency of only one CD24 allele is sufficient to suppress almost completely polyp formation in the APCMin/+ mice that normally develop hundreds of polyps. 2. Although the mechanism/s underlying the pathophysiologic role of CD24 in the development of CRC are still unknown, these studies suggest that CD24 plays a critical role in the proliferation and progression of CRC and is a potential important target for the prevention and treatment of intestinal neoplasia. 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 1338. doi:1538-7445.AM2012-1338