Eyal Sagiv

Targeting CD24 for Treatment of Colorectal and Pancreatic Cancer by Monoclonal Antibodies or Small Interfering RNA

CD24 is a potential oncogene reported to be overexpressed in a large variety of human malignancies. We have shown that CD24 is overexpressed in 90% of colorectal tumors at a fairly early stage in the multistep process of carcinogenesis. Anti-CD24 monoclonal antibodies (mAb) induce a significant growth inhibition in colorectal and pancreatic cancer cell lines that express the protein. This study is designed to investigate further the effects of CD24 down-regulation using mAb or small interfering RNA in vitro and in vivo. Western blot analysis showed that anti-CD24 mAb induced CD24 protein down-regulation through lysosomal degradation. mAb augmented growth inhibition in combination with five classic chemotherapies. Xenograft models in vivo showed that tumor growth was significantly reduced in mAb-treated mice. Similarly, stable growth inhibition of cancer cell lines was achieved by down-regulation of CD24 expression using short hairpin RNA (shRNA). The produced clones proliferated more slowly, reached lower saturation densities, and showed impaired motility. Most importantly, down-regulation of CD24 retarded tumorigenicity of human cancer cell lines in nude mice. Microarray analysis revealed a similar pattern of gene expression alterations when cells were subjected to anti-CD24 mAb or shRNA. Genes in the Ras pathway, mitogen-activated protein kinase, or BCL-2 family and others of oncogenic association were frequently down-regulated. As a putative new oncogene that is overexpressed in gastrointestinal malignancies early in the carcinogenesis process, CD24 is a potential target for early intervention in the prevention and treatment of cancer.

Targeting the active β-catenin pathway to treat cancer cells

The adenomatous polyposis coli or β-catenin genes are frequently mutated in colorectal cancer cells, resulting in oncogenic activation of β-catenin signaling. We tried to establish in vitro and in vivo models for selectively killing human cancer cells with an activated β-catenin/T-cell factor (Tcf) pathway. We used a recombinant adenovirus that carries a lethal gene [p53-up-regulated modulator of apoptosis (PUMA)] under the control of a β-catenin/Tcf–responsive promoter (AdTOP-PUMA) to selectively target human colorectal cancer cells (SW480, HCT116, DLD-1, and LS174T), hepatocellular carcinoma (HepG2), and gastric cancer cells (AGS) in which the β-catenin/Tcf pathway is activated, and compared its efficiency in killing cancer cells in which this pathway is inactive or only weakly active. AdFOP-PUMA, carrying a mutant Tcf-binding site, was used as control virus. The combined effect of AdTOP-PUMA with several chemotherapeutic agents (5-florouracil, doxorubicin, and paclitaxel) was also evaluated. The effect of AdTOP-PUMA on colorectal cancer cells was also examined in nude mice: SW480 cells were infected with the AdTOP-PUMA and AdFOP-PUMA, and then inoculated s.c. into nude mice. The TOP-PUMA adenovirus inhibited cell growth in a dose-dependent fashion, depending on the signaling activity of β-catenin. The growth of cells displaying high levels of active β-catenin/Tcf signaling was inhibited after infection with AdTOP-PUMA, whereas that of cells with low levels of β-catenin signaling was not. Growth inhibition was associated with induction of apoptosis. Chemotherapy synergistically enhanced the effect of AdTOP-PUMA. A combination of the adenovirus system with standard therapy may improve the efficacy and reduce the toxicity of therapy in humans. [Mol Cancer Ther 2006;5(11):2861–71]

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.

Gene expression analysis proposes alternative pathways for the mechanism by which celecoxib selectively inhibits the growth of transformed but not normal enterocytes.

Purpose: Cyclooxygenase-2 inhibitor (celecoxib, Pfizer) is a promising chemopreventive agent, yet its long-term use may be limited due to increased cardiovascular toxicity. This study was aimed to identify genes and pathways involved in colorectal tumorigenesis and affected by celecoxib. Experimental Design: Normal rat enterocytes (IEC18 cells) and their Ras-transformed derivatives (R1) were exposed for 72 h or over 6 months to celecoxib and analyzed for gene expression pattern using Genechip (RG-U34). Cluster and pathway analyses were done using GeneSpring software and Gene Ontology database. Cyclin D1 was overexpressed in IEC18 cells using stable transfection; cell cycle and prostaglandin synthesis were assessed. Results: Five hundred thirty-eight genes were differentially expressed after transformation, and 70 and 126 genes, respectively, were affected by short and long treatments with celecoxib. Clusters of expression showed different expression in the transformed cells that revert to normal after treatment; they included Ras/Erk/Ral-B, Jagged2/Notch, calcineurin, lysyl-oxidase, etc. Cyclin D1 is up-regulated under the Ras pathway and is down-regulated by celecoxib. Thus, we showed that cyclin D1–transformed cells are resistant to inhibition by celecoxib. Celecoxib was also shown to work via cyclooxygenase-2 inhibition in transformed cells. Conclusions: Celecoxib selectively affects transformed and not normal enterocytes by targeting genes and pathways that are involved in the transformation. Thus, an alternative mechanism is proposed for the cancer-preventive role of celecoxib other than the classic mechanism of inhibiting prostaglandin synthesis, stressing mainly the role of cyclin D1. These data may help in the development of safer and more effective preventive drugs.

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.

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.

992 Unraveling Novel Mechanisms By Which the CD24 Oncogene Serves As a Target for Colorectal and Pancreatic Cancer Treatment, Via Down-Regulation By Monoclonal Antibodies or SIRNA

Background: We have previously shown that CD24 is a potential oncogene in the gut, being highly expressed in 90% of malignant and pre-malignant lesions from the entire GI tract, and only in 17% of normal epithelium (N=389). A year ago we confirmed that CD24 is indeed an important oncogene, based on the fact that stably expressed CD24 siRNA reduced the malignant phenotype of colorectal (CRC) and pancreatic (PC) cancer cell lines, and we were able to show the potential of anti-CD24 monoclonal antibodies (MAb) in CRC and PC treatment In Vitro. Aim: To further explore the therapeutic potential of CD24 MAb and siRNA silencing In Vivo and to unravel possible downstream cellular mechanisms. Methods: 1. HT29 (CRC) and Colo357 (PC) cell lines, expressing high levels of CD24, were used as human xenograft models in nude mice bearing: A. Subcutaneous tumors (5x106 cells) N= 20. B. Splenic tumors (1x106 cells) N=20. In each experiment, the mice were randomly divided into two groups; treated IV twice weekly with either MAb or saline. Animals were killed when tumor volume reached 15 mm, or after 18 days in the splenic model. Primary and metastatic tumors were dissected, fixed and stained for HE functional analysis was carried out based on Gene Ontology (GO) groups. Results: 1. MAb treatment significantly inhibited subcutaneous tumor growth and formation in the spleen, and metastases shedding. 2. MAb treatment induced lysosomal-mediated degradation of CD24 protein 3. Altered gene expression (>3) following MAb therapy (n=581) and stable expression of CD24 siRNA (N= 1572) was found. siRNA expression in cells mainly affected the Ras and MAPK pathways. The expression of 139 genes was similarly altered by both treatment modalities, including genes of the BCl-2 super-family and p21 WAF1/CIP1. Pathway analysis revealed that genes associated with cell cycle regulation were mostly involved. Conclusions: 1. CD24 is a potential useful target for early intervention and treatment of CRC and PC. 2. Anti-CD24 MAb therapy involves degradation of CD24 through the lysosomal pathway. 3. CD24 initiated a signal transduction pathway that affects cell cycle progression.