Richard B. Arenas

YKL-40, a secreted glycoprotein, promotes tumor angiogenesis.

Tumor angiogenesis is of paramount importance in solid tumor development. Elevated serum levels of YKL-40, which is a secreted heparin-binding glycoprotein, have been associated with a worse prognosis from various advanced human cancers. Yet the role of YKL-40 activity in these cancers is still missing. In this study, we showed that ectopic expression of YKL-40 in MDA-MB-231 breast cancer cells and in HCT-116 colon cancer cells led to larger tumor formation with an extensive angiogenic phenotype than did control cancer cells in mice. Affinity-purified recombinant YKL-40 protein promoted vascular endothelial cell angiogenesis in vitro, the effects of which are similar to the activities observed using MDA-MB-231 and HCT-116 cell-conditioned medium after transfection with YKL-40. Furthermore, YKL-40 was found to induce coordination of membrane-bound receptor syndecan-1 and integrin αvβ3 and to activate an intracellular signaling cascade, including focal adhesion kinase and mitogen-activated protein kinase extracellular signal-related kinase1/2 in endothelial cells. Moreover, blockade of YKL-40 using small-interfering RNA gene knockdown suppressed tumor angiogenesis in vitro and in vivo. Immunohistochemical analysis of human breast cancer showed a correlation between YKL-40 expression and blood vessel density. These findings provide novel insights into angiogenic activities and molecular mechanisms of YKL-40 in cancer development.

Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice

Background:An effective cancer therapeutic must selectively target tumours with minimal systemic toxicity. Expression of a cytotoxic protein using Salmonella typhimurium would enable spatial and temporal control of delivery because these bacteria preferentially target tumours over normal tissue.Methods:We engineered non-pathogenic S. typhimurium to secrete murine TNF-related apoptosis-inducing ligand (TRAIL) under the control of the prokaryotic radiation-inducible RecA promoter. The response of the RecA promoter to radiation was measured using fluorometry and immunoblotting. TRAIL toxicity was determined using flow cytometry and by measuring caspase-3 activation. A syngeneic murine tumour model was used to determine bacterial accumulation and the response to expressed TRAIL.Results:After irradiation, engineered S. typhimurium secreted TRAIL, which caused caspase-3-mediated apoptosis and death in 4T1 mammary carcinoma cells in culture. Systemic injection of Salmonella and induction of TRAIL expression using 2u2009Gy γ-irradiation caused a significant delay in mammary tumour growth and reduced the risk of death by 76% when compared with irradiated controls. Repeated dosing with TRAIL-bearing Salmonella in conjunction with radiation improved the 30-day survival from 0 to 100%.Conclusion:These results show the pre-clinical utility of S. typhimurium as a TRAIL expression vector that effectively reduces tumour growth and extends host survival.

Incidence and therapeutic implications of synchronous colonic pathology in colorectal adenocarcinoma

BACKGROUNDnThe presence of synchronous benign and malignant colonic pathology may influence the magnitude of surgery for colorectal adenocarcinoma. The aim of this prospective study was to quantitate the need for a more extensive surgical procedure because of synchronous pathology in colorectal cancer patients.nnnMETHODSnBetween 1984 and 1996, 235 consecutive patients were treated for colorectal adenocarcinoma. Preoperative survey of the colon in 228 patients included colonoscopy (91%) and double contrast barium enema (35.7%). Seven patients were excluded for incomplete preoperative survey because of perforating or obstructing colon carcinoma or acute ulcerative colitis.nnnRESULTSnOne hundred four patients (45.6%) had the following synchronous colonic lesions: benign polyps (68 patients, 29.8%), diverticular disease (30, 13.1%), ulcerative colitis (10, 4.4%), synchronous adenocarcinoma (8, 3.5%), and Crohns colitis (3, 1.3%). Pathologic examination demonstrated three additional synchronous adenocarcinomas for a total of 11 patients (4.9%). Twenty-five (11%) required more extensive surgery than dictated by the primary cancer. Of these 25 patients, 17 had a benign or premalignant condition associated with their carcinoma and 8 had a synchronous carcinoma. Seventeen patients underwent a sphincter-saving procedure. Of the remaining eight patients requiring sphincter ablation, seven were needed because of a synchronous nonmalignant lesion, rather than because of the primary tumor.nnnCONCLUSIONSnIn our patient population, the incidence of synchronous colorectal lesions was 45.6%. Synchronous colorectal cancer occurred in 4.9%. In 11%, the presence of synchronous colorectal lesions made the surgical procedure more extensive than that dictated by the primary cancer, and in 3%, the need for a sphincter ablating procedure was dictated by a synchronous nonmalignant lesion.

Adjuvant therapy for colorectal cancer

Approximately 141,000 patients will develop colorectal cancer each year and 45% will have positive lymph nodes, one of the most significant predictors of survival. Now there is evidence that systemic chemotherapy with fluorouracil (FU) and leucovorin, levamisole, or FU and leucovorin with decrease recurrence and increase survival for patients with Dukes C colon cancer. In patients with Dukes B or C rectal cancer, combined radiation and FU increase survival and decrease local and distal recurrence.

Breast cancer expression of YKL-40 correlates with tumour grade, poor differentiation, and other cancer markers

Background:Serum levels of a secreted glycoprotein YKL-40 are elevated in patients with a wide range of cancers including breast, colorectal, and ovarian cancers. Furthermore, these increased levels correlate with poorer survival of cancer patients, suggesting that serum levels of YKL-40 might be a prognostic biomarker. However, the tissue expression of YKL-40 and its relationship with clinical outcomes and other potential markers are poorly understood.Methods:Tissue samples from invasive breast cancers, breast ductal carcinoma in situ (DCIS), and cancer-free reduction mammoplasty were enrolled. YKL-40 expression was measured using immunohistochemistry and evaluated by a semi-quantification assay. Statistical analyses explored the relationship of YKL-40 with clinical outcome and other breast cancer biomarkers.Results:Breast ductal carcinoma in situ expressed low and moderate levels of YKL-40. In the subset of 203 patients with invasive cancer, YKL-40 levels were positively correlated with tumour grade (P<0.0001) and Her2/neu (P<0.01), but negatively correlated with oestrogen (P<0.0001) and progesterone receptor (P<0.0001). YKL-40 levels were inversely correlated with expressions of GATA3 (P=0.0137) and E-cadherin (P=0.0417).Conclusion:These data demonstrate that expression levels of YKL-40 are associated with tumour grade, poor differentiation, and other breast cancer markers, highlighting that tissue levels of YKL-40 serve as a valuable biomarker for breast cancer diagnosis and prognosis.

Introduction of human adenomatous polyposis coli gene into Min mice via cationic liposomes

BACKGROUNDnThe adenomatous polyposis coli (APC) gene is a tumor-suppressor gene involved in familial polyposis coli (FAP), a hereditary disease heralded by the development of hundreds of colorectal adenomas. A mouse model for FAP, the multiple intestinal neoplasia (Min) mouse, develops multiple adenomatous polyps of the large and small intestine similar to their human counterparts. To test the feasibility of introducing normal human APC as a means of either preventing or reversing polyp formation, we describe a method of in vivo transfection of APC into colonic epithelium of the Min mouse.nnnMETHODSnAnesthetized young (4 weeks) Min mice were treated with enemas containing lipofectant and a normal human APC cDNA plasmid every 72 hours for 2 months and then euthanized at 24, 48, and 72 hours after the last treatment. Polymerase chain reaction (PCR) was used to detect the presence of the plasmid DNA.nnnRESULTSnPCR on the extracted colonic epithelial DNA showed the presence of plasmid DNA up to 72 hours after the last treatment. Expression of the plasmid construct was confirmed by reverse transcriptase-PCR.nnnCONCLUSIONSnWe have demonstrated the repeated introduction and detection of normal human APC in the colonic epithelium of the Min Mouse in vivo during an extended period of time with no toxic side effects by means of our prolonged therapy.

Selective expression of carcinoembryonic antigen promoter in cancer cell lines

PURPOSE: This study was designed to characterize the mechanisms regulating the expression of the human carcinoembryonic antigen promoter (pCEA), in terms of tissue-specific targeting for gene therapy. The promoter was subcloned to a luciferase reporter gene (pCEA/Luc) in our laboratory and compared with a virally controlled luciferase vector (pSV40/Luc). METHODS: Four human cancer cell lines (HeLa, SW480, Caco2, and SW1116) were transfected with either pCEA/Luc or pSV40/Luc. Cells were treated with interferon-gamma and assayed at 72 hours after treatment. Carcinoembryonic antigen level was measured by enzyme immunoassay. Luciferase expression was measured at 48 hours and one week after transfection by luminometry. RESULTS: Luciferase activity after transfection with pCEA/Luc was higher in CEA-positive cells than in CEA-negative cells (P<0.0001). pCEA/Luc demonstrated higher activity than pSV40/Luc in CEA-positive cells (P<0.0001), but not in CEA-negative cells. In Caco2 cells, which before confluence are CEA-negative, luciferase expression increased on reaching confluence (P<0.0001). Well to moderately differentiated cells responded to the interferon-gamma treatment, but the increase in CEA secretion did not correspond to an increase in pCEA/Luc expression. CONCLUSIONS: The expression of pCEA correlates well with the CEA production by the specific cell line offering a potential tissue-specific targeting strategy for colon cancer gene therapy. Furthermore, the tissue-specific CEA promoter has a higher and more persistent activity in CEA-positive human cancer cells than a viral promoter. The lack of response to interferon-gamma treatment suggests a different mechanism of action for interferon-gamma other than directly interacting with the promoter.

Gene therapy of the gut: introduction of the APC tumor-suppressor gene for cancer prevention or treatment

Abstract Liposomal delivery of gene therapy to the colonic epithelium has been evaluated in a rodent model. The use of β-galactosidase as a reporter gene demonstrated uptake and expression in virtually 100% of the epithelial cells which contacted the liposome-gene mixture. Expression was transient, persisting for 2–3 days, diminishing in accordance with normal epithelial turnover. Similar results were seen in both rats and mice. The method was used to deliver the APC tumor suppressor gene to rat colonic epithelium. Expression of RNA was verified by means of RNAse protection, as well as by an exon-connection strategy utilizing reverse-transcription. A 6-week period of bi-weekly retreatment established safety and minimal toxicity of this route; in particular, there were no obvious changes in cell growth or differentiation with prolonged administration of the APC gene. The potential use of this method for delivery of gene therapy, and the specific application of the APC gene for cancer prevention or treatment, are discussed.

Alterations in intestinal epithelial cell proliferation and apoptosis after APC gene therapy and selective COX-2 inhibition in the Min mouse

Purpose: The Multiple intestinal Neoplasia (Min) mouse has been proved to be an ideal model for intestinal tumorigenesis. Mutated for the murine homolog of the Adenomatous Polyposis Coli (APC) gene, these mice develop polyps throughout the intestinal tract. Min mice studies have shown alterations in apoptosis and cell migration in areas of the intestinal epithelium that do not harbor polyp formation that may be mediated through increased cyclooxygenase 2 (COX-2) activity. Our laboratory has successfully reduced polyp formation in the Min mouse through introduction of a human APC construct transorally via liposomes. We describe our results on intestinal cell proliferation and apoptosis in the Min mouse after APC gene therapy and selective COX-2 inhibition.