P-183 Abrogation of alcohol dehydrogenase-1B expression by CD90+ stromal cells supports tumor-promoting inflammation in colorectal cancer

Abstract

Introduction: Members of the alcohol dehydrogenase (ADH) enzyme family metabolize a wide variety of substrates, including C2-C4 alcohols and alcohol form of Vitamin A, retinol (RO). ADH1B, a major isoform expressed in normal colon, is implicated in the conversion of vitamin A to retinoic acid (RA), an oxidized form of RO known to be a potent anti-inflammatory molecule.We and others have recently reported a dramatic decrease in ADH1B mRNA expression in colorectal cancer (CRC). However, the pathophysiological role of downregulation of ADH1B in cancer development and progression remains unknown. Thus, in this work, we determined the cell-type specific source associated with ADH1B decrease and hypothesize that a suppression of RO-metabolizing enzyme, ADH1B, promotes tumor-associated inflammatory responses in CRC. Methods: Human tissue samples were collected from patients undergoing screening or colectomy for CRC under IRB-approved human protocols at the University of Texas Medical Branch, University of New Mexico Health Sciences Center, and Legacy Research Tumor Bank (Portland, OR). Real-time RT-PCR, confocal microscopy, flow cytometry and cytokine luminex arrays were used to evaluate alterations in gene expression and its relevance to the tumor associated inflammation. Silencing of ADH1B mRNA expression was performed using primary cultures of human colonic CD90+ myo-/fibroblasts (CMFs). Results: Using confocal microscopy, we confirmed a dramatic decrease in ADH1B expression in sporadic CRC tumors. Downregulation of ADH1B was associated with malignant neoplastic transformation: a dramatic decrease in this enzyme expression in adenoma? but not hyperplastic polyps ? while almost complete shutdown was observed in T1-T4 CRC tumors.We also observed that colonic CD90+ CMFs are the major cellular source of ADH1B expression in normal colonic mucosa, and its transformation to cancer-associated fibroblasts (CAFs) in tubular adenoma and CRC results in the abrogation of ADH1B expression. We recently reported that CAFs are major source of tumor-promoting inflammatory cytokine IL-6. Silencing of the ADH1B by using gene specific siRNA in normal CMFs reproduced a pro-inflammatory CAF phenotype and results in high production of the LPS-inducible IL-6. Both RO and RA were able to downregulate expression of an LPS inducible IL-6 in normal CMFs in a dose dependent manner, with the strongest effect at 25 μM concentration. In contrast, a lack of ADH1B expression in CAFs prevented the downregulation of a basal and inducible IL-6 expression by RO. Conclusion: Our study suggests that suppression of the ADH1B is an early event associated with neoplasia of the colonic tissue. However, this is not observed in hyperplastic lesions. CMFs are the major source of ADH1B in the normal colonic mucosa, and decrease of the ADH1B expression in CMFs during adenoma-carcinoma sequence promotes IL-6-driven tumor-promoting inflammation and involves disruption of the vitamin A-mediated regulation of IL-6. Taken together, these results support the notion that abrogation of ADH1B expression in CAFs may be considered as an early stage biomarker of carcinogenesis and a target for treating or preventing CRC.