Phosphatidylinositol 3-Kinase Mediates the Ability of Retinol to Decrease Colorectal Cancer Cell Invasion Jennifer N. Griffin Lengyel, Eun Young Park, Anna R. Brunson, Daniel Pinali, and Michelle A. Lane School of Family and Consumer Sciences, Nutrition and Foods Program, Texas State University—San Marcos, San Marcos, Texas, USA Previously, we showed that retinol (vitamin A) decreased both colorectal cancer cell invasion and phosphatidylinositol 3-kinase (PI3K) activity through a retinoic acid receptor–independent mechanism. Here, we determined if these phenomena were related by using parental HCT-116 cells that harbor 1 allele of wild-type PI3K and 1 allele of constitutively active (ca) PI3K and 2 mutant HCT-116 cell lines homozygous for caPI3K. In vitro, treatment of parental HCT-116 cells with 10 mM retinol reduced cell invasion whereas treatment of mutant HCT-116 cell lines with retinol did not. Treatment with 10 mM retinol also decreased the activity of matrixmetalloproteinase-9 and increased tissue inhibitor of matrixmetalloproteinase-I levels in parental, but not mutant, HCT-116 cells. Finally, parental or mutant cells were intrasplenically injected into athymic mice consuming diets with or without supplemental vitamin A. As expected, vitamin A supplementation tended (P D 0.18) to reduce the incidence of metastases in mice injected with the parental cell line and consuming the supplemented diet. In contrast, metastatic incidence was not affected (P D 1.00) by vitamin A supplementation in mice injected with mutant cells. These data indicate that the capacity of retinol to inhibit PI3K activity confers its ability to decrease colorectal cancer metastasis. INTRODUCTION Retinoids have been shown to exhibit cancer chemopreven- tative and chemotherapeutic properties. Most research in this area focuses on the efficacy and mechanism of action of all- trans retinoic acid (ATRA); however as cancer progresses, tumors frequently become resistant to the actions of ATRA, diminishing the effectiveness of ATRA as cancer chemother- apy (1). In addition, the diet contains very little ATRA (2). Rather, animal-derived foods in the diet contain retinyl esters that are cleaved to yield retinol (vitamin A) in the intestinal lumen; thus, colonocytes are primarily exposed to retinol. In addition, vitamin A is stored in the liver as retinyl esters. Ele- vated intestinal lumen and hepatic concentrations of retinol have been attained by dietary vitamin A supplementation (3–5). Recently, our laboratory showed that dietary vitamin A sup- plementation decreases colorectal cancer cell metastasis in a nude mouse xenograft model (3). We have also shown that ret- inol decreases the invasion of cultured ATRA-resistant human colon cancer cells via a novel ATRA and retinoic acid receptor (RAR)-independent mechanism in vitro (6). In addition, reti- nol favorably alters the activity and levels of invasion-related proteins. Specifically, retinol decreases matrix metalloprotei- nase (MMP)-9 activity and protein levels while increasing tis- sue inhibitor of matrixmetalloproteinase (TIMP)-1 protein concentrations (6). Both MMP-9 and TIMP-1 are downstream of phosphatidylinositol 3-kinase (PI3K) (7,8). Previous work in our laboratory also established that PI3K activity is inhib- ited by retinol in vitro (9). Elevated PI3K activity is associated with increased colon cancer cell invasion and metastasis (for review, see Ref. 10). Increased cell invasion following activation of the PI3K path- way has been associated with increased expression of MMP-9 in various carcinomas (7,11–13). MMP-9 is a secreted gelati- nase that promotes metastatic progression via degradation of extracellular matrix (ECM) proteins (14–18). MMP-9 is over- expressed in colorectal carcinomas (19). Increased levels of MMP-9 expression in colorectal cancer, when compared with normal mucosa, have been associated with significantly shorter disease-free and overall survival times (20). Reports have also shown that patients with colon carcinoma have a sig- nificant increase in levels of MMP-9 protein and mRNA in tumor areas when compared with noninvolved regions (21,22). In addition to our work with retinol, other retinoids, specifically ATRA, have been shown to decrease MMP-9 activity and protein levels in several tumor types potentially by altering PI3K signaling (23–25). TIMP-1 specifically inhibits MMP-9 activity by binding to pro-MMP-9 thereby preventing the conversion of pro-MMP-9 Submitted 4 December 2013; accepted in final form 12 August 2014. Jennifer N. Griffin Lengyel is now with the Houston Independent School District, Houston, TX. Eun Young Park is now with the Bio- Therapeutic Laboratory, Samsung, Seoul, Korea. Address correspondence to Michelle A. Lane, School of Family and Consumer Sciences, Nutrition and Foods Program, Texas State University, 601 University Dr., San Marcos, TX 78666, USA. Phone: 1-512-245-4654. Fax: 1-512-245-3829. E-mail: ML48@txstate.edu 1352 Nutrition and Cancer, 66(8), 1352–1361 Copyright Ó 2014, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2014.956258