Gastrin-releasing peptide (GRP) and its receptor (GRPR) act as morphogens when expressed in colorectal cancer (CRC), promoting the assumption of a better differentiated phenotype by regulating cell motility in the context of remodeling and retarding tumor cell metastasis by enhancing cell-matrix attachment. Although we have shown that these processes are mediated by focal adhesion kinase (FAK), the downstream target(s) of GRP-induced FAK activation are not known. Since osteoblast differentiation is mediated by FAK-initiated upregulation of ICAM-1 (Nakayamada S, Okada Y, Saito K, Tamura M, Tanaka Y. J Biol Chem 278: 45368-45374, 2003), we determined whether GRP-induced activation of FAK alters ICAM-1 expression in CRC and, if so, determined the contribution of ICAM-1 to mediating GRP's morphogenic properties. Caco-2 and HT-29 cells variably express GRP/GRPR. These cells only express ICAM-1 when GRPR are present. In human CRC, GRPR and ICAM-1 are only expressed by better differentiated tumor cells, with ICAM-1 located at the basolateral membrane. ICAM-1 expression was only observed subsequent to GRPR signaling via FAK. To study the effect of ICAM-1 expression on tumor cell motility, CRC cells expressing GRP, GRPR, and ICAM-1 were cultured in the presence and absence of GRPR antagonist or monoclonal antibody to ICAM-1. CRC cells engaged in directed motility in the context of remodeling and were highly adherent to the extracellular matrix, only in the absence of antagonist or ICAM-1 antibody. These data indicate that GRP upregulation of ICAM-1 via FAK promotes tumor cell motility and attachment to the extracellular matrix.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are aberrantly up-regulated in colon cancer. When expressed, they act as morphogens, retaining tumor cells in a better differentiated state and retarding metastasis. To identify targets activated in response to GRPR signaling we studied Caco-2 and HT-29 cells, colon cancer cell lines that expresses GRPR as a function of confluence. Total cell protein was extracted from pre-confluent cells (expressing GRP/GRPR) cultured in serum-free media in the presence or absence of GRPR-specific antagonist; as well as from confluent cells that do not express GRPR. Overall, we identified 5 proteins that are specifically down-regulated after GRP/GRPR expression: Bach2, creatine kinase B, p47, and two that could not be identified; and 6 proteins that are up-regulated: gephyrin, HSP70, HP1, ICAM-1, ACAT, and one that could not be identified. These findings suggest that the mechanism(s) by which GRP/GRPR mediate its morphogenic effects in colon cancer involve the actions of a number of hitherto unappreciated proteins.

Epithelial cells lining the adult colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, GRP/GRPR can be aberrantly expressed in colon cancer where they are associated with improved patient survival rates. However, the mechanism of action whereby these proteins mediate their beneficial effects is not known. Heterochromatin protein 1 is an epigenetic modifier of gene transcription for which three different isoforms exist in humans: HP1(Hsα), HP1(Hsβ), and HP1(Hsγ). In breast cancer and melanoma, respectively, HP1(Hsα) and HP1(Hsβ) have been shown to modulate the aggressiveness of tumor cells in vivo. In contrast, the role of HP1 in colon cancer has not been elucidated, and a mechanism of regulating the expression of any HP1 isoform in any context has not yet been identified. In this article we demonstrate that abrogating GRP/GRPR signaling specifically down-regulates HP1(Hsβ) expression and that inhibiting GRPR signaling, or ablating HP1(Hsβ) expression, increases colon cancer cell invasiveness in vitro. These findings identify for the first time a signaling pathway regulating heterochromatin protein expression and suggest a mechanism whereby aberrantly expressed GRPR might alter the outcome of patients with colorectal cancer.

Epithelial cells lining the adult human colon do not normally express gastrin releasing peptide (GRP) or its receptor (GRPR), but both can be up regulated post malignant transformation. However, controversy exists as to the contribution these proteins make to tumor cell behavior once present. Since GRPR activation promotes proliferation, it has been assumed that their aberrant expression promotes colon cancer (CC) growth and progression. Yet we have contended that when expressed, GRP/GRPR benefits the host since in vitro studies demonstrate they enhance tumor cell attachment to the extracellular matrix and promote CC cytolysis by natural killer lymphocytes. Thus the aim of this study was to ascertain the effect of aberrant GRP/GRPR expression on patient survival. To do this we identified all CC diagnosed at a single institution from 1998 to 2002 that were classified as AJCC stage II or III (n = 88); of these 50 (57%) had sufficient tissues remaining for study. GRP/GRPR expression and natural killer cell density were determined immunohistochemically at the leading edge of each CC, and survival assessed by Kaplan Meier analysis. Expression of high levels of GRPR alone, or both GRP and GRPR, was associated with delayed CC recurrence (14.1-17.0 months, respectfully; P = 0.005) and increased survival (10.1-13.1 months, respectfully; P = 0.0124). CC expressing GRP/GRPR were associated with significantly fewer lymph node metastases than tumors not expressing these proteins, and contained significantly more CD16 + natural killer cells, than tumors not expressing these proteins. These findings demonstrate that patients whose CC express GRPR are associated with a survival advantage as compared to those whose CC do not express these proteins.