Gastrin-releasing peptide signaling alters colon cancer invasiveness via heterochromatin protein 1Hsß.

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(Hsß), and HP1(Hsγ). In breast cancer and melanoma, respectively, HP1(Hsα) and HP1(Hsß) 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(Hsß) expression and that inhibiting GRPR signaling, or ablating HP1(Hsß) 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.

DDB2 suppresses epithelial-to-mesenchymal transition in colon cancer.

Colon cancer is one of the deadliest cancers worldwide because of its metastasis to other essential organs. Metastasis of colon cancer involves a complex set of events, including epithelial-to-mesenchymal transition (EMT) that increases invasiveness of the tumor cells. Here, we show that the xeroderma pigmentosum group E (XPE) gene product, damaged DNA-binding protein (DDB)-2, is downregulated in high-grade colon cancers, and it plays a dominant role in the suppression of EMT of the colon cancer cells. Depletion of DDB2 promotes mesenchymal phenotype, whereas expression of DDB2 promotes epithelial phenotype. DDB2 constitutively represses genes that are the key activators of EMT, indicating that DDB2 is a master regulator of EMT of the colon cancer cells. Moreover, we observed evidence that DDB2 functions as a barrier for EMT induced by hypoxia and TGF-ß. Also, we provide evidence that DDB2 inhibits metastasis of colon cancer. The results presented here identify a transcriptional regulatory pathway of DDB2 that is directly linked to the mechanisms that suppress metastasis of colon cancer.

Identification of ChIP-seq mapped targets of HP1ß due to bombesin/GRP receptor activation.

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 human colorectal cancer (CRC) including Caco-2 cells. We have previously shown that GRPR activation results in the up-regulation of HP1ß, an epigenetic modifier of gene transcription. The aim of this study was to identify the genes whose expression is altered by HP1ß subsequent to GRPR activation. We determined HP1ß binding positions throughout the genome using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). After exposure to GRP, we identified 9,625 genomic positions occupied by HP1ß. We performed gene microarray analysis on Caco-2 cells in the absence and presence of a GRPR specific antagonist as well as siRNA to HP1ß. The expression of 97 genes was altered subsequent to GRPR antagonism, while the expression of 473 genes was altered by HP1ß siRNA exposure. When these data were evaluated in concert with our ChIP-seq findings, 9 genes showed evidence of possible altered expression as a function of GRPR signaling via HP1ß. Of these, genomic PCR of immunoprecipitated chromatin demonstrated that GRPR signaling affected the expression of IL1RAPL2, FAM13A, GBE1, PLK3, and SLCO1B3. These findings provide the first evidence by which GRPR aberrantly expressed in CRC might affect tumor progression.

Expression of GRP and its receptor is associated with improved survival in patients with colon 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.