Growing evidence has shown that ZIC1 is involved in the progression of several tumours [3–8]. It appears that ZIC1 is aberrantly expressed in certain types of cancer and differentially functions as a tumour suppressor or oncogenic gene. For instance, ZIC1 expression was reported to be low or absent in gastrointestinal and lung cancer cell lines, and was found to suppress gastrointestinal cancer cell proliferation [7, 8, 29]. In contrast, overexpression of ZIC1 in liposarcoma was found to promote cell proliferation and invasion . We and others have demonstrated that the epigenetic modulations including DNA methylation and histone remodeling, and genetic mutations may contribute to its differential expression patterns in cancers [3, 4, 7, 8]. It is becoming clear that as a zinc finger transcription factor, ZIC1 may modulate multiple downstream genes in neural tissue, colorectal cancer and liposarcoma cells [2, 3, 7, 9]. However, little is known about the mechanism underlying ZIC1 function in the development and progression of gastric cancer. Underscoring the key pathways and downstream targets regulated by ZIC1 may facilitate our understanding of its roles in tumorigenesis. Here, we have demonstrated that overexpression of ZIC1 results in significant inhibition of cell survival and impairment of cell migration. ZIC1 suppresses the Shh , PI3K and MAPK signaling pathways which are critical for the regulation of cell-cycle distributions and cell migration in gastric cancer. Moreover, ZIC1 inhibits cell cycle regulatory kinases, p21, p27 and cyclin D1, thus leading to G1/S cell cycle transit in gastric cancer cells. ZIC1 suppresses the Shh signaling pathway which is critical for the regulation of cell-cycle distributions and cell migration in gastric cancer.
MAPK and PI3K pathways play critical roles in cell proliferation, differentiation, and progression in a variety of human cancers . Recently, it was also reported that the activation of both pathways are essential to induce cell cycle entry [30, 31]. During this process, cyclin dependent kinase inhibitors p21, p27 and cyclin D/E participate in the regulation of p53 induced cell-cycle arrest [24, 32]. The activation of MAPK and its downstream kinase-Erk could not only lead to the induction of cyclin D1 and pass through G1/S checkpoint, but also the accumulation of p21 that inhibits cyclin E/CDK2 complexes to block S-phase entry . PI3K/Akt pathway could inactivate the Gsk3-β and FOXO transcription factors, thus inhibit cyclin D1 while induce p27 and p21 in the regulation of cell-cycle entry . We have shown that re-expression of ZIC1 effectively inactivate the phosphorylated Akt and Erk1/2 in AGS, MKN28, BGC823 and SGC7901 gastric cancer cell lines. In this regard, the CDK1 inhibitor p21 was activated, while cyclin D1 inactivated, after overexpression of ZIC1 in gastric cancer cells. Though the results need to be validated in future studies, our miroarray data have revealed that other key components of cell cycle kinase regulators including TP53INPI and CDKN2B, are deregulated with forced expression of ZIC1 in an individual MKN28 gastric cancer cell line (Figure 5 A and B). Therefore, we propose that ZIC1 regulates G1/S transit mainly through PI3K and MAPK pathways and downstream cell-cycle regulator kinases in gastric cancer cells.
Another major finding in our present study is that ZIC1 transcriptionally regulates Sonic hedgehog (Shh) signaling in gastric cancer cells. Aside from its central role on regulating gastric gland morphogenesis in human stomach, Shh signaling is also involved in the pathogenesis of gastric cancer [16, 33]. Shh is frequently activated in advanced gastric adenocarcinomas and associated with aggressive tumour behavior [13, 15, 33]. Previous studies have shown that Shh signaling promotes the motility and invasiveness of gastric cancer cells through TGF-β-ALK5-Smad3 pathway . Shh signaling could also regulate the expression of p21 and cyclin D1 in a Gli-dependent pathway [16, 25]. We have observed that inhibition of Shh signaling by administration with cyclopamine suppresses AGS, BGC823 and SGC7901 gastric cancer cell migration, and regulates the expression of p21 and cyclin D1 (Figure 4). These results are consistent with previously reported studies [25, 34]. Thus, we have revealed that ZIC1 plays important roles in gastric cancer progression by regulation of the Shh signaling pathway.
ZIC1 may regulate target genes in both sequence-specific and independent manners . ZIC1 could regulate the transcriptional expression of targets including cyclin D1, p27, Wnt1 and Wnt7a, and modulate Notch and BMP pathways in neural development [2, 9]. ZIC1 could counteract GLI by binding to GC-rich sequences, and suppress the expression of GLI-binding sequence directed reporter genes [9, 35]. We identified several ZIC potential target genes in gastric cancer cells by microarray analysis. These targets are closely related to cell cycle, cell proliferation and migration (Figure 5B). The association between ZIC and downstream targets might be a clue for understanding the potential perspective of ZIC proteins in the progression of gastric cancer.