Grhl3 Induces Human Epithelial Tumor Cell Migration And Invasion Via Downregulation Of E-cadherin

Background and ObjectiveGrainyhead family factors have been shown to play important roles in wound healing, epidermal formation, and the mechanistically related process of embryonic neural tube closure. Among these proteins, Grainyhead-like 3 (Grhl3, also known as Getl), a transcription factor expressed in the differentiated suprabasal layers, plays important roles in epidermal differentiation and barrier formation by controlling multiple genes that regulate the differentiation program in skin. These proteins also determine some structural proteins, enzymes, and intercellular adhesion molecules. Grhl3 was recently shown to suppress squamous cell carcinoma due to its activation of phosphatase and tensin homolog (PTEN) expression, and previous studies have shown that Grhl3 plays important roles in epithelial migration during early embryogenesis. Mammalian eyelid development and fusion follows a stereotypical stepwise pathway:first, the primitive eyelid root forms, then, a leading edge of keratinocytes extends centripetally from the rim of the eyelid and ultimately covers the eye, depending on the migration of the keratinocyte sheet. The eye-open phenotype appears in the Grhl3-/-mice because of a defect in the migration of the epithelial sheet. This process is also similar to the upregulation of Grhl3 in wound-front keratinocytes in adult skin. Grhl3 regulates fate specification of surface ectoderm in the neural fold. Grhl3-/-mice often have neural tube defects,such as thoraco-lumbo-sacral spina bifida and fully penetrant exencephaly. Grhl3 promotes epidermal migration during wound healing, upregulated in wound-front keratinocytes in adult skin.It has been proposed that the movement and migration of the epithelial cells can be greatly limited by some cellular contacts. E-cadherin, an essential molecule for the formation of adhesion junction, is critical to form the intercellular contacts and frequently downregulated in the later stage of tumorigenesis. Some previous studies indicated that Grainyhead family factors are the activators of E-cadherin in the physiological context. Since Grhl3 also represses the expression of some target genes, we hypothesized that Grhl3 may regulate cancer cell migration and invasion by the downregulation of E-cadherin.MethodsTo investigate the relationship between Grhl3 and E-cadherin, the expression of Grhl3 and E-cadherin was analyzed by western blotting in highly metastatic mammary carcinoma cell line MDA-MB-231, low invasive human skin squamous carcinoma cell line A431 and human breast cancer cell line MCF7, and human immortalized keratinocyte cell line Hacat. To investigate the ability of Grhl3 in regulating E-cadherin expression in cancer cells, less invasive cell lines that express higher level of E-cadherin, including A431 and MCF7, were used. An expression vector containing Flag tag fused to the N-terminal of human Grhl3 cDNA was generated and overexpressed in both MCF7 and A431 cells. The expression of Grhl3 and E-cadherin was detected by immunoblot analysis and immunofluorescence analysis. To determine whether knockdown of Grhl3 affects the expression of E-cadherin in the invasive MDA-MB-231 cells, recombinant GV287 construct with Grhl3 short hairpin RNA cassette (Grhl3-shRNA 229/65,66,67, or 68) or a scrambled control short hairpin RNA cassette (ConshRNA) was transfected into 293 T cells to identify the interference sequences to Grhl3. The recombinant vector which have interference sequences to Grhl3 was transfected into MDA-MB-231 cells. Stable transfectants were generated after selection with with 1 μg/ml puromycin. pCMV-2B-Grhl3 was transiently transfected into the stable transfectants to rescue Grhl3. Immunoblotting analysis was performed to detect Grhl3 and E-cadherin expression in MDA-MB-231 cells following stable transfection with Grhl3-shRNA and rescue with pCMV-2B-Grhl3. Wound-healing assays were performed to qualitatively determine the effect of Grhl3 on cell motility. To investigate the invasive ability of Grhl3-transfected cells, an in vitro transwell invasion assay was performed. To further detect whether knockdown of Grhl3 inhibits cell migration or cell invasion, the Boyden chamber motility assay and transwell invasion assay were performed with MDA-MB-231 cells. To determine the mechanism of E-cadherin repression by Grhl3, and in particular, whether it interacts directly with a specific region of the E-cadherin promoter, the ability of this transcription factor to interfere with the function of the-178 to+92 fragment of the human E-cadherin promoter was analyzed by Dual Luciferase Reporter Assay System. To determine whether the E-cadherin promoter is a direct target for repression by Grhl3, ChIP analysis was conducted. Chromatin from A431-Grhl3 was immunoprecipitated with an anti-FLAG antibody or nonimmune-IgG antibody and then analyzed by qPCR using the indicated ChIP primers.ResultsThe results of Western blotting showed that the higher expression of E-cadherin protein in most of these cell lines is associated with relatively lower level of Grhl3. And the Grhl3 is dominantly expressed in the E-cadherin-negative epithelial tumor cell line, MDA-MB-231, a highly metastatic mammary carcinoma cell line. Grhl3 overexpression in A431-Grhl3 and MCF7-Grhl3 cells was observed by western blot analysis, with the expression of E-cadherin undetectable in A431-Grhl3 cells: Additionally, E-cadherin protein was totally or substantially reduced in MCF7-Grhl3 cells. Immunofluorescence analysis revealed that E-cadherin staining was restricted to the MCF7-vector cells and completely disappeared in the Grhl3-transfected MCF7 cells. In contrast, control cells exhibited a high level of E-cadherin. These effects were also observed in the A431 cell line. And Grhl3 knockdown significantly increased the expression of E-cadherin in MDA-MB-231 cells.Grhl3 expression could be rescued and E-cadherin expression could also be inversely decreased after transiently transfected pCMV-2B-Grhl3 into the stable transfectant. The results of wound-healing assay showed that Grhl3-transfected A431 cells or MCF7 cells showed a significant (P < 0.01)increase in motility compared with the controls. The results of invasion assays exhibited the Grhl3-transfected cells, including A431 cells and MCF7 cells, showed a significant (P< 0.05) increase in the number of invading cells compared with the controls. The number of migrating cells (P< 0.01) or invading cells (P< 0.001) was significantly decreased when cells were transfected with Grhl3-shRNA compared with cells transfected with Con-shRNA. To further explore the mechanism by which Grhl3 regulated E-cadherin expression, an E-cadherin promoter report analysis was performed and results showed that Luc activity was efficiently repressed when cotransfected with the Grhl3 expression vector, but not with the empty control vector (P< 0.001). To further define the elements inside the human E-cadherin promoter that are involved in this repression, the-178 to+92 fragmentwas analyzed for the presence of putative Grhl3-binding sites. There were three consensus sequence repeats (CACCTG) previously described as E-boxes. The abilities of Grhl3 to repress different reporter constructs carrying combinations of mutated putative Grhl3-binding sites were compared (P< 0.001). The results from these experiments prompt us to conclude that the three boxes cooperate in the Grhl3-mediated E-cadherin repression and that the furthest downstream sequence (E-box 3) has the strongest repressive activity. The data of ChIP analysis showed a strong enrichment of the PCR signal using the anti-Flag antibody compared with nonimmune IgG or primers set that represented an unrelated region of the genome, such as promoter of gapdh (at positions-1700 to-1589) and promoter of afp (at positions -395 to-228).ConclusionThere is an inverse relationship between Grainyhead-like 3 (Grhl3) and E-cadherin expression in some epithelial tumor cell lines. Grhl3 generats a transcriptional blockage of the E-cadherin gene and induces human epithelial tumor cell migration and cell invasion. The molecular mechanism is that Grhl3 repressed E-cadherin gene expression by directly or indirectly binding to the E-boxes present in the proximal E-cadherin promoter.