(A) KLF5 expression in normal thyroid and papillary thyroid carcinoma (PTC) tissues

(A) KLF5 expression in normal thyroid and papillary thyroid carcinoma (PTC) tissues. subtype of PTC. Abstract The Krppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1 overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1 in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1 in PTC cells. We also identified KLF5-binding sites in the HIF-1 promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC. 0.001; Figure 1A). The detailed association between KLF5 expression and the clinico-pathological parameters of patients with PTC is presented in Table 1. KLF5 overexpression was noted in 65.1% Mouse monoclonal to Ki67 (793/1219) of PTC cases (Figure 1B), which was significantly associated with tall cell variant ( 0.0001), extra-thyroidal extension (= 0.0003), lymph node metastasis ( 0.0001) and stage IVA tumors (= 0.0003). The protein levels of KLF5 were also significantly correlated with HIF-1 expression (= 0.0492). A significant association was also noted between KLF5 overexpression and mutation ( 0.001). Interestingly, overexpression of KLF5 was significantly Bax channel blocker associated with disease-free survival in univariate analysis (= 0.0066; Table 1, Figure 1C), although significance was not retained upon multivariate analysis, after Bax channel blocker adjusting for confounding factors such as age, Bax channel blocker gender, histology, extra-thyroidal extension, and stage of tumor. Open in a separate window Figure 1 Bax channel blocker Immunohistochemical analysis of KLF5 and HIF-1 expression in Papillary Thyroid Cancer (PTC) TMA. (A) KLF5 expression in normal thyroid and papillary thyroid carcinoma (PTC) tissues. A significant difference in expression levels was noted between normal thyroid (= 225) and PTC tissues (= 1219) ( 0.001). (B) PTC array spot showing overexpression of KLF5 (a) and HIF-1 (c). In contrast, another PTC tissue array spot showing low expression of KLF5 (b) and HIF-1 (d). 20X/0.70 objective on an Olympus BX 51 microscope. (Olympus America Inc, Center Valley, PA, USA) with the inset showing a 40X 0.85 aperture magnified view of the same TMA spot. (C) KaplanCMeier survival analysis for the prognostic significance of KLF5 expression in PTC. PTC patients with overexpression of KLF5 had reduced disease-free survival at 5 years on univariate analysis compared to tumors showing low expression of KLF5 (= 0.0066). Table 1 Clinico-pathological associations of KLF5 protein expression in PTC. promoter. For the ChIP assay, the KLF5-binding regions on promoter were identified. PTC cells were treated with and without ML264 (5 and 10 m) for 6 h, fixed with formaldehyde, and cross-linked, and then chromatin was isolated. The chromatin was immunoprecipitated (IP) with an anti-KLF5 antibody or control mouse IgG. The KLF5 binding to the promoters was analyzed by regular PCR (E) or quantitative real-time PCR (F) with a primer specific to the KLF5-binding regions in promoter. The data represent the percent input and are normalized to each control. GAPDH was used as a loading control. (G) Silencing of KLF5 inhibits HIF-1. PTC cells were transfected with scrambled siRNA and siRNA (50 and 100 nM). After 48 h, Bax channel blocker cells were lysed and proteins were immunoblotted with antibodies against KLF5, HIF-1 and GAPDH. (H) ML264 treatment down-regulates the expression of KLF5 and HIF-1 in PTC cells. PTC cells were treated with indicated doses of ML264 for 48 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to.