Krüppel-like factor 8 (KLF8) is a transcriptional factor critical for metastatic progression of breast cancer. human breast cancer cells and patient tumors. Ectopic overexpression of KLF8 in the non-invasive MCF-10A cells induced the EPSTI1 expression whereas KLF8 knockdown from the invasive MDA-MB-231 cells decreased the EPSTI1 expression. Promoter activation and binding analyses LY 2183240 indicated that KLF8 promoted the EPSTI1 expression by directly acting on the EPSTI1 gene promoter. EPSTI1 knockdown dramatically reduced the KLF8-promoted MCF-10A cell invasion and ectopic expression of EPSTI1 in the non-invasive MCF-7 cells is sufficient to induce the cell LY 2183240 invasion. Experiments using nude mice demonstrated that the ectopic EPSTI1 granted the MCF-7 cells capability of both invasive growth in the breasts and metastasis to the lungs. Using co-immunoprecipitation coupled with mass spectrometry we discovered that EPSTI1 interacts with the valosin containing protein (VCP) resulting in the degradation of IκBα and subsequent activation of NF-κB in the nucleus. These findings suggest a novel KLF8 to EPSTI1 to VCP to NF-κB signaling mechanism potentially critical for breast cancer invasion and LY 2183240 metastasis. (19 20 molecular mechanisms that are responsible for the aberrant high expression of EPSTI1 in the breast cancer cells and whether and how EPSTI1 plays a role for breast cancer progression have not been reported to date. In this study we provide strong evidence showing that EPSTI1 is a novel target of transcriptional activation by KLF8 and plays a critical role in the progression of invasive growth and metastasis of breast cancer through VCP-dependent activation of NF-κB. Results KLF8 and EPSTI1 are highly co-overexpressed in human metastatic breast tumor cell lines and patient tumors LY 2183240 Our cDNA microarray analysis has shown that EPSTI1 is one of the highly upregulated genes in the 10A-iK8 cells (8) when KLF8 manifestation is induced as compared to the uninduced cells (submitted elsewhere). In addition we have also recently demonstrated a co-overexpression of KLF8 and EPSTI1 in the MDA-MB-231 cells (21). Since both KLF8 and EPSITI1 have been shown to be aberrantly LY 2183240 high in breast tumor cells or cells (8 9 14 19 20 we asked if EPSTI1 is definitely upregulated in breast tumor cell lines and tumor specimens known to communicate high levels of KLF8. We 1st examined the manifestation of EPSTI1 inside a panel of human being breast tumor cell lines in which the KLF8 manifestation was determined in our earlier work (8 9 14 The results clearly indicated that there is a high manifestation of EPSTI1 at both the mRNA and protein levels in the invasive cell lines including the MDA-MB-231 Hs578T and BT549 cells as compared to the barely detectable manifestation in the non-invasive cancer cell collection MCF-7 and the immortalized non-tumorigenic human being mammary epithelial cell collection MCF-10A (Number 1A). Number 1 Correlated upregulation of KLF8 and EPSTI1 manifestation in human being metastatic breast tumor cell lines and patient tumors. (a) Aberrant high manifestation of EPSTI1 in invasive breast cancer cells is definitely well correlated with KLF8 manifestation. Total RNA and protein … We have recently performed cells array analysis of KLF8 protein manifestation in individual tumors and correlated it with the invasive potential of the tumors (8). To test if manifestation of EPSTI1 protein is also correlated with that of KLF8 protein and/or the tumor invasive potential in the same set of cells array we carried out related IHC staining for EPSTI protein. The result showed that 75% of the KLF8 positive tumors communicate EPSTI1 (Number 1B and 1C). Among the KLF8-bad samples 59 communicate EPSTI1 suggesting that in the absence of KLF8 additional factors Mouse monoclonal to SCGB2A2 could play a role for the upregulation of EPSTI1 (Number 1C). EPSTI1 expresses in 77% of the invasive tumors but only 20% of the non-invasive tumors (Number 1D).. These results strongly suggested that EPSTI1 manifestation is definitely positively controlled by KLF8 in the invasive breast tumor cells. EPSTI1 is a direct target of transcriptional activation by KLF8 We further validated the microarray results by qRT-PCR and western blotting and verified the high manifestation of EPSTI1 in the 10A-iK8 cells only when the KLF8 manifestation was induced (Number 2A compare lanes 2 with 1). Conversely when.