Epithelial to mesenchymal transition (EMT) and extracellular matrix degradation are critical for the initiation and progression of tumor invasion. We have recently identified Krüppel-like factor 8 (KLF8) as a critical inducer of EMT and invasion. KLF8 induces EMT primarily by repressing E-cadherin transcription. However, how KLF8 promotes invasion is unknown. Here we report a novel KLF8-to-MMP9 signaling that promotes human breast cancer invasion. To identify the potential KLF8 regulation of MMPs in breast cancer, we established two inducible cell lines that allow either KLF8 overexpression in MCF-10A or knockdown in MDA-MB-231 cells. KLF8 overexpression induced a strong increase in MMP9 expression and activity as determined by quantitative real-time PCR and zymography. This induction was well correlated with the MMP inhibitor-sensitive Matrigel invasion. Conversely, KLF8 knockdown caused the opposite changes that could be partially prevented by MMP9 overexpression. Promoter-reporter assays and chromatin and oligonucleotide precipitations determined that KLF8 directly bound and activated the human MMP9 gene promoter. Three-dimensional (3D) glandular culture showed that KLF8 expression disrupted the normal acinus formation which could be prevented by the MMP inhibitor, whereas KLF8 knockdown corrected the abnormal 3D architecture which could be protected by MMP9 overexpression. KLF8 knockdown promoted MDA-MB-231 cell aggregation in suspension culture which could be prevented by MMP9 overexpression. KLF8 knockdown inhibited the lung metastasis of MDA-MB-231 cells in nude mice. Immunohistochemical staining strongly correlated the co-expression of KLF8 and MMP9 with the patient tumor invasion, metastasis and poor survival. Taken together, this work identified the KLF8 activation of MMP9 as a novel and critical signaling mechanism underlying human breast cancer invasion and metastasis.

Acute kidney injury (AKI), which involves the loss of kidney function caused by damage to renal tubular cells, is an important public health concern. We previously showed that sirtuin (SIRT)3 protects the kidneys against mitochondrial damage by inhibiting the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome, attenuating oxidative stress, and downregulating proinflammatory cytokines. In this article, we investigated the role of autophagy, mediated by a mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), in the protective effect of SIRT3, against sepsis-induced AKI, in a mouse model of cecal ligation and puncture (CLP). The AKI in CLP mice was associated with the upregulation of autophagy markers; this effect was abolished in SIRT3− mice in parallel with the downregulation of phospho (p)-AMPK and the upregulation of p-mTOR. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) or AMPK inhibitor compound isotonic saline (C), exacerbated AKI. SIRT3 overexpression promoted autophagy, upregulated p-AMPK and downregulated p-mTOR in CLP mice, attenuating sepsis-induced AKI, tubular cell apoptosis, and inflammatory cytokine accumulation in the kidneys. The blockage of autophagy induction largely abolished the protective effect of SIRT3 in sepsis-induced AKI. These findings indicate that SIRT3 protects against CLP-induced AKI by inducing autophagy through regulation of the AMPK/mTOR pathway.

Krüppel-like factor 8 (KLF8) regulates critical cellular processes including cell cycle progression, transformation, epithelial-to-mesenchymal transition, migration and invasion by either repressing or activating target gene promoters. As a repressor, KLF8 recruits the CtBP co-repressor via its PVDLS repression motif. However, how KLF8 acts as an activator has not been determined. Here we report the identification of both the KLF8 activation domain and associated co-activators. By site-directed mutagenesis and cyclin D1 promoter reporter assays using both mouse fibroblasts and human epithelial cells, we determined that deletion of residues 100–260 or mutation of Q118-Q248 abolished KLF8 transactivity. This transactivity was dramatically reduced in p300−/−, CBP−/− or PCAF−/− cells and could be restored by re-expressing p300 or PCAF, but not CBP. Co-immunoprecipitation analyses demonstrated that KLF8 interacted with these co-activators whereas the Q118N-Q248N mutant did not. Chromatin immunoprecipitation experiments showed that KLF8 promoted histone acetylation at the promoter whereas the Q118N-Q248N mutant had a dramatic loss of this function. Western blotting revealed that unlike wild-type KLF8 the Q118N-Q248N was no longer able to upregulate cyclin D1 protein level. BrdU incorporation assays showed that the Q118N-Q248N mutant also lost the ability to promote DNA synthesis. Taken together, these results identified the KLF8 activation domain located between residues 101–260 where the well-conserved Q118 and Q248 are essential for recruiting p300 and PCAF to activate target gene transcription.

The development of Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC) is highly linked to exposure to acidic bile salts due to chronic gastroesophageal reflux disease (GERD). In this study, we investigated the role of Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/REF-1) in STAT3 activation in response to acidic bile salts. Our results indicate that APE1 is constitutively overexpressed in EAC, whereas its expression is transiently induced in response to acidic bile salts in non-neoplastic BE. Using overexpression or shRNA knockdown of APE1, we found that APE1 is required for phosphorylation, nuclear localization, and transcriptional activation of STAT3. By using an APE1 redox-specific mutant (C65A) and APE1 redox inhibitor (E3330), we demonstrate that APE1 activates STAT3 in a redox-dependent manner. By using pharmacologic inhibitors and genetic knockdown systems, we found that EGFR is a required link between APE1 and STAT3. EGFR phosphorylation (Y1068) was directly associated with APE1 levels and redox function. Co-immunoprecipitation and proximity ligation assays indicated that APE1 coexists and interacts with the EGFR-STAT3 protein complex. Consistent with these findings, we demonstrated a significant induction in mRNA expression levels of STAT3 target genes (IL-6, IL-17A, BCL-xL, Survivin, and c-MYC) in BE and EAC cells, following acidic bile salts treatment. ChIP assays indicated that acidic bile salts treatment enhances binding of STAT3 to the promoter of its target genes, Survivin and BCL-xL. Inhibition of APE1/REF-1 redox activity using E3330 abrogated STAT3 DNA binding and transcriptional activity. The induction of APE1-STAT3 axis in acidic bile salts conditions provided a survival advantage and promoted cellular proliferation. In summary, our study provides multiple pieces of evidence supporting a critical role for APE1 induction in activating the EGFR-STAT3 signaling axis in response to acidic bile salts, the main risk factor for Barrett's carcinogenesis.