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.

KLF8 (Krüppel-like factor 8) is a member of the Krüppel transcription factor family that binds CACCC elements in DNA and activates or represses their target genes in a context-dependent manner. Here we present sumoylation as a novel mechanism that regulates KLF8 post-translationally. We found that KLF8 can be covalently modified by small ubiqitin-like modifier (SUMO)-1, SUMO-2, and SUMO-3 in vivo. We showed that KLF8 interacted with the PIAS family of SUMO E3 ligases PIAS1, PIASy, and PIASx␣ but not with E2 SUMO-conjugating enzyme Ubc9. Furthermore, we demonstrated that the E2 and E3 ligases enhanced the sumoylation of KLF8. In addition, site-directed mutagenesis identified lysine 67 as the major sumoylation site on KLF8. Lysine 67 to arginine mutation strongly enhanced activity of KLF8 as a repressor or activator to its physiological target promoters and as an inducer of the G 1 cell cycle progression. Taken together, our results demonstrated that sumoylation of KLF8 negatively regulates its transcriptional activity and cellular functions.Post-translational modifications of proteins, such as phosphorylation, acetylation, methylation, ubiquitination, and sumoylation, play crucial roles in many cellular processes due to their ability to cause rapid changes in the conformation and functions of preexisting proteins. Small ubiqitin-like modifier (SUMO) 2 -1, -2, and -3 are ubiquitin-like proteins that can be covalently attached to a large number of proteins through the formation of isopeptide bonds between the C terminus of mature SUMOs and the -amino group of a lysine in the acceptor proteins (1, 2). The conjugated forms of SUMO-2 and SUMO-3 differ from one another only by three N-terminal residues. They form a distinct subfamily known as SUMO-2/3 and are 50% identical in sequence to SUMO-1 (2, 3). Sumoylation of proteins proceeds via a multi-enzymatic pathway that shares similarity with the ubiquitin-conjugation system but uses a SUMO-specific enzymatic machinery (4).Like ubiquitin, SUMOs are expressed in inactive precursors that have to be processed by SUMO-specific proteases to expose a C-terminal double glycine motif that is required for SUMO conjugation. The processed form of SUMO is specifically activated in an ATP-dependent manner by an E1-activating enzyme consisting of an SAE1 (AOS1)-SAE2 (UBA2) heterodimer. Activated SUMO is transferred to Ubc9, the E2-conjugating enzyme, and is subsequently attached to the -amino group of a specific lysine in the target protein (4, 5). In Saccharomyces cerevisiae, sumoylation involves an E3 ligase (e.g. Siz1 and Siz2) for ligation of SUMO to its substrates. In contrast, E3 enzyme is not required for the addition of SUMO to its target proteins in mammals, although specific E3 ligases can promote the conjugation of SUMO from the E2 to its target protein (6 -8). To date, three types of SUMO E3 ligases, the nucleoporin RanBP2 (4, 9), the PIAS proteins (10), and the polycomb group protein Pc2 (11), have been described. The PIAS proteins were initially described as protein inh...

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Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer affecting humans worldwide. To determine the potential mechanisms by which chronic tobacco and alcohol abuse lead to HNSCC of the oral cavity, we have used both the 4-nitroquinoline-1-oxide (4-NQO) murine oral carcinogenesis and the Meadows-Cook alcohol models. In this study, we treated mice with 4-NQO in drinking water for 10 weeks and then administered 20% (w:v) ethanol (EtOH) for another 10 weeks. We observed increased levels and/or activation of signaling proteins [p38 mitogen-activated protein kinase (MAPK), β-catenin and Erk 1/2] that are typically altered during HNSCC initiation in humans. We found that EtOH administration alone increased the expression of p38 MAPK but not Erk 1/2 MAPK. Total β-catenin levels in the tongues increased by 2- to 3-fold after 4-NQO treatment, with or without EtOH. However, EtOH combined with 4-NQO reduced phosphorylated β-catenin levels, whereas 4-NQO treatment alone did not. These data implicate EtOH as a regulator of β-catenin signaling in this HNSCC model. We also utilized K14-CreER(TAM); ROSA26 mice to mark permanently stem/progenitor cells in the tongue epithelia. We found that 4-NQO alone and 4-NQO plus EtOH treatment resulted in massive, horizontal expansion of stem/progenitor cell populations arising from single stem cells in the basal layer of the epithelia. This expansion is consistent with carcinogen-associated, symmetric division of stem/progenitor cells. Our data suggest that specific therapeutic targets for prevention of HNSCC of the oral cavity associated with both alcohol and tobacco use are p38 MAPK and β-catenin.