Alison M. Urvalek

KLF8 promotes human breast cancer cell invasion and metastasis by transcriptional activation of MMP9

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- matrix metalloproteinase (MMP)9 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.

Sumoylation delimits KLF8 transcriptional activity associated with the cell cycle regulation

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 PIASxα 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 G1 cell cycle progression. Taken together, our results demonstrated that sumoylation of KLF8 negatively regulates its transcriptional activity and cellular functions.

Activation of KLF8 transcription by focal adhesion kinase in human ovarian epithelial and cancer cells

KLF8 (Krüppel-like factor 8) is a transcription factor downstream of focal adhesion kinase (FAK) important in the regulation of the cell cycle and also plays a critical role in oncogenic transformation and epithelial to mesenchymal transition. Here we report the mechanisms by which FAK regulates KLF8 expression in human ovarian epithelial and cancer cells. We show that the overexpression of both KLF8 and FAK in the human ovarian cancer cells as compared with the normal human ovarian surface epithelial cells is critical for cell growth. Using promoter luciferase reporter assays, we demonstrate that exogenous FAK strongly promotes the activity of the KLF8 promoter, and knockdown of FAK inhibits it. KLF8 promoter activity and mRNA levels are induced by expression of constitutively active (CA) phosphatidylinositol 3-kinase (PI3K) or CA-Akt but are repressed by dominant negative Akt or the PI3K inhibitor LY294002. Disruption of an Sp1 binding site in the KLF8 promoter abolishes the FAK- or Sp1-mediated promoter activation. Sp1 knockdown prevents the KLF8 promoter from being activated by Sp1 or CA-Akt, and expression of CA-Akt enhances Sp1 expression in SKOV3ip1 cells. Chromatin immunoprecipitation and oligonucleotide precipitation results show that Sp1 binds to the KLF8 promoter. Taken together, our data suggest that FAK induces KLF8 expression in human ovarian cancer cells by activating the PI3K-Akt signaling pathway, leading to the activation of KLF8 promoter by Sp1.

KLF8 recruits the p300 and PCAF co-activators to its amino terminal activation domain to activate transcription

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.

Identification of Poly (ADP-ribose) Polymerase-1 (PARP-1) as a Novel Krüppel-like Factor 8-interacting and -regulating Protein

Krüppel-like factor 8 (KLF8) regulates critical gene transcription and cellular events associated with cancer. However, KLF8-interacting proteins remain largely unidentified. Using co-immunoprecipitation (co-IP), mass spectrometry, and GST pulldown assays, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel KLF8-interacting protein. Co-IP and Western blotting indicated that KLF8 is also a PARP-1 substrate. Mutation of the cysteines in the zinc finger domain of KLF8 abolished PARP-1 interaction. Surprisingly, immunofluorescent staining revealed a cytoplasmic mislocalization of KLF8 in PARP-1−/− cells or when the interaction was disrupted. This mislocalization was prevented by either PARP-1 re-expression or inhibition of CRM1-dependent nuclear export. Interestingly, co-IP indicated competition between PARP-1 and CRM1 for KLF8 binding. Cycloheximide chase assay showed a decrease in the half-life of KLF8 protein when PARP-1 expression was suppressed or KLF8-PARP-1 interaction was disrupted. Ubiquitination assays implicated KLF8 as a target of ubiquitination that was significantly higher in PARP-1−/− cells. Promoter reporter assays and chromatin immunoprecipitation assays showed that KLF8 activation on the cyclin D1 promoter was markedly reduced when PARP-1 was deleted or inhibited or when KLF8-PARP-1 interaction was disrupted. Overall, this work has identified PARP-1 as a novel KLF8-binding and -regulating protein and provided new insights into the mechanisms underlying the regulation of KLF8 nuclear localization, stability, and functions.

KLF8 and FAK cooperatively enrich the active MMP14 on the cell surface required for the metastatic progression of breast cancer

Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with cancer. The underlying mechanisms, however, remain largely unidentified. We have recently demonstrated that KLF8 expression enhances the activity but not expression of matrix metalloproteinase-2 (MMP2), the target substrate of MMP14. Here, we report a novel KLF8 to MMP14 signaling that promotes human breast cancer invasion and metastasis. Using cell lines for inducible expression and knockdown of KLF8, we demonstrate that KLF8 promotes MMP14 expression at the transcriptional level. Knocking down KLF8 expression inhibited the breast cancer cell invasion both in vitro and in vivo as well as the lung metastasis in mice, which could be rescued by ectopic expression of MMP14. Promoter reporter assays and oligonucleotide and chromatin immunoprecipitations determined that KLF8 activates the human MMP14 gene promoter by both directly acting on the promoter and indirectly via promoting the nuclear translocation of β-catenin, the expression of T-cell factor-1 (TCF1) and subsequent activation of the promoter by the β-catenin/TCF1 complex. Inhibition of focal adhesion kinase (FAK) using pharmacological inhibitor, RNA interference or knockout showed that the cell surface presentation of active MMP14 downstream of KLF8 depends on FAK expression and activity. Taken together, this work identified novel signaling mechanisms by which KLF8 and FAK work together to promote the extracellular activity of MMP14 critical for breast cancer metastasis.

Transformation of human ovarian surface epithelial cells by Krüppel-like factor 8

We have previously demonstrated that Krüppel-like factor 8 (KLF8) participates in oncogenic transformation of mouse fibroblasts and is highly overexpressed in human ovarian cancer. In this work, we first correlated KLF8 overexpression with the aggressiveness of ovarian patient tumors and then tested if KLF8 could transform human ovarian epithelial cells. Using the immortalized non-tumorigenic human ovarian surface epithelial cell line T80 and retroviral infection, we generated cell lines that constitutively overexpress KLF8 alone or its combination with the known ovarian oncogenes c-Myc, Stat3c and/or Akt and examined the cell lines for anchorage-independent growth and tumorigenesis. The soft agar clonogenic assay showed that T80/KLF8 cells formed significantly more colonies than the mock cells. Interestingly, the cells expressing both KLF8 and c-Myc formed the largest amounts of colonies, greater than the sum of colonies formed by the cells expressing KLF8 and c-Myc alone. These results suggested that KLF8 might be a weak oncogene that works cooperatively with c-Myc to transform ovarian cells. Surprisingly, overexpression of KLF8 alone was sufficient to induce tumorigenesis in nude mice resulting in short lifespan irrespective of whether the T80/KLF8 cells were injected subcutaneously, intraperitoneally or orthotopically into the ovarian bursa. Histopathological studies confirmed that the T80/KLF8 tumors were characteristic of human serous ovarian carcinomas. Comparative expression profiling and functional studies identified the cell cycle regulators cyclin D1 and USP44 as primary KLF8 targets and effectors for the T80 transformation. Overall, we identified KLF8 overexpression as an important factor in human ovarian carcinoma pathogenesis.

Abstract 4985: Identification of PARP-1 as a novel KLF8 interacting and regulating protein

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC KLF8 transcription factor regulates important genes including cyclin D1 and E-cadherin and plays a critical role in cell cycle progression, oncogenic transformation and EMT. Transcription factors need to interact with both DNA and other proteins in order to regulate transcription. However, little is known about KLF8-protein interaction. Using mass spectrometry and co-immunoprecipitation (co-IP), we identified PARP-1, a chromatin-associated enzyme that catalyzes protein poly(ADP-ribosyl)ation (PARylation), as one of novel proteins that interact with KLF8. Co-IP and western blotting indicated that KLF8 is a PARylation substrate. Mutation of the cystein residues in the first and second zinc-finger motifs of KLF8 abolishes its interation with PARP1. Luciferase assays showed KLF8 activation on the cyclin D1 promoter was significantly reduced when PARP1 was physically deleted or functionally inhibited or when KLF8-PARP-1 interation was disrupted. Surprisingly, immunofluorescent staining demonstrated that KLF8 was localized in the nuclei of the wild-type mouse embryonic fibroblasts (MEF), but was mislocalized in the perinuclear cytoplasm of the PARP-1−/− MEFs, which could be rescued by re-expression of PARP1. Interestingly, while the PARP1-interation deficient KLF8 mutant was also mislocalized in the cytoplasm of the wild-type MEFs, inhibition of PARP1 activity in the MEFs did not affect the nuclear localization of wild-type KLF8. Cychloheximade (CHX)-chase assay revealed the stability of KLF8 dramatically reduced by loss of PARP-1 or when KLF8-PARP-1 interaction was disrupted. Ubiquitination assay showed heavier ubiquitin chains in PARP-1−/− MEFs or when KLF8-PARP-1 interaction was disrupted. Furthermore, DNA damage assay under Doxorubin treatment indicated KLF8 involved in PARP-1-dependent DNA damage repair pathway. Those results suggest that PARP-1 work as a co-activator in KLF8s transcriptional activity on Cyclin D1 promoter, keep KLF8s nuclear localization, inhibit KLF8s ubiquitination and contribute to KLF8s stability. This work has identified PARP1 as a novel binding partner and post-translational modifier of KLF8 and a critical regulator of KLF8 localization, function and stability in the cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4985.

Abstract P4-06-23: KLF8 Is Regulated by Its Novel Interacting Protein PARP-1

Kruppel-like factor 8 (KLF8) plays an important role in the progression of breast cancer invasion and metastasis. Using mass spectrometry and co-immunoprecipitation, we identified PARP-1, a chromatin-associated enzyme that catalyzes protein poly(ADP-ribosyl)ation (PARylation), as a novel KLF8 interacting protein. Co-IP and western blotting indicated that KLF8 is a PARylation substrate. Mutation of the cystein residues in the first and second zinc-finger motifs of KLF8 abolishes its interaction with PARP-1. Luciferase assays showed KLF8 activation on the cyclin D1 promoter was significantly reduced when PARP-1 was physically deleted or functionally inhibited or when KLF8-PARP-1 interaction was disrupted. Surprisingly, immunofluorescent staining demonstrated that KLF8 was localized in the nuclei of wide type MEFs, but was mislocalized in the perinuclear cytoplasm of the PARP-1 -/- cells, which could be rescued by re-expression of PARP-1. The detection of γH2A.X, comet assay and clonogenic assay indicated KLF8 play a DNA repair role in a PARP-1- dependent matter. Cychloheximade-chase assay revealed the stability of KLF8 dramatically reduced by loss of PARP-1 or when KLF8-PARP-1 interaction was disrupted, meanwhile ubiquitination assay showed heavier ubiquitin chains of KLF8. This work has identified PARP-1 as a novel binding partner and post-translational modifier of KLF8 and a critical regulator of KLF8 localization, functions and stability in the cells and suggested a critical role of the interaction between KLF8 and PARP1 during metastatic progression of breast cancer. Support by: NIH, ACS and Komen grants to J.Z. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-23.

Abstract P4-06-24: KLF8 Induction of Mammary Epithelial Stem Cell Formation and Tumorigenesis by Regulating EMT-Associated MicroRNAs

Although it is well established that the epithelial-mesenchymal transition (EMT) generates cells with properties of stem cells, the molecularmechanisms responsible for this change remain largely undefined. We set out to identify the mechanisms by examining the expression of stem cell markers as well as microRNAs in MCF-10A cells during EMT induction. By immunostaining and FACS analysis, we found that during EMT induction by either TGF-β treatment or overexpression of KLF8, both the expression of CD44high/CD24low and the activity of aldehyde dehydrogenase (ALDH) were dramatically increased. Using microRNA PCR array, we identified a microRNA signature associated with the EMT. Using microRNA inhibitor, We further demonstrated that one of the microRNAs, miR-146a, is upregulated and plays an important role in keeping the stem cell percentage during EMT induced by both TGF-β and KLF8, as revealed by manipulation of miR-146a expression, analysis of the stem cell markers and mammosphere growth. Taken together, these results highlight a novel role for miR-146a in regulating the stem-like cell induction from MCF-10A cells by EMT. Experiments are in progress to investigate the contribution of KLF8-regulated expression of miR146a and its targets to the stem cell induction and transformation. Supported by: NIH, ACS, Komen and NYSTEM grants to J.Z. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-24.