Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

Infection-induced signals generated at the plasma membrane epigenetically regulate Wnt signaling in vitro and in vivo.

Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in ß-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20-34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and ß-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased ß-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.

Dietary Interventions Ameliorate Infectious Colitis by Restoring the Microbiome and Promoting Stem Cell Proliferation in Mice.

Decreases in short-chain-fatty-acids (SCFAs) are linked to inflammatory bowel disease (IBD). Yet, the mechanisms through which SCFAs promote wound healing, orchestrated by intestinal stem cells, are poorly understood. We discovered that, in mice with Citrobacter rodentium (CR)-induced infectious colitis, treatment with Pectin and Tributyrin diets reduced the severity of colitis by restoring Firmicutes and Bacteroidetes and by increasing mucus production. RNA-seq in young adult mouse colon (YAMC) cells identified higher expression of Lgr4, Lgr6, DCLK1, Muc2, and SIGGIR after Butyrate treatment. Lineage tracing in CR-infected Lgr5-EGFP-IRES-CreERT2/ROSA26-LacZ (Lgr5-R) mice also revealed an expansion of LacZ-labeled Lgr5(+) stem cells in the colons of both Pectin and Tributyrin-treated mice compared to control. Interestingly, gut microbiota was required for Pectin but not Tributyrin-induced Lgr5(+) stem cell expansion. YAMC cells treated with sodium butyrate exhibited increased Lgr5 promoter reporter activity due to direct Butyrate binding with Lgr5 at -4.0 Kcal/mol, leading to thermal stabilization. Upon ChIP-seq, H3K4me3 increased near Lgr5 transcription start site that contained the consensus binding motif for a transcriptional activator of Lgr5 (SPIB). Thus, a multitude of effects on gut microbiome, differential gene expression, and/or expansion of Lgr5(+) stem cells seem to underlie amelioration of colitis following dietary intervention.

Depletion of tumor suppressor Kank1 induces centrosomal amplification via hyperactivation of RhoA.

Chromosome instability, frequently found in cancer cells, is caused by a deficiency in cell division, including centrosomal amplification and cytokinesis failure, and can result in abnormal chromosome content or aneuploidy. The small GTPase pathways have been implicated as important processes in cell division. We found that knockdown of a tumor suppressor protein Kank1 increases the number of cells with a micronucleus or bi-/multi-nuclei, which was likely caused by centrosomal amplification. Kank1 interacts with Daam1, known to bind to and activate a small GTPase, RhoA, in actin assembly. Knockdown of Kank1 or overexpression of Daam1, respectively, hyperactivates RhoA, potentially leading to the modulation of the activity of Aurora-A, a key regulator of centrosomal functions, eventually resulting in centrosomal amplification. Kank1 is also associated with contractile ring formation in collaboration with RhoA, and its deficiency results in the interruption of normal daughter cell separation, generating multinucleate cells. Such abnormal segregation of chromosomes may cause further chromosomal instability and abnormal gene functions, leading to tumorigenesis. Thus, Kank1 plays a crucial role in regulating the activity of RhoA through retrieving excess Daam1 and balancing the activities of RhoA and its effectors.

Tissue-Specific Effects of Reduced ß-catenin Expression on Adenomatous Polyposis Coli Mutation-Instigated Tumorigenesis in Mouse Colon and Ovarian Epithelium.

Adenomatous polyposis coli (APC) inactivating mutations are present in most human colorectal cancers and some other cancers. The APC protein regulates the ß-catenin protein pool that functions as a co-activator of T cell factor (TCF)-regulated transcription in Wnt pathway signaling. We studied effects of reduced dosage of the Ctnnb1 gene encoding ß-catenin in Apc-mutation-induced colon and ovarian mouse tumorigenesis and cell culture models. Concurrent somatic inactivation of one Ctnnb1 allele, dramatically inhibited Apc mutation-induced colon polyposis and greatly extended Apc-mutant mouse survival. Ctnnb1 hemizygous dose markedly inhibited increases in ß-catenin levels in the cytoplasm and nucleus following Apc inactivation in colon epithelium, with attenuated expression of key ß-catenin/TCF-regulated target genes, including those encoding the EphB2/B3 receptors, the stem cell marker Lgr5, and Myc, leading to maintenance of crypt compartmentalization and restriction of stem and proliferating cells to the crypt base. A critical threshold for ß-catenin levels in TCF-regulated transcription was uncovered for Apc mutation-induced effects in colon epithelium, along with evidence of a feed-forward role for ß-catenin in Ctnnb1 gene expression and CTNNB1 transcription. The active ß-catenin protein pool was highly sensitive to CTNNB1 transcript levels in colon cancer cells. In mouse ovarian endometrioid adenocarcinomas (OEAs) arising from Apc- and Pten-inactivation, while Ctnnb1 hemizygous dose affected ß-catenin levels and some ß-catenin/TCF target genes, Myc induction was retained and OEAs arose in a fashion akin to that seen with intact Ctnnb1 gene dose. Our findings indicate Ctnnb1 gene dose exerts tissue-specific differences in Apc mutation-instigated tumorigenesis. Differential expression of selected ß-catenin/TCF-regulated genes, such as Myc, likely underlies context-dependent effects of Ctnnb1 gene dosage in tumorigenesis.

An ornamental plant targets epigenetic signaling to block cancer stem cell-driven colon carcinogenesis.

Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea(AR; Hollyhock) is an ornamental plant with known anti-inflammatory properties. This study explored its role as an anticancer agent. The AR seed extract (AR extract) inhibited proliferation and colony formation in a dose- and time-dependent manner and promoted apoptosis as was evidenced by cleavage of PARP and increased expression of Bax accompanying reduced levels of BCL-xl protein in HCT116 and SW480 cells, respectively. In addition, AR extract-arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. AR extract-treated cells exhibited reduced number and size of colonospheres in a dose-dependent manner concomitant with decreases in cancer stem cell (CSC) markers ALDH1A1 and Dclk1. Relative levels of ß-catenin, Notch-ICD, Hes1 and EZH2 were also attenuated by AR extract. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover, WIF1 (a Wnt antagonist) promoter activity increased dramatically following treatment with AR extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown.In vivo, AR extract attenuated tumor growth due probably to reduced levels of EZH2, ß-catenin, CyclinD1 and Ki-67 along with reduced levels of CSC markers. Since partial purification via HPLC yielded a prominent peak, efforts are underway to identify the active ingredient(s). Taken together, the results clearly suggest that AR extract/active component(s) can be an effective preventative/therapeutic agent to target colon cancer.

RNA binding protein RBM3 increases ß-catenin signaling to increase stem cell characteristics in colorectal cancer cells.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. It arises from loss of intestinal epithelial homeostasis and hyperproliferation of the crypt epithelium. In order to further understand the pathogenesis of CRC it is important to further understand the factors regulating intestinal epithelial proliferation and more specifically, regulation of the intestinal epithelial stem cell compartment. Here, we investigated the role of the RNA binding protein RBM3 in stem cell homeostasis in colorectal cancers. Using a doxycycline (Dox) inducible RBM3 overexpressing cell lines HCT 116 and DLD-1, we measured changes in side population (SP) cells that have high xenobiotic efflux capacity and increased capacity for self-renewal. In both cell lines, RBM3 induction showed significant increases in the percentage of side population cells. Additionally, we observed increases in spheroid formation and in cells expressing DCLK1, LGR5 and CD44Hi . As the Wnt/ß-catenin signaling pathway is important for both physiologic and cancer stem cells, we next investigated the effects of RBM3 overexpression on ß-catenin activity. RBM3 overexpression increased levels of nuclear ß-catenin as well as TCF/LEF transcriptional activity. In addition, there was inactivation of GSK3ß leading to decreased ß-catenin phosphorylation. Pharmacologic inhibition of GSK3ß using (2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO) also recapitulates the RBM3 induced ß-catenin activity. In conclusion, we see that RNA binding protein RBM3 induces stemness in colorectal cancer cells through a mechanism involving suppression of GSK3ß activity thereby enhancing ß-catenin signaling. © 2015 Wiley Periodicals, Inc.

Evidence of functional cross talk between the Notch and NF-κB pathways in nonneoplastic hyperproliferating colonic epithelium.

The Notch and NF-κB signaling pathways regulate stem cell function and inflammation in the gut, respectively. We investigate whether a functional cross talk exists between the two pathways during transmissible murine colonic hyperplasia (TMCH) caused by Citrobacter rodentium (CR). During TMCH, NF-κB activity and subunit phosphorylation in colonic crypts of NIH Swiss mice at days 6 and 12 were associated with increases in downstream target CXC chemokine ligand (CXCL)-1/keratinocyte-derived chemokine (KC) expression. Blocking Notch signaling acutely for 5 days with the Notch blocker dibenzazepine (DBZ) failed to inhibit crypt NF-κB activity or CXCL-1/KC expression. Chronic DBZ administration for 10 days, however, blocked Notch and NF-κB signaling in the crypts and abrogated hyperplasia. Intriguingly, chronic Notch inhibition was associated with significant increases in IL-1α, granulocyte colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 2, and KC in the crypt-denuded lamina propria or whole distal colon, with concomitant increases in myeloperoxidase activity. In core-3(-/-) mice, which are defective in intestinal mucin, DBZ administration replicated the results of NIH Swiss mice; in Apc(Min/+) mice, which are associated with CR-induced elevation of NF-κB-p65(276) expression, DBZ reversed the increase in NF-κB-p65(276), which may have blocked rapid proliferation of the mutated crypts. DBZ further blocked reporter activities involving the NF-κB-luciferase reporter plasmid or the Toll-like receptor 4/NF-κB/SEAPorter HEK-293 reporter cell line, while ectopic expression of Notch-N(ICD) reversed the inhibitory effect. Dietary bael (Aegle marmelos) extract (4%) and curcumin (4%) restored Notch and NF-κB cross talk in NIH Swiss mice, inhibited CR/DBZ-induced apoptosis in the crypts, and promoted crypt regeneration. Thus functional cross talk between the Notch and NF-κB pathways during TMCH regulates hyperplasia and/or inflammation in response to CR infection.

SIGIRR Mutation in Human Necrotizing Enterocolitis (NEC) Disrupts STAT3-Dependent microRNA Expression in Neonatal Gut.

BACKGROUND & AIMS: Single immunoglobulin interleukin-1-related receptor (SIGIRR) is a major inhibitor of Toll-like receptor signaling. Our laboratory identified a novel SIGIRR stop mutation (p.Y168X) in an infant who died of severe necrotizing enterocolitis (NEC). Herein, we investigated the mechanisms by which SIGIRR mutations induce Toll-like receptor hyper-responsiveness in the neonatal gut, disrupting postnatal intestinal adaptation. METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was used to generate transgenic mice encoding the SIGIRR p.Y168X mutation. Ileal lysates, mouse intestinal epithelial cell (IEC) lysates, and intestinal sections were used to assess inflammation, signal transducer and activator of transcription 3 (STAT3) phosphorylation, microRNA (miRNA), and interleukin-1-related-associated kinase 1 (IRAK1) expression. Western blot, quantitative reverse-transcription polymerase chain reaction(qRT-PCR), and luciferase assays were performed to investigate SIGIRR-STAT3 signaling in human intestinal epithelial cells (HIEC) expressing wild-type or SIGIRR (p.Y168X) plasmids. RESULTS: SigirrTg mice showed increased intestinal inflammation and nuclear factor-κB activation concomitant with decreased IEC expression of miR-146a and miR-155. Mechanistic studies in HIECs showed that although SIGIRR induced STAT3-mediated expression of miR-146a and miR-155, the p.Y168X mutation disrupted SIGIRR-mediated STAT3-dependent miRNA expression. Chromatin immunoprecipitation and luciferase assays showed that SIGIRR activation of STAT3-induced miRNA expression is dependent on IRAK1. Both in HIECs and in the mouse intestine, decreased expression of miR-146a observed with the p.Y168X mutation increased expression of IRAK1, a protein whose down-regulation is important for postnatal gut adaptation. CONCLUSIONS: Our results uncover a novel pathway (SIGIRR-STAT3-miRNA-IRAK1 repression) by which SIGIRR regulates postnatal intestine adaptation, which is disrupted by a SIGIRR mutation identified in human NEC. These data provide new insights into how human genetic mutations in SIGIRR identified in NEC result in loss of postnatal intestinal immune tolerance.

DCLK1 isoforms and aberrant Notch signaling in the regulation of human and murine colitis.

Alternative promoter usage generates long and short isoforms (DCLK1-L and DCLK1-S) of doublecortin-like kinase-1 (DCLK1). Tight control of Notch signaling is important to prevent and restitute inflammation in the intestine. Our aim was to investigate whether Notch1-DCLK1 axis regulates the mucosal immune responses to infection and whether this is phenocopied in human models of colitis. In the FFPE (formalin-fixed paraffin-embedded) sections prepared from the colons of ulcerative colitis (UC) and immune-mediated colitis (IRAEC) patients, expression of DCLK1 isoforms correlated positively with Notch1 and negatively with a transcriptional repressor, FoxD3 (Forkhead Box D3). DCLK1 protein staining in these sections was predominantly sub-epithelial (stromal) wherein DCLK1 co-localized with NICD, CD68, CD11c, and neutrophil elastase (NE). NE also co-stained with Citrullinated-H3 indicating the presence of neutrophil extracellular traps. In human neutrophils, elevated levels of DCLK1-S, CXCL-10, Ly6G, MPO, NE, and Notch1/2 in LPS-treated cells were inhibited when LPS was added in conjunction with Notch blocker dibenzazepine (DBZ; LPS + DBZ group). In CR-infected Rag1-/- mice, higher levels of DCLK1 in the colonic crypts were inhibited when mice received DBZ for 10 days coincident with significant dysbiosis, barrier disruption, and colitis. Concurrently, DCLK1 immunoreactivity shifted toward the stroma in CR + DBZ mice with predominance of DCLK1-S that coincided with higher Notch1 levels. Upon antibiotic treatment, partial restoration of crypt DCLK1, reduction in MPO activity, and increased survival followed. When intestinal epithelial cell-specific Dclk1-knockout (Dclk1ΔIEC) or Dclk1ΔIEC;Rag1-/- double knockout (DKO) mice were infected with CR and given a single dose of DBZ, they developed barrier defect and severe colitis with higher levels of stromal DCLK1-S, Ly6G, NE, and Notch1. We therefore propose that, by regulating the mucosal immune responses, the Notch-DCLK1 axis may be integral to the development of murine or human colitis.

Co-localization of autophagy-related protein p62 with cancer stem cell marker dclk1 may hamper dclk1's elimination during colon cancer development and progression.

Autophagy may play a critical role in colon cancer stem cells (CCSCs)-related cancer development. Here, we investigate whether accumulation of infection/injury-induced CCSCs due to impaired autophagy influences colon cancer development and progression. When Apc++ mice were infected with Citrobacter rodentium (CR; 109CFUs), we discovered presence of autophagosomes with increases in Beclin-1, LC3B and p62 staining during crypt hyperplasia. Apc1638N/+ mice when infected with CR or subjected to CR+AOM treatment, exhibited increased colon tumorigenesis with elevated levels of Ki-67, ß-catenin, EZH2 and CCSC marker Dclk1, respectively. AOM/DSS treatment of Apc1638N/+ mice phenocopied CR+AOM treatment as colonic tumors exhibited pronounced changes in Ki-67, EZH2 and Dclk1 accompanied by infiltration of F4/80+ macrophages, CD3+ lymphocytes and CD3/ß-catenin co-localization. Intestinal and colonic tumors also stained positive for migrating CSC markers CD110 and CDCP1 wherein, colonic tumors additionally exhibited stromal positivity. In tumors from CR-infected, CR+AOM or AOM/DSS-treated Apc1638N/+ mice and surgically-resected colon tumor/metastatic liver samples, significant accumulation of p62 and it's co-localization with LC3B and Dclk1 was evident. ApcMin/+ mice when infected with CR and BLT1-/-;ApcMin/+ mice, exhibited similar co-localization of p62 with LC3B and Dclk1 within the tumors. Studies in HCT116 and SW480 cells further confirmed p62/Dclk1 co-localization and Chloroquin/LPS-induced increases in Dclk1 promoter activity. Thus, co-localization of p62 with Dclk1 may hamper Dclk1's elimination to impact colon cancer development and progression.

Enteric infection coupled with chronic Notch pathway inhibition alters colonic mucus composition leading to dysbiosis, barrier disruption and colitis.

Intestinal mucus layer disruption and gut microflora modification in conjunction with tight junction (TJ) changes can increase colonic permeability that allows bacterial dissemination and intestinal and systemic disease. We showed previously that Citrobacter rodentium (CR)-induced colonic crypt hyperplasia and/or colitis is regulated by a functional cross-talk between the Notch and Wnt/ß-catenin pathways. In the current study, mucus analysis in the colons of CR-infected (108 CFUs) and Notch blocker Dibenzazepine (DBZ, i.p.; 10µmol/Kg b.w.)-treated mice revealed significant alterations in the composition of trace O-glycans and complex type and hybrid N-glycans, compared to CR-infected mice alone that preceded/accompanied alterations in 16S rDNA microbial community structure and elevated EUB338 staining. While mucin-degrading bacterium, Akkermansia muciniphila (A. muciniphila) along with Enterobacteriaceae belonging to Proteobacteria phyla increased in the feces, antimicrobial peptides Angiogenin-4, Intelectin-1 and Intelectin-2, and ISC marker Dclk1, exhibited dramatic decreases in the colons of CR-infected/DBZ-treated mice. Also evident was a loss of TJ and adherens junction protein immuno-staining within the colonic crypts that negatively impacted paracellular barrier. These changes coincided with the loss of Notch signaling and exacerbation of mucosal injury. In response to a cocktail of antibiotics (Metronidazole/ciprofloxacin) for 10 days, there was increased survival that coincided with: i) decreased levels of Proteobacteria, ii) elevated Dclk1 levels in the crypt and, iii) reduced paracellular permeability. Thus, enteric infections that interfere with Notch activity may promote mucosal dysbiosis that is preceded by changes in mucus composition. Controlled use of antibiotics seems to alleviate gut dysbiosis but may be insufficient to promote colonic crypt regeneration.

Microbiome, Metabolome and Inflammatory Bowel Disease.

Inflammatory Bowel Disease (IBD) is a multifactorial disorder that conceptually occurs as a result of altered immune responses to commensal and/or pathogenic gut microbes in individuals most susceptible to the disease. During Crohn's Disease (CD) or Ulcerative Colitis (UC), two components of the human IBD, distinct stages define the disease onset, severity, progression and remission. Epigenetic, environmental (microbiome, metabolome) and nutritional factors are important in IBD pathogenesis. While the dysbiotic microbiota has been proposed to play a role in disease pathogenesis, the data on IBD and diet are still less convincing. Nonetheless, studies are ongoing to examine the effect of pre/probiotics and/or FODMAP reduced diets on both the gut microbiome and its metabolome in an effort to define the healthy diet in patients with IBD. Knowledge of a unique metabolomic fingerprint in IBD could be useful for diagnosis, treatment and detection of disease pathogenesis.

Genome-wide RNAi screening identifies TMIGD3 isoform1 as a suppressor of NF-κB and osteosarcoma progression.

The ability of cancer cells to survive and grow in anchorage- and serum-independent conditions is well correlated with their aggressiveness. Here, using a human whole-genome shRNA library, we identify TMIGD3 isoform1 (i1) as a factor that suppresses this ability in osteosarcoma (OS) cells, mainly by inhibiting NF-κB activity. Knockdown of TMIGD3 increases proliferation, tumour formation and metastasis of OS cells. Overexpression of TMIGD3 isoform1 (i1), but not isoform3 (i3) which shares a common C-terminal region, suppresses these malignant properties. Adenosine A3 receptor (A3AR) having an identical N-terminal region shows similar biological profiles to TMIGD3 i1. Protein expression of TMIGD3 and A3AR is lower in human OS tissues than normal tissues. Mechanistically, TMIGD3 i1 and A3AR commonly inhibit the PKA-Akt-NF-κB axis. However, TMIGD3 i1 only partially rescues phenotypes induced by A3AR knockdown, suggesting the presence of distinct pathways. Our findings reveal an unappreciated role for TMIGD3 i1 as a suppressor of NF-κB activity and OS progression.

Differential effects of ß-catenin and NF-κB interplay in the regulation of cell proliferation, inflammation and tumorigenesis in response to bacterial infection.

Both ß-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that ß-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4(-/-) (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total ß-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) ß-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3ß and not loss of wild type APC protein accounted for ß-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/ß-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to ß-catenin (si-ß-Cat), almost complete loss of nuclear ß-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-ß-Cat treatment to Apc(Min/+) mice attenuated CR-induced increases in ß-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant ß-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, ß-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.

Involvement of LKB1 in epithelial-mesenchymal transition (EMT) of human lung cancer cells.

Epithelial-mesenchymal transition (EMT) is a critical phenotypic alteration of cancer cells that triggers invasion and metastasis. Lung cancer cells often show mesenchymal phenotypes; however, a causative genetic alteration for the induction of EMT in lung cancer cells remains unknown. Recent studies have shown that the LKB1 gene is mutated in up to one-third of lung adenocarcinomas. Therefore, to pursue the possible involvement of LKB1 inactivation in the induction of EMT in lung carcinogenesis, we generated immortalized lung epithelial cells and lung adenocarcinoma cells with stable or transient LKB1 knockdown. LKB1 knockdown increased cell motility and invasiveness, and induced the expression of several mesenchymal marker proteins accompanied by the expression of ZEB1, a transcriptional repressor for E-cadherin and an EMT inducer. In agreement with the recent findings, expression of miR-200a/c was inversely correlated with that of ZEB1 in LKB1 knockdown clones with mesenchymal phenotype. Furthermore, transient knockdown of LKB1 induced ZEB1 mRNA and increased cell motility, and this motility was suppressed by ZEB1 repression. These results strongly indicate that LKB1 inactivation triggers EMT in lung cancer cells through the induction of ZEB1.

Kank attenuates actin remodeling by preventing interaction between IRSp53 and Rac1.

In this study, insulin receptor substrate (IRS) p53 is identified as a binding partner for Kank, a kidney ankyrin repeat-containing protein that functions to suppress cell proliferation and regulate the actin cytoskeleton. Kank specifically inhibits the binding of IRSp53 with active Rac1 (Rac1(G12V)) but not Cdc42 (cdc42(G12V)) and thus inhibits the IRSp53-dependent development of lamellipodia without affecting the formation of filopodia. Knockdown (KD) of Kank by RNA interference results in increased lamellipodial development, whereas KD of both Kank and IRSp53 has little effect. Moreover, insulin-induced membrane ruffling is inhibited by overexpression of Kank. Kank also suppresses integrin-dependent cell spreading and IRSp53-induced neurite outgrowth. Our results demonstrate that Kank negatively regulates the formation of lamellipodia by inhibiting the interaction between Rac1 and IRSp53.

Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K-Akt signaling.

Phosphoinositide-3 kinase (PI3K)/Akt signaling is activated by growth factors such as insulin and epidermal growth factor (EGF) and regulates several functions such as cell cycling, apoptosis, cell growth, and cell migration. Here, we find that Kank is an Akt substrate located downstream of PI3K and a 14-3-3-binding protein. The interaction between Kank and 14-3-3 is regulated by insulin and EGF and is mediated through phosphorylation of Kank by Akt. In NIH3T3 cells expressing Kank, the amount of actin stress fibers is reduced, and the coexpression of 14-3-3 disrupted this effect. Kank also inhibits insulin-induced cell migration via 14-3-3 binding. Furthermore, Kank inhibits insulin and active Akt-dependent activation of RhoA through binding to 14-3-3. Based on these findings, we hypothesize that Kank negatively regulates the formation of actin stress fibers and cell migration through the inhibition of RhoA activity, which is controlled by binding of Kank to 14-3-3 in PI3K-Akt signaling.

Alternative splicing of the human Kank gene produces two types of Kank protein.

The human Kank gene encodes an ankyrin repeat domain-containing protein which regulates actin polymerization. There are at least two types of Kank protein depending on cell type, likely due to differences in transcription. Here, to examine the transcriptional initiation and genomic organization of the human Kank gene, we performed 5'-RACE (rapid amplification of cDNA ends) using total RNA from normal kidney and a human kidney cancer cell line, VMRC-RCW cells. The results suggest that the human Kank gene has several alternative first exons. While mRNA from VMRC-RCW cells encoded Kank protein (referred to as Kank-S) as reported previously, mRNA from the normal kidney tissue encoded a novel type of Kank protein (referred to as Kank-L), which contained an additional N-terminal sequence 158 amino acids long. Promoter activity and the expression of the Kank variants in normal and cancer tissues were examined.