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Machine learning (ML)- and deep learning (DL)-based imaging modalities have exhibited the capacity to handle extremely high dimensional data for a number of computer vision tasks. While these approaches have been applied to numerous data types, this capacity can be especially leveraged by application on histopathological images, which capture cellular and structural features with their high-resolution, microscopic perspectives. Already, these methodologies have demonstrated promising performance in a variety of applications like disease classification, cancer grading, structure and cellular localizations, and prognostic predictions. A wide range of pathologies requiring histopathological evaluation exist in gastroenterology and hepatology, indicating these as disciplines highly targetable for integration of these technologies. Gastroenterologists have also already been primed to consider the impact of these algorithms, as development of real-time endoscopic video analysis software has been an active and popular field of research. This heightened clinical awareness will likely be important for future integration of these methods and to drive interdisciplinary collaborations on emerging studies. To provide an overview on the application of these methodologies for gastrointestinal and hepatological histopathological slides, this review will discuss general ML and DL concepts, introduce recent and emerging literature using these methods, and cover challenges moving forward to further advance the field.

Oncogenic RAS is a critical driver for the initiation and progression of several types of cancers. However, effective therapeutic strategies by targeting RAS, in particular RASG12D and RASG12V, and associated downstream pathways have been so far unsuccessful. Treatment of oncogenic RAS-ravaged cancer patients remains a currently unmet clinical need. Consistent with a major role in cancer metabolism, oncogenic RAS activation elevates both reactive oxygen species (ROS)-generating NADPH oxidase (NOX) activity and ROS-scavenging glutathione biosynthesis. At a certain threshold, the heightened oxidative stress and antioxidant capability achieve a higher level of redox balance, on which cancer cells depend to gain a selective advantage on survival and proliferation. However, this prominent metabolic feature may irrevocably render cancer cells vulnerable to concurrent inhibition of both NOX activity and glutathione biosynthesis, which may be exploited as a novel therapeutic strategy. In this report, we test this hypothesis by treating the HRASG12V-transformed ovarian epithelial cells, mutant KRAS-harboring pancreatic and colon cancer cells of mouse and human origins, as well as cancer xenografts, with diphenyleneiodonium (DPI) and buthionine sulfoximine (BSO) combination, which inhibit NOX activity and glutathione biosynthesis, respectively. Our results demonstrate that concomitant targeting of NOX and glutathione biosynthesis induces a highly potent lethality to cancer cells harboring oncogenic RAS. Therefore, our studies provide a novel strategy against RAS-bearing cancers that warrants further mechanistic and translational investigation.

The Th17 cell lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggestive of additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor Atoh1 in Lgr5(+) intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1(+) cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the crosstalk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

Yersinia pseudotuberculosis is a foodborne pathogen that subverts immune function by translocation of Yersinia outer protein (Yop) effectors into host cells. As adaptive γδ T cells protect the intestinal mucosa from pathogen invasion, we assessed whether Y. pseudotuberculosis subverts these cells in mice and humans. Tracking Yop translocation revealed that the preferential delivery of Yop effectors directly into murine Vγ4 and human Vδ2+ T cells inhibited anti-microbial IFNγ production. Subversion was mediated by the adhesin YadA, injectisome component YopB, and translocated YopJ effector. A broad anti-pathogen gene signature and STAT4 phosphorylation levels were inhibited by translocated YopJ. Thus, Y. pseudotuberculosis attachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of immune subversion in mice and humans. This study uncovered a conserved Y. pseudotuberculosis pathway that subverts adaptive γδ T cell function to promote pathogenicity., Author summary Unconventional γδ T cells are a dynamic immune population important for mucosal protection of the intestine against invading pathogens. We determined that the foodborne pathogen Y. pseudotuberculosis preferentially targets an adaptive subset of these cells to subvert immune function. We found that direct injection of Yersinia outer proteins (Yop) into adaptive γδ T cells inhibited their anti-pathogen functions. We screened all Yop effectors and identified YopJ as the sole effector to inhibit adaptive γδ T cell production of IFNγ. We determined that adaptive γδ T cell subversion occurred by limiting activation of the transcription factor STAT4. When we infected mice with Y. pseudotuberculosis expressing an inactive YopJ, this enhanced the adaptive γδ T cell response and led to greater cytokine production from this subset of cells to aid mouse recovery. This mechanism of immune evasion appears conserved in humans as direct injection of Y. pseudotuberculosis YopJ into human γδ T cells inhibited cytokine production. This suggested to us that Y. pseudotuberculosis actively inhibits the adaptive γδ T cell response through YopJ as a mechanism to evade immune surveillance at the site of pathogen invasion.

Inflammatory bowel disease (IBD) is a chronic immune-mediated disease of the gastrointestinal tract. While therapies exist, response can be limited within the patient population. Researchers have thus studied mouse models of colitis to further understand pathogenesis and identify new treatment targets. Flow cytometry and RNA-sequencing can phenotype immune populations with single-cell resolution but provide no spatial context. Spatial context may be particularly important in colitis mouse models, due to the simultaneous presence of colonic regions that are involved or uninvolved with disease. These regions can be identified on hematoxylin and eosin (H&E)-stained colonic tissue slides based on the presence of abnormal or normal histology. However, detection of such regions requires expert interpretation by pathologists. This can be a tedious process that may be difficult to perform consistently across experiments. To this end, we trained a deep learning model to detect ‘Involved’ and ‘Uninvolved’ regions from H&E-stained colonic tissue slides. Our model was trained on specimens from controls and three mouse models of colitis–the dextran sodium sulfate (DSS) chemical induction model, the recently established intestinal epithelium-specific, inducible Klf5ΔIND (Villin-CreERT2;Klf5fl/fl) genetic model, and one that combines both induction methods. Image patches predicted to be ‘Involved’ and ‘Uninvolved’ were extracted across mice to cluster and identify histological classes. We quantified the proportion of ‘Uninvolved’ patches and ‘Involved’ patch classes in murine swiss-rolled colons. Furthermore, we trained linear determinant analysis classifiers on these patch proportions to predict mouse model and clinical score bins in a prospectively treated cohort of mice. Such a pipeline has the potential to reveal histological links and improve synergy between various colitis mouse model studies to identify new therapeutic targets and pathophysiological mechanisms.

Rationale: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator–activated receptor) that regulate cardiac lipid metabolism. Objective: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. Methods and Results: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase ( NOX )4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. Conclusions: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.

Reduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn's disease (CD). PCs reside in proximity to Lgr5Ileal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60In patients with CD and TNFWe provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.

Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-CreER to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21Waf1/Cip1 in Bmi1-CreER cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21Waf1/Cip1 mRNA translation, which promotes exit of the Bmi1-CreER cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1+ cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21Waf1/Cip1, MSI1 and KLF4 regulates Bmi1-CreER cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.

Background Podocyte injury is the hallmark of proteinuric kidney diseases, such as FSGS and minimal change disease, and destabilization of the podocyte’s actin cytoskeleton contributes to podocyte dysfunction in many of these conditions. Although agents, such as glucocorticoids and cyclosporin, stabilize the actin cytoskeleton, systemic toxicity hinders chronic use. We previously showed that loss of the kidney-enriched zinc finger transcription factor Kruppel-like factor 15 (KLF15) increases susceptibility to proteinuric kidney disease and attenuates the salutary effects of retinoic acid and glucocorticoids in the podocyte. Methods We induced podocyte-specific KLF15 in two proteinuric murine models, HIV-1 transgenic ( Tg26 ) mice and adriamycin (ADR)-induced nephropathy, and used RNA sequencing of isolated glomeruli and subsequent enrichment analysis to investigate pathways mediated by podocyte-specific KLF15 in Tg26 mice. We also explored in cultured human podocytes the potential mediating role of Wilms Tumor 1 (WT1), a transcription factor critical for podocyte differentiation. Results In Tg26 mice, inducing podocyte-specific KLF15 attenuated podocyte injury, glomerulosclerosis, tubulointerstitial fibrosis, and inflammation, while improving renal function and overall survival; it also attenuated podocyte injury in ADR-treated mice. Enrichment analysis of RNA sequencing from the Tg26 mouse model shows that KLF15 induction activates pathways involved in stabilization of actin cytoskeleton, focal adhesion, and podocyte differentiation. Transcription factor enrichment analysis, with further experimental validation, suggests that KLF15 activity is in part mediated by WT1. Conclusions Inducing podocyte-specific KLF15 attenuates kidney injury by directly and indirectly upregulating genes critical for podocyte differentiation, suggesting that KLF15 induction might be a potential strategy for treating proteinuric kidney disease.

Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors that are found in many species. Recent studies have shown that KLFs play a fundamental role in regulating diverse biological processes such as cell proliferation, differentiation, development and regeneration. Of note, several KLFs are also crucial for maintaining pluripotency and, hence, have been linked to reprogramming and regenerative medicine approaches. Here, we review the crucial functions of KLFs in mammalian embryogenesis, stem cell biology and regeneration, as revealed by studies of animal models. We also highlight how KLFs have been implicated in human diseases and outline potential avenues for future research.

Krüppel-like factor 5 (KLF5), a member of the SP/KLF family of zinc finger transcription factors, is overexpressed in human colorectal cancer specimens and this overabundance is associated with aggressive cancer development and progression. We demonstrated that mice haploinsufficient for Klf5 had reduced intestinal tumor burden in the background of germline mutation in Apc, a gate keeper of intestinal tumorigenesis. Based on a high-throughput screening strategy, we developed ML264, a small molecule compound that inhibits KLF5 , and showed that it inhibits growth of colorectal cancer (CRC) in vitro and in vivo. Through optimization efforts based on structure of ML264, we have now identified a new lead compound, SR18662. We find that treatment with SR18662 significantly reduces growth and proliferation of CRC cells as compared to treatment with vehicle control, ML264, or SR15006 (a less optimized analogue from SAR efforts leading to SR18662). SR18662 showed improved efficacy in reducing viability of multiple CRC cell lines. Flow cytometry analysis following SR18662 treatment showed an increase in cells captured in either S or G2/M phases of the cell cycle and a significant increase in the number of apoptotic cells, the latter a unique property compared to ML264 or SR15006. SR18662 treatment also reduces the expression of cyclins and components of MAPK and WNT signaling pathways. Importantly, we observed a significant dose-dependent inhibition of xenograft growth in mice following SR18662 treatment that exceeded the effect of ML264 at equivalent doses. These findings support further development of SR18662 and its analogues for colorectal cancer therapy.

Colitis associated cancer (CAC) is chronic inflammation driven colon cancer, prevalent among individuals with Inflammatory Bowel Disease. Matrix-metalloproteinase (MMP9) is one of the essential regulators of extra cellular matrix components. We have shown that MMP9 is protective in CAC contrary to its inflammatory role in acute-colitis. Aim of our study is to identify the mechanism of the protective role of epithelial derived-MMP9 in CAC. We used homozygous transgenic mice constitutively-expressing MMP9 in colonic-epithelium (TgM9) and wild-type (WT) littermates for in vivo experiments. Stably-transfected HCT116 with/without MMP9, and mouse embryonic-fibroblasts (WT and MMP9-/-, MEFs) were used for in vitro experiments. TgM9 mice exhibited less tumor burden, increased apoptosis, and increased expressions of active-Notch1, p53, p21WAF1/Cip1, caspase-3 and cyclin E in CAC compared to WTs. These results were supported by MEFs data. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation, S-phase cell-cycle arrest and less DNA damage compared to vector. MMP9-/- mice showed attenuation of MMP9 was directly associated with p19ARF. Our study identifies the tumor suppressor role of epithelial derived-MMP9 in CAC via novel mechanistic pathway "MMP9-Notch1-ARF-p53 axis" regulating apoptosis, cell-cycle arrest and DNA damage implying, that MMP9 expression might be a natural/biological way to suppress colonic ulceration due to chronic inflammation.

Podocyte injury is the inciting event in primary glomerulopathies, such as minimal change disease and primary FSGS, and glucocorticoids remain the initial and often, the primary treatment of choice for these glomerulopathies. Because inflammation is not readily apparent in these diseases, understanding the direct effects of glucocorticoids on the podocyte, independent of the immunomodulatory effects, may lead to the identification of targets downstream of glucocorticoids that minimize toxicity without compromising efficacy. Several studies showed that treatment with glucocorticoids restores podocyte differentiation markers and normal ultrastructure and improves cell survival in murine podocytes. We previously determined that Krüppel–like factor 15 (KLF15), a kidney–enriched zinc finger transcription factor, is required for restoring podocyte differentiation markers in mice and human podocytes under cell stress. Here, we show that in vitro treatment with dexamethasone induced a rapid increase of KLF15 expression in human and murine podocytes and enhanced the affinity of glucocorticoid receptor binding to the promoter region of KLF15. In three independent proteinuric murine models, podocyte-specific loss of Klf15 abrogated dexamethasone–induced podocyte recovery. Furthermore, knockdown of KLF15 reduced cell survival and destabilized the actin cytoskeleton in differentiated human podocytes. Conversely, overexpression of KLF15 stabilized the actin cytoskeleton under cell stress in human podocytes. Finally, the level of KLF15 expression in the podocytes and glomeruli from human biopsy specimens correlated with glucocorticoid responsiveness in 35 patients with minimal change disease or primary FSGS. Thus, these studies identify the critical role of KLF15 in mediating the salutary effects of glucocorticoids in the podocyte.

Pathologic glomerular epithelial cell (GEC) hyperplasia is characteristic of both rapidly progressive glomerulonephritis (RPGN) and subtypes of focal segmental glomerulosclerosis (FSGS). Although initial podocyte injury resulting in activation of STAT3 signals GEC proliferation in both diseases, mechanisms regulating this are unknown. Here, we show that the loss of Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, enhances GEC proliferation in both RPGN and FSGS due to dysregulated STAT3 signaling. We observed that podocyte-specific knockdown of Klf4 (C57BL/6J) increased STAT3 signaling and exacerbated crescent formation after nephrotoxic serum treatment. Interestingly, podocyte-specific knockdown of Klf4 in the FVB/N background alone was sufficient to activate STAT3 signaling, resulting in FSGS with extracapillary proliferation, as well as renal failure and reduced survival. In cultured podocytes, loss of KLF4 resulted in STAT3 activation and cell-cycle reentry, leading to mitotic catastrophe. This triggered IL-6 release into the supernatant, which activated STAT3 signaling in parietal epithelial cells. Conversely, either restoration of KLF4 expression or inhibition of STAT3 signaling improved survival in KLF4-knockdown podocytes. Finally, human kidney biopsy specimens with RPGN exhibited reduced KLF4 expression with a concomitant increase in phospho-STAT3 expression as compared with controls. Collectively, these results suggest the essential role of KLF4/STAT3 signaling in podocyte injury and its regulation of aberrant GEC proliferation.

Specificity proteins (SPs) and Krüppel-like factors (KLFs) belong to the family of transcription factors that contain conserved zinc finger domains involved in binding to target DNA sequences. Many of these proteins are expressed in different tissues and have distinct tissue-specific activities and functions. Studies demonstrate that SPs and KLFs regulate not only physiological processes such as growth, development, differentiation, proliferation, and embryogenesis, but pathogenesis of many diseases, including cancer and inflammatory disorders. Consistently, these proteins have been shown to regulate normal functions and pathobiology in the digestive system. We review recent findings on the tissue- and organ-specific functions of SPs and KLFs in the digestive system including the oral cavity, esophagus, stomach, small and large intestines, pancreas, and liver. We provide a list of agents under development to target these proteins.

Kruppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell growth, proliferation, and differentiation. Since its discovery in 1996, KLF4 has been gaining a lot of attention, particularly after it was shown in 2006 as one of four factors involved in the induction of pluripotent stem cells (iPSCs). Here we review the current knowledge about the different functions and roles of KLF4 in various tissue and organ systems.

Hypoxia-inducible factor 1α (HIF-1α) and p53 are pivotal regulators of tumor growth. Lysophosphatidic acid (LPA) is a lipid mediator that functions as a mitogen by acting through LPA receptors. We have shown previously that LPA stimulates HIF-1α expression in colon cancer cells. To determine the mechanism of HIF-1α induction by LPA, we compared the effect of LPA on HIF-1α in several colon cancer cell lines. LPA transcriptionally induced HIF-1α in colon cancer cells. HIF-1α induction was observed in cells expressing WT p53, where LPA decreased p53 expression. However, LPA failed to induce HIF-1α when the p53 gene was mutated. A decrease in p53 expression was dependent on induction of p53-specific E3 ubiquitin ligase Mdm2 by LPA. Kruppel-like factor 5 (KLF5) is an effector of LPA-induced proliferation of colon cancer cells. Because HIF-1α was necessary for LPA-induced growth of colon cancer cells, we determined the relationship between KLF5 and HIF-1α by a loss-of-function approach. Silencing of KLF5 inhibited LPA-induced HIF-1α induction, suggesting that KLF5 is an upstream regulator of HIF-1α. KLF5 and p53 binding to the Hif1α promoter was assessed by ChIP assay. LPA increased the occupancy of the Hif1α promoter by KLF5, while decreasing p53 binding. Transfection of HCT116 cells with KLF5 or p53 attenuated the binding of the other transcription factor. These results identify KLF5 as a transactivator of HIF-1α and show that LPA regulates HIF-1α by dynamically modulating its interaction with KLF5 and p53. Background: LPA induces HIF-1α expression, but the underlying mechanism remains unknown. Results: LPA induced HIF-1α by decreasing p53 expression, and additionally, KLF5 transactivated HIF-1α expression. Conclusion: KLF5 and p53 reciprocally regulate HIF-1α expression. Significance: This study provides a new dimension to understanding how LPA promotes colon cancer.

Summary The irreversibility of developmental processes in mammalian cells has been challenged by rising evidence that de-differentiation of hepatocytes occurs in adult liver. However, whether reversibility exists in mesenchymal stromal cell (MSC)-derived hepatocytes (dHeps) remains elusive. In this study, we find that hepatogenic differentiation (HD) of MSCs is a reversible process and is modulated by DNA methyltransferases (DNMTs). DNMTs are regulated by transforming growth factor β1 (TGFβ1), which in turn controls hepatogenic differentiation and de-differentiation. In addition, a stepwise reduction in TGFβ1 concentrations in culture media increases DNMT1 and decreases DNMT3 in primary hepatocytes (Heps) and confers Heps with multi-differentiation potentials similarly to MSCs. Hepatic lineage reversibility of MSCs and lineage conversion of Heps are regulated by DNMTs in response to TGFβ1. This previously unrecognized TGFβ1-DNMTs-MSC-HD axis may further increase the understanding the normal and pathological processes in the liver, as well as functions of MSCs after transplantation to treat liver diseases., Graphical Abstract, Highlights • TGFβ1-DNMT axis modulates reversibility of hepatogenic differentiation in MSCs • Primary hepatocytes can be converted into MSC-like cells by TGFβ1 • Respectively, DNMT1 represses and DNMT3a promotes hepatogenic differentiation • DNMT1 knockdown shortened hepatogenic differentiation time from 28 to 14 days, Lee and colleagues demonstrate that through TGFβ1-regulated DNA methyltransferase axis, adult hepatocytes and MSC-derived hepatocyte-like cells can revert to an undifferentiated state without forced ectopic gene expression.

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), significantly increases the risk for development of colorectal cancer. Specifically, dysplasia and cancer associated with IBD (colitis-associated cancer or CAC) develop as a result of repeated cycles of injury and healing in the intestinal epithelium. Animal models are utilized to examine the mechanisms of CAC, the role of epithelial and immune cells in this process, as well as the development of novel therapeutic targets. These models typically begin with the administration of a carcinogenic compound, and inflammation is caused by repeated cycles of colitis-inducing agents. This review describes a common CAC model that utilizes the pro-carcinogenic compound azoxymethane (AOM) followed by dextran sulfate sodium (DSS) which induces the inflammatory insult.

Recent advancements in understanding the role of both genetics and molecular pathways in the formation and progression of colorectal cancer have allowed the identification of factors that may be targeted for drug discovery. During the past decade, various approaches have been developed to target specific steps or components of these pathways in order to prevent the development and progression of colorectal cancer and to treat this disease. The innovation and optimization of high-throughput screening methods, as well as the recent emphasis from the NIH on translational sciences, have enabled rapid progress in drug discovery in many fields, including colorectal cancer. Here we present a summary of the recent efforts of targeted high-throughput drug discovery directed at pathways affected in colorectal cancer.

Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age. Although FAP is involved in only 1% of all colon cancer cases, over 80% of sporadic cancers harbor somatic mutations of APC. We show here that bromo-noscapine (EM011), a rationally designed synthetic derivative of a natural nontoxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G(2) /M arrest in wild type murine embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with small interfering RNA. Furthermore, both β-catenin levels and activity fell to half the original levels with a concomitant reduction of cell proliferation-inducing cyclin D1, c-Myc, and induction of cytostatic protein p21 before caspase-3 activation. Additionally, we show a statistically significant reduction in the number of newly emerging intestinal polyps (to 35% compared with untreated mice) as well as the mean size of polyps (to 42% compared with untreated mice) in EM011-treated Apc(Min/+) mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of Apc(Min/+) mice showed evidence of elevated apoptosis as revealed by immunohistochemistry. We failed to detect any evidence of histopathological and hematological toxicities following EM011 treatment. Taken together, our data are persuasive that a clinical trial of EM011 is possible for the prevention/amelioration of polyposis in FAP patients.

The zinc finger transcription factor Krüppel-like factor 5 (KLF5) is regulated posttranslationally. We identified SMAD ubiquitination regulatory factor 2 (SMURF2), an E3 ubiquitin ligase, as an interacting protein of KLF5 by yeast two-hybrid screen, coimmunoprecipitation, and indirect immunofluorescence studies. The SMURF2-interacting domains in KLF5 were mapped to its carboxyl terminus, including the PY motif of KLF5 and its zinc finger DNA-binding domain. KLF5 protein levels were reduced significantly upon overexpression of SMURF2 but not catalytically inactive SMURF2-C716A mutant or SMURF1. SMURF2 alone reduced the protein stability of KLF5 as shown by cycloheximide chase assay, indicating that SMURF2 specifically destabilizes KLF5. In contrast, KLF5(1-165), a KLF5 amino-terminal construct that lacks the PY motif and DNA binding domain, was not degraded by SMURF2. The degradation of KLF5 by SMURF2 was blocked by the proteasome inhibitor MG132, and SMURF2 efficiently ubiquitinated both overexpressed and endogenous KLF5. In contrast, knocking down SMURF2 by siRNAs significantly enhanced KLF5 protein levels, reduced ubiquitination of KLF5, and increased the expression of cyclin D1 and PDGF-A, two established KLF5 target genes. In consistence, SMURF2, but not the E3 ligase mutant SMURF2-C716A, significantly inhibited the transcriptional activity of KLF5, as demonstrated by dual luciferase assay using the PDGF-A promoter, and suppressed the ability of KLF5 to stimulate cell proliferation as measured by BrdU incorporation. Hence, SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by targeting it for proteasomal degradation.

Background & Aims Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell–derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice. Methods C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl- S -phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9 −/− mice and HCT116 cells that were stably transfected with MMP-9. Results Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21 WAF1/Cip1 , Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9 −/− mice on γ-radiation–induced damage of DNA. Conclusions MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.

Fecal microbiota transplantation (FMT) has emerged as an effective treatment for C. difficile infection (CDI) refractory to antibiotic therapy alone. Given the important roles of microbiota in the function of the gastrointestinal tract and other aspects of human physiology, there is also growing interest in studying FMT for other clinical indications. The U.S. Food and Drug Administration has determined that FMT constitutes a drug and is an investigational agent; as such an investigational new drug application (IND) is encouraged in treatment of recurrent CDI and required to administer FMT for any other indications. Most physicians and investigators do not have the regulatory experience necessary to navigate the IND process easily. Our aim is to provide guidance to others who are interested in obtaining an IND to perform or study FMT.

We have previously shown that a non-toxic noscapinoid, EM011 binds tubulin without altering its monomer/polymer ratio. EM011 is more active than the parent molecule, noscapine, in inducing G2/M arrest, inhibiting cellular proliferation and tumor growth in various human xenograft models. However, the mechanisms of mitotic-block and subsequent cell death have remained elusive. Here, we show that EM011-induced attenuation of microtubule dynamics was associated with impaired association of microtubule plus-end tracking proteins, such as EB1 and CLIP-170. EM011 treatment then led to the formation of multipolar spindles containing ‘real’ centrioles indicating drug-induced centrosome amplification and persistent centrosome declustering. Centrosome amplification was accompanied by an upregulation of Aurora A and Plk4 protein levels, as well as a surge in the kinase activity of Aurora A, suggesting a deregulation of the centrosome duplication cycle. Cell-cycle phase-specific experiments showed that the ‘cytotoxicity-window’ of the drug encompasses the late S-G2 period. Drug-treatment, excluding S-phase, not only resulted in lower sub-G1 population but also attenuated centrosome amplification and spindle multipolarity, suggesting that drug-induced centrosome amplification is essential for maximal cell death. Subsequent to a robust mitotic arrest, EM011-treated cells displayed diverse cellular fates suggesting a high degree of intraline variation. Some ‘apoptosisevasive’ cells underwent aberrant cytokinesis to generate rampant aneuploidy that perhaps contributed to drug-induced cell death. These data indicate that spindle multipolarity induction by means of centrosome amplification has an exciting chemotherapeutic potential that merits further investigation. Cell Death and Differentiation (2011) 18, 632–644; doi:10.1038/cdd.2010.133; published online 5 November 2010

Background: The zinc finger transcription factor Krüppel-like factor 4 (KLF4) regulates numerous physiologic processes, including proliferation, differentiation, and development. Studies also showed that KLF4 is involved in tumorigenesis and somatic cell reprogramming. Here, we aimed to assess whether KLF4 is a prognostic indicator for colon cancer. Methods: Levels of KLF4 were measured by immunohistochemical analysis of a tissue microarray containing 367 independent colon cancer sections. Univariate data analysis was done in addition to construction of multivariate models with several clinicopathologic factors to evaluate KLF4 as an independent predictor of survival and cancer recurrence (disease-free survival). Results: Colon cancer tissues had significantly overall lower KLF4 levels compared with noncancer tissues (P < 0.0001). Using logistic regression, a trend was noted for decreased odds of KLF4 expression in higher stages of tumors. In univariate and multivariate analyses, KLF4 was a significant predictor of survival and recurrence. Conclusions: KLF4 expression is significantly downregulated in colon cancer, and loss of KLF4 is an independent predictor of survival and recurrence. Impact: These findings suggest that KLF4 may serve as a prognostic biomarker for colon cancer. Cancer Epidemiol Biomarkers Prev; 19(10); 2631–8. ©2010 AACR.

The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), regulates numerous biological processes, including proliferation, differentiation, and embryonic stem cell self-renewal. Although the DNA sequence to which KLF4 binds is established, the mechanism by which KLF4 controls transcription is not well defined. Small ubiquitin-related modifier (SUMO) is an important regulator of transcription. Here we show that KLF4 is both SUMOylated at a single lysine residue and physically interacts with SUMO-1 in a region that matches an acidic and hydrophobic residue-rich SUMO-interacting motif (SIM) consensus. The SIM in KLF4 is required for transactivation of target promoters in a SUMO-1-dependent manner. Mutation of either the acidic or hydrophobic residues in the SIM significantly impairs the ability of KLF4 to interact with SUMO-1, activate transcription, and inhibit cell proliferation. Our study provides direct evidence that SIM in KLF4 functions as a transcriptional activation domain. A survey of transcription factor sequences reveals that established transactivation domains of many transcription factors contain sequences highly related to SIM. These results, therefore, illustrate a novel mechanism by which SUMO interaction modulates the activity of transcription factors.

Background & Aims Activating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)—a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar-to-ductal metaplasia (ADM). We investigated the role of the transcription factor Kruppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs. Methods We performed studies in adult mice with conditional disruption of Klf5 ( Klf5 fl/fl ) and/or expression of Kras G12D ( LSL-Kras G12D ) via Cre ERTM recombinase regulated by an acinar cell–specific promoter ( Ptf1a ). Activation of Kras G12D and loss of KLF5 was achieved by administration of tamoxifen. Pancreatitis was induced in mice by administration of cerulein; pancreatic tissues were collected, analyzed by histology and immunohistochemistry, and transcriptomes were compared between mice that did or did not express KLF5. We performed immunohistochemical analyses of human tissue microarrays, comparing levels of KLF5 among 96 human samples of PDAC. UN-KC-6141 cells (pancreatic cancer cells derived from Pdx1-Cre;LSL-Kras G12D mice) were incubated with inhibitors of different kinases and analyzed in proliferation assays and by immunoblots. Expression of KLF5 was knocked down with small hairpin RNAs or CRISPR/Cas9 strategies; cells were analyzed in proliferation and gene expression assays, and compared with cells expressing control vectors. Cells were subcutaneously injected into flanks of syngeneic mice and tumor growth was assessed. Results Of the 96 PDAC samples analyzed, 73% were positive for KLF5 (defined as nuclear staining in more than 5% of tumor cells). Pancreata from Ptf1a-Cre ERTM ; LSL-Kras G12D mice contained ADM and PanIN lesions, which contained high levels of nuclear KLF5 within these structures. In contrast, Ptf1a-Cre ERTM ; LSL-Kras G12D ; Klf5 fl/fl mice formed fewer PanINs. After cerulein administration, Ptf1a-Cre ERTM ; LSL-Kras G12D mice formed more extensive ADM than Ptf1a-Cre ERTM ;LSL- Kras G12D ; Klf5 fl/fl mice. Pancreata from Ptf1a-Cre ERTM ; LSL-Kras G12D ; Klf5 fl/fl mice had increased expression of the tumor suppressor NDRG2 and reduced phosphorylation (activation) of STAT3, compared with Ptf1a-Cre ERTM ; LSL-Kras G12D mice. In UN-KC-6141 cells, PI3K and MEK signaling increased expression of KLF5; a high level of KLF5 increased proliferation. Cells with knockdown of Klf5 had reduced proliferation, compared with control cells, had reduced expression of ductal markers, and formed smaller tumors (71.61 ± 30.79 mm 3 vs 121.44 ± 34.90 mm 3 from control cells) in flanks of mice. Conclusion Levels of KLF5 are increased in human PDAC samples and in PanINs of Ptf1a-Cre ERTM ; LSL-Kras G12D mice, compared with controls. KLF5 disruption increases expression of NDRG2 and reduces activation of STAT3 and reduces ADM and PanINs formation in mice. Strategies to reduce KLF5 activity might reduce progression of acinar cells from ADM to PanIN and pancreatic tumorigenesis.

Colorectal cancer is one of the leading causes of cancer mortality and morbidity worldwide. Previous studies indicate that the zinc finger-containing transcription factor Krüppel-like factor 5 (KLF5) positively regulates proliferation of intestinal epithelial cells and colorectal cancer cells. Importantly, inhibition of KLF5 expression in intestinal epithelial cells and colorectal cancer cells by pharmacologic or genetic means reduces their rate of proliferation. To identify additional and novel small molecules that inhibit KLF5 expression and thus colorectal cancer proliferation, we developed a reporter assay using colorectal cancer cell line (DLD-1) that stably expressed a luciferase reporter gene directed by 1,959 bp of the human KLF5 promoter upstream of the ATG start codon and performed a cell-based high-throughput screen with the Library of Pharmacologically Active Compounds that contains 1,280 biologically active compounds. The screen identified 8 potential inhibitors and 6 potential activators of the KLF5 promoter. Three potential inhibitors, wortmannin, AG17, and AG879, were further evaluated by secondary analyses. All three significantly reduced both KLF5 promoter-luciferase activity and protein level in DLD-1 cells in a dose- and time-dependent manner when compared with controls. They also significantly reduced the rate of proliferation of DLD-1 and two other colorectal cancer cell lines, HCT116 and HT29. Our results show the principle of using high-throughput screening to identify small-molecule compounds that modulate KLF5 activity and consequently inhibit colorectal cancer proliferation. [Mol Cancer Ther 2009;8(3):563–70]

The zinc finger-containing transcription factor, Krüppel-like factor 4 (KLF4), inhibits cell proliferation. An in vivo tumor-suppressive role for KLF4 is shown by the recent finding that Klf4 haploinsufficiency in ApcMin/+ mice promotes intestinal tumorigenesis. Studies also show that KLF4 is required for the terminal differentiation of goblet cells in the mouse intestine. The Notch signaling pathway suppresses goblet cell formation and is up-regulated in intestinal tumors. Here, we investigated the relationship between Notch signaling and KLF4 expression in intestinal epithelial cells. The rate of proliferation of HT29 human colon cancer cells was reduced when treated with the γ-secretase inhibitor dibenzazepine to inhibit Notch signaling or small interfering RNA directed against Notch. KLF4 levels were increased in dibenzazepine-treated or Notch small interfering RNA-treated cells. Conversely, overexpression of Notch in HT29 cells reduced KLF4 levels, suppressed KLF4 promoter activity, and increased proliferation rate. Treatment of ApcMin/+ mice with dibenzazepine resulted in a 50% reduction in the number of intestinal adenomas compared with the vehicle-treated group (P < 0.001). Both the normal-appearing intestinal mucosa and adenomas obtained from dibenzazepine-treated ApcMin/+ mice had increased goblet cell numbers and Klf4 staining accompanied by reduced cyclin D1 and Ki-67 staining when compared with those from vehicle-treated mice. Results of these studies indicate that Notch signaling suppresses KLF4 expression in intestinal tumors and colorectal cancer cells. Inhibition of Notch signaling increases KLF4 expression and goblet cell differentiation and reduces proliferation and tumor formation. KLF4 is therefore a potential mediator for the antitumor effect of Notch inhibitors such as dibenzazepine. (Mol Cancer Res 2008;6(12):1920–7)

SUMOylation is a form of post-translational modification shown to control nuclear transport. Krüppel-like factor 5 (KLF5) is an important mediator of cell proliferation and is primarily localized to the nucleus. Here we show that mouse KLF5 is SUMOylated at lysine residues 151 and 202. Mutation of these two lysines or two conserved nearby glutamates results in the loss of SUMOylation and increased cytoplasmic distribution of KLF5, suggesting that SUMOylation enhances nuclear localization of KLF5. Lysine 151 is adjacent to a nuclear export signal (NES) that resembles a consensus NES. The NES in KLF5 directs a fused green fluorescence protein to the cytoplasm, binds the nuclear export receptor CRM1, and is inhibited by leptomycin and site-directed mutagenesis. SUMOylation facilitates nuclear localization of KLF5 by inhibiting this NES activity, and enhances the ability of KLF5 to stimulate anchorage-independent growth of HCT116 colon cancer cells. A survey of proteins whose nuclear localization is regulated by SUMOylation reveals that SUMOylation sites are frequently located in close proximity to NESs. A relatively common mechanism for SUMOylation to regulate nucleocytoplasmic transport may lie in the interplay between neighboring NES and SUMOylation motifs.

The presence of somatic β-catenin mutations in some prostate cancers implies that aberrant WNT signaling is involved in the cancer development. Although β-catenin stability is regulated by a multicomponent destruction complex, mutational alterations of β-catenin or other components of the destruction complexes are rare in prostate tumors. Therefore, β-catenin may be regulated by another protein in the prostate. In fact, recent linkage and somatic deletion analyses in prostate cancers reveal a 1.4-Mb candidate tumor suppressor locus on 8p23.1, which includes the Sox7 gene. Here we show that Sox7 protein expression was indeed down-regulated in 47% (15 of 32) of prostate adenocarcinomas. In addition, Sox7 mRNA was down-regulated in 60% of snap-frozen tumors. This down-regulation was found to be due to tumor-specific promoter hypermethylation, which was present in 48% (10 of 21) of primary prostate tumors and 44% (11 of 25) of prostate cancer cell lines/xenografts. We discovered that Sox7 protein physically interacts with β-catenin and suppresses β-catenin–mediated transcription by depleting active β-catenin. Furthermore, in HCT116 colorectal cancer cell lines with Sox7 inactivation, ectopic Sox7 expression suppressed cell proliferation and inhibited transcription that was activated by an endogenous mutant β-catenin. Although nearly all colorectal cancers contain mutations in β-catenin or adenomatous polyposis coli/axin, epigenetic silencing of Sox7 was still observed. These data suggest that Sox7 is a tumor suppressor that functions as an independent checkpoint for β-catenin transcriptional activity. Inactivation of Sox7 could promote the development of a majority of colorectal tumors and approximately half of prostate tumors. (Mol Cancer Res 2008;6(9):1421–10)

The mitotic checkpoint is a mechanism that arrests the progression to anaphase until all chromosomes have achieved proper attachment to mitotic spindles. In cancer cells, satisfaction of this checkpoint is frequently delayed or prevented by various defects, some of which have been causally implicated in tumorigenesis. At the same time, deliberate induction of mitotic arrest has proved clinically useful, as antimitotic drugs that interfere with proper chromosome-spindle interactions are effective anticancer agents. However, how mitotic arrest contributes to tumorigenesis or antimitotic drug toxicity is not well defined. Here, we report that mitotic chromosomes can acquire DNA breaks during both pharmacologic and genetic induction of mitotic arrest in human cancer cells. These breaks activate a DNA damage response, occur independently of cell death, and subsequently manifest as karyotype alterations. Such breaks can also occur spontaneously, particularly in cancer cells containing mitotic spindle abnormalities. Moreover, we observed evidence of some breakage in primary human cells. Our findings thus describe a novel source of DNA damage in human cells. They also suggest that mitotic arrest may promote tumorigenesis and antimitotic toxicity by provoking DNA damage. [Cancer Res 2007;67(24):11487–92]

Lysophosphatidic acids (LPA) exert growth factor-like effects through specific G protein-coupled receptors. The presence of different LPA receptors often determines the specific signaling mechanisms and the physiological consequences of LPA in different environments. Among the four members of the LPA receptor family, LPA(2) has been shown to be overexpressed in colon cancer suggesting that the signaling by LPA(2) may potentiate growth and survival of tumor cells. In this study, we examined the effect of LPA on survival of colon cancer cells using Caco-2 cells as a cell model system. LPA rescued Caco-2 cells from apoptosis elicited by the chemotherapeutic drug, etoposide. This protection was accompanied by abrogation of etoposide-induced stimulation of caspase activity via a mechanism dependent on Erk and PI3K. In contrast, perturbation of cellular signaling mediated by the LPA(2) receptor by knockdown of a scaffold protein NHERF2 abrogated the protective effect of LPA. Etoposide decreased the expression of Bcl-2, which was reversed by LPA. Etoposide decreased the phosphorylation level of the proapoptotic protein Bad in an Erk-dependent manner, without changing Bad expression. We further show that LPA treatment resulted in delayed activation of Erk. These results indicate that LPA protects Caco-2 cells from apoptotic insult by a mechanism involving Erk, Bad, and Bcl-2.

The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently downregulated in colorectal cancer. Previous studies showed that KLF4 is a tumor suppressor in the intestinal tract and plays an important role in DNA damage-repair mechanisms. Here, the in vivo effects of Klf4 deletion were examined from the mouse intestinal epithelium (Klf4ΔIS) in a genetic or pharmacological setting of colonic tumorigenesis: ApcMin/+ mutation or carcinogen treatment with azoxymethane (AOM), respectively. Klf4ΔIS/ApcMin/+ mice developed significantly more colonic adenomas with 100% penetrance as compared with ApcMin/+ mice with intact Klf4 (Klf4fl/fl/ApcMin/+). The colonic epithelium of Klf4ΔIS/ApcMin/+ mice showed increased mTOR pathway activity, together with dysregulated epigenetic mechanism as indicated by altered expression of HDAC1 and p300. Colonic adenomas from both genotypes stained positive for γH2AX, indicating DNA double-strand breaks. In Klf4ΔIS/ApcMin/+ mice, this was associated with reduced nonhomologous end joining (NHEJ) repair and homologous recombination repair (HRR) mechanisms as indicated by reduced Ku70 and Rad51 staining, respectively. In a separate model, following treatment with AOM, Klf4ΔIS mice developed significantly more colonic tumors than Klf4fl/fl mice, with more Klf4ΔIS mice harboring K-Ras mutations than Klf4fl/fl mice. Compared with AOM-treated Klf4fl/fl mice, adenomas of treated Klf4ΔIS mice had suppressed NHEJ and HRR mechanisms, as indicated by reduced Ku70 and Rad51 staining. This study highlights the important role of KLF4 in suppressing the development of colonic neoplasia under different tumor-promoting conditions. Implications: The study demonstrates that KLF4 plays a significant role in the pathogenesis of colorectal neoplasia. Mol Cancer Res; 14(4); 385–96. ©2016 AACR.

Colorectal cancer is one of the leading causes of cancer mortality in Western civilization. Studies have shown that colorectal cancer arises as a consequence of the modification of genes that regulate important cellular functions. Deregulation of the WNT and RAS/MAPK/PI3K signaling pathways has been shown to be important in the early stages of colorectal cancer development and progression. Krüppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in the proliferating intestinal crypt epithelial cells. Previously, we showed that KLF5 is a mediator of RAS/MAPK and WNT signaling pathways under homeostatic conditions and that it promotes their tumorigenic functions during the development and progression of intestinal adenomas. Recently, using an ultrahigh-throughput screening approach we identified a number of novel small molecules that have the potential to provide therapeutic benefits for colorectal cancer by targeting KLF5 expression. In the current study, we show that an improved analogue of one of these screening hits, ML264, potently inhibits proliferation of colorectal cancer cells in vitro through modifications of the cell-cycle profile. Moreover, in an established xenograft mouse model of colon cancer, we demonstrate that ML264 efficiently inhibits growth of the tumor within 5 days of treatment. We show that this effect is caused by a significant reduction in proliferation and that ML264 potently inhibits the expression of KLF5 and EGR1, a transcriptional activator of KLF5. These findings demonstrate that ML264, or an analogue, may hold a promise as a novel therapeutic agent to curb the development and progression of colorectal cancer. Mol Cancer Ther; 15(1); 72–83. ©2015 AACR.

Krüppel-like factor 4 (KLF4; also known as gut-enriched Krüppel-like factor or GKLF) is known to exhibit checkpoint function during the G1/S and G2/M transitions of the cell cycle. The mechanism by which KLF4 exerts these effects is not fully established. Here we investigated the expression profile of KLF4 in an inducible system over a time course of 24 h. Using oligonucleotide microarrays, we determined that the fold changes relative to control in expression levels of KLF4 exhibited a time-dependent increase from 3- to 20-fold between 4 and 24 h following KLF4 induction. During this period and among a group of 473 cell cycle regulatory genes examined, 96 were positively correlated and 86 were negatively correlated to KLF4's expression profile. Examples of upregulated cell cycle genes include those encoding tumor suppressors such as MCC and FHIT, and cell cycle inhibitors such as CHES1 and CHEK1. Examples of downregulated genes include those that promote the cell cycle including several cyclins and those required for DNA replication. Unexpectedly, several groups of genes involved in macromolecular synthesis, including protein biosynthesis, transcription, and cholesterol biosynthesis, were also significantly inhibited by KLF4. Thus, KLF4 exerts a global inhibitory effect on macromolecular biosynthesis that is beyond its established role as a cell cycle inhibitor.

Wnts are morphogens with well recognized functions during embryogenesis. Aberrant Wnt signaling has been demonstrated to be important in colorectal carcinogenesis. However, the role of Wnt in regulating normal intestinal epithelial cell proliferation is not well established. Here we determine that Wnt11 is expressed throughout the mouse intestinal tract including the epithelial cells. Conditioned media from Wnt11-secreting cells stimulated proliferation and migration of IEC6 intestinal epithelial cells. Co-culture of Wnt11-secreting cells with IEC6 cells resulted in morphological transformation of the latter as evidenced by the formation of foci, a condition also accomplished by stable transfection of IEC6 with a Wnt11-expressing construct. Treatment of IEC6 cells with Wnt11 conditioned media failed to induce nuclear translocation of beta-catenin but led to increased activities of protein kinase C and Ca(2+)/calmodulin-dependent protein kinase II. Inhibition of protein kinase C resulted in a decreased ability of Wnt11 to induce foci formation in IEC6 cells. Finally, E-cadherin was redistributed in Wnt11-treated IEC6 cells, resulting in diminished E-cadherin-mediated cell-cell contact. We conclude that Wnt11 stimulates proliferation, migration, cytoskeletal rearrangement, and contact-independent growth of IEC6 cells by a beta-catenin-independent mechanism. These findings may help understand the molecular mechanisms that regulate proliferation and migration of intestinal epithelial cells.

Background & Aims: Familial adenomatous polyposis because of germline mutation of the adenomatous polyposis coli gene is characterized by development of colorectal adenomas and, ultimately, colorectal cancer. The usefulness of colorectal mucosal compounds to predict the effect on adenoma development of primary chemoprevention with the nonsteroidal anti-inflammatory drug sulindac was evaluated. Methods: A randomized, double-blind, placebo-controlled study of 41 subjects genotypically affected with familial adenomatous polyposis but phenotypically unaffected was conducted. Patients received either sulindac or placebo for 48 months, and development of new adenomas was evaluated. The levels of 5 prostanoids, ornithine decarboxylase, and polyamines were measured serially in normal-appearing rectal mucosa. Results: There were no statistically significant differences between treatment groups in baseline levels of prostanoids, ornithine decarboxylase, or polyamines. At conclusion of the study, 4 of 5 prostaglandin levels were statistically significantly lower in the sulindac group than in the placebo group. Among the subset of patients taking sulindac, 3 of 5 prostaglandin levels were statistically significantly lower in patients who were polyp free than in those who developed polyps. By contrast, there were no statistically significant differences in ornithine decarboxylase or polyamines between treatment groups or in those on sulindac who were polyp free compared with those who developed polyps. Conclusions: Colorectal mucosal prostaglandin levels, but not ornithine decarboxylase or polyamines, may be valuable biomarkers to assess appropriate drug dosage and medication compliance in patients undergoing primary chemoprevention therapy with sulindac. Reduction of mucosal prostaglandin levels may be necessary to achieve chemopreventive benefit from this agent.

The gut-enriched Krüppel-like factor (KLF4) and the ligand-bound thyroid hormone receptor (TR) have each been shown to play a critical role in mammalian gut development and differentiation. We investigated an interrelationship between these two presumably independent pathways using the differentiation marker gene, intestinal alkaline phosphatase (IAP). Transient transfections were performed in Cos-7 cells using luciferase reporter plasmids containing a 2.5 kb segment of the proximal human IAP 5' regulatory region, as well as multiple deletions. Cells were cotransfected with TR and/or KLF4 expression vectors and treated+/-100 nmol/L thyroid hormone (T3). IAP reporter gene transactivation was increased independently by KLF4 (ninefold) and ligand-bound TR beta 1 (sevenfold). Cells cotransfected with KLF4 and TR beta 1 in the presence of T3 showed synergistic activation (70-fold). A similar pattern was seen with the other T3 receptor isoform, TR alpha 1. The synergistic effect was lost with deletions of the T3 and KLF4 response elements in the IAP promoter and was completely or partially abolished in the case of mutant KLF4 expression vectors. The thyroid hormone receptor complex and KLF4 synergistically activate the enterocyte differentiation marker gene IAP, suggesting a previously unrecognized interrelationship between these two transcription factor pathways.

Krüppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor, the expression of which is enriched in the postmitotic cells of the intestinal epithelium. KLF4 is a target gene of the tumor suppressor adenomatous polyposis coli (APC). We sought to determine the role of KLF4 in suppressing the tumorigenecity of RKO colon cancer cells, which do not express KLF4. We utilized an established system in RKO cells, in which an inducible promoter controls expression of KLF4. Four independent assays were used to assess the effects of KLF4 induction on tumor cells. We find that KLF4 overexpression reduces colony formation, cell migration and invasion, and in vivo tumorigenecity. The mechanism of action of KLF4 does not involve apoptosis. These findings, along with our previous findings that KLF4 induces G1/S arrest, suggest that KLF4 is a cell cycle checkpoint protein that can reduce tumorigenecity of colon cancer cells.

Krüppel-like factor 4 (KLF4) is an epithelially enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Constitutive expression of KLF4 inhibits G1/S transition of the cell cycle but the manner by which it accomplishes this effect is unclear. To better understand the biochemical function of KLF4, we identified its target genes using cDNA microarray analysis in an established human cell line containing inducible KLF4. RNA extracted from induced and control cells were hybridized differentially to microarray chips containing 9600 human cDNAs. In all, 84 genes with significantly increased expression and 107 genes with significantly reduced expression due to KLF4 induction were identified. The affected genes are sorted to several clusters on the basis of functional relatedness. A major cluster belongs to genes involved in cell-cycle control. Within this cluster, many up-regulated genes are inhibitors of the cell cycle and down-regulated genes are promoters of the cell cycle. Another up-regulated gene cluster includes nine keratin genes, of which seven are located in a specific region on chromosome 12. The results indicate that KLF4 is involved in the control of cell proliferation and does so by eliciting changes in expression of numerous cell-cycle regulatory genes in a concerted manner. Furthermore, KLF4 regulates expression of a group of epithelial-specific keratin genes in a manner consistent with a potential locus control region function.

APC gene mutation is an early alteration in most colorectal tumors. In an attempt to determine its role in tumor development, we asked whether reintroducing wild-type APC into colorectal cancer cells with mutant APC affected cell cycle progression.Using transient transfection, a plasmid containing the APC complementary DNA and DNA encoding the green fluorescent protein was expressed in SW480 cells. In addition, several other constructs were co-expressed with APC to determine their combined effects.We report that colorectal cancer cell lines transfected with wild-type APC arrest in the G(1)- phase of the cell cycle and that this arrest is abrogated by cotransfecting constitutively active beta-catenin or cyclin D1 and cMYC together. This APC-induced cell cycle arrest involves the disruption of beta-catenin-mediated transcription and depends on components of the G(1)/S regulatory machinery, as overexpression of E1a or E2F-1, -2, or -3 overrides the G(1) arrest. Consistent with this, APC transfection inhibits RB phosphorylation and reduces levels of cyclin D1.Our results suggest that APC functions upstream of RB in the G(1)/S regulatory pathway, cyclin D1 and cMYC affect APC-mediated arrest equivalently to oncogenic beta-catenin, and most colon tumors disrupt control of G(1)/S progression by APC mutation.

Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Kruppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.

A large body of research has contributed to our understanding of the pathophysiology of diabetic nephropathy. Yet, many questions remain regarding the progression of a disease that accounts for nearly half the patients entering dialysis yearly. Several murine models of diabetic nephropathy secondary to Type 2 diabetes mellitus (T2DM) do exist, and some are more representative than others, but all have limitations. In this study, we aimed to identify a new mouse model of diabetic nephropathy secondary to T2DM in a previously described T2DM model, the MKR (MCK-KR-hIGF-IR) mouse. In this mouse model, T2DM develops as a result of functional inactivation of insulin-like growth factor-1 receptor (IGF-1R) in the skeletal muscle. These mice are lean, with marked insulin resistance, hyperinsulinemia, hyperglycemia, and dyslipidemia and thus are representative of nonobese human T2DM. We show that the MKR mice, when under stress (high-fat diet or unilateral nephrectomy), develop progressive diabetic nephropathy with marked albuminuria and meet the histopathological criteria as defined by the Animal Models of Diabetic Complications Consortium. Finally, these MKR mice are fertile and are on a common background strain, making it a novel model to study the progression of diabetic nephropathy.

Kruppel-like factor 5 (KLF5) is a zinc finger transcription factor that is highly expressed in the crypt epithelial cells of the intestine and plays a critical role in regulating proliferation of both normal intestinal epithelial cells and colorectal cancer cells. Stability of the KLF5 is mediated by proteasomal degradation via phosphorylation by glycogen synthase kinase 3β (GSK3β) and recognition by F-box and WD repeat domain-containing 7 (FBW7) of a phosphodegron sequence surrounding serine 303 in KLF5. A genomic analysis of colorectal cancer tissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence. We hypothesized that due to its close proximity to the phosphodegron sequence, the P301S mutation may affect signaling that is involved in proper KLF5 degradation. We demonstrated that the P301S KLF5 mutant has a longer half-life than wild type (WT) KLF5. Furthermore, P301S KLF5 has a higher transcriptional activity than WT KLF5 as demonstrated by luciferase assays using cyclin D1 and CDC2 promoter constructs. In contrast to WT KLF5, P301S KLF5 does not physically interact with FBW7α. Concomitantly, the P301S KLF5 mutant displays reduced levels of phosphorylation at serine 303 in comparison with WT KLF5. These results of our study indicate that amino acid residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence by FBW7α and that the P301S mutation inhibits this recognition, leading to a degradation-resistant protein with elevated levels and enhanced transcriptional activity. These findings raise a potentially oncogenic role for the P301S KLF5 mutant in colorectal cancer.

Gut-enriched Krüppel-like factor (GKLF or KLF4) is a zinc finger-containing, epithelial-specific transcription factor, that functions as a suppressor of cell proliferation. We previously showed that GKLF expression is decreased in intestinal and colonic adenomas, respectively, from multiple intestinal neoplasia (Min) mice and familial adenomatous polyposis (FAP) patients. This study shows that GKLF is induced upon activation of the adenomatous polyposis coli (APC) gene. However, among several human colon cancer cell lines surveyed, expression of GKLF is lowest in RKO, a line with wild-type APC and beta-catenin. RKO contains a mutated allele that encodes the putative tumor suppressor homeodomain protein, CDX2. We show that wild-type CDX2 activates the GKLF promoter and that the mutated CDX2 has a dominant negative effect on wild-type function. Our results may help explain the exceedingly low levels of GKLF expression detected in this cell line, which may in turn contribute to the tumor phenotype.

The gut-enriched Krüppel-like factor (GKLF) is a newly identified zinc finger-containing transcription factor. Recent studies indicate that GKLF binds to a core DNA sequence of 5'-(G/A)(G/A)GG(C/T)G(C/T)-3', which is found in an endogenous cis element, the basic transcription element (BTE) of the cytochrome P-450IA1 (CYP1A1) promoter. The present study characterizes the ability of GKLF to regulate CYP1A1 expression. By electrophoretic mobility gel shift assay (EMSA) and methylation interference assay, GKLF was found to bind BTE in a manner similar to several other transcription factors known to interact with BTE including Sp1 and BTEB. Cotransfection studies in Chinese hamster ovary cells showed that GKLF inhibited the CYP1A1 promoter in a dose- and BTE-dependent manner. The same experiments also revealed that BTE was responsible for a significant portion of the CYP1A1 promoter activity. EMSA of nuclear extracts from Chinese hamster ovary cells showed that Sp1 and Sp3 were two major proteins that interacted with BTE. Additional cotransfection studies showed that GKLF inhibited Sp1-mediated activation of the CYP1A1 promoter. In contrast, GKLF enhanced Sp3-dependent suppression of the same promoter. Moreover, the ability of GKLF to inhibit Sp1-dependent transactivation was in part due to physical interaction of the two proteins. These findings indicate that GKLF is a negative regulator of the CYP1A1 promoter in a BTE-dependent fashion and that this inhibitory effect is in part mediated by physical interaction with Sp1.