The intestinal epithelium is a tissue that undergoes continuous self-renewal initiated at the bottom of the crypts, which harbor the intestinal stem cell (ISC) pool. The ISC pool is sub-divided into crypt base columnar (CBC) cells at the crypt bottom and label retention cells (LRC) at position +4 from the crypt bottom. CBC cells are marked by Leucine-rich repeat-containing G-protein coupled receptor (Lgr5) while LRC cells are identified by several markers including Bmi1, mTert, Hopx, Lrig1, and Sox9. Krüppel-like factors (KLFs) belong to a family of transcription factors that exert important physiological function in various tissues. In the intestine, KLF4 is predominantly expressed in the terminally differentiated, non-proliferating cells lining the villus. Its deletion in the adult mouse intestine results in perturbed homeostasis. In contrast, KLF5 is expressed in actively proliferating cells of the intestinal crypt, including CBC cells and transit amplifying (TA) cells. We recently investigated the effect of Klf5 deletion specifically from the Lgr5-expressing CBC cells in adult mouse intestine using an inducible Cre recombinase system. Shortly (3–5 days) after Cre induction, proliferation of both CBC and TA cells ceased, which was accompanied by an increase in apoptosis in the crypt. Beginning at two weeks following Cre induction, both Klf5 expression and proliferation re-appeared but without the re-emergence of Lgr5-positive CBC cells, which were eventually depleted by four months following induction. These findings indicate that KLF5 plays an important role in regulating proliferation and survival of CBC stem cells in the intestine.
intestinal epithelium; stem cell; Lgr5; Krüppel-like factors
Cells engage sophisticated programs of DNA damage response (DDR) and repair to guard against genetic mutations. While there is significant knowledge concerning DDR in interphase cells, much less is known about these processes in mitosis. Direct interaction between MDC1, a master DDR organizer, and a marker of DNA damage, histone γH2AX, is required to trigger robust repair. Here we show that the DNA damage-induced interaction between MDC1 and γH2AX is attenuated in mitosis. Furthermore, inhibition in the activity of the core mitotic regulator CDK1, either by pharmacological inhibition or siRNA attenuation, enhances MDC1-γH2AX colocalization in mitosis. Our findings offer key new insights into how DDR is controlled during mitosis.
Mitosis; CDK1; MDC1; γH2AX; DDR; DSB
The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the NIH’s public domain, the Molecular Libraries Probe Production Centers Network (MLPCN) library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was performed in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC50<10 µM for KLF5 inhibition and EC50>10 µM for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized based on computational, Western blot, and cell viability analyses. Both of these compounds and two newly-synthesized structural analogs of CID 5951923 significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results demonstrate the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5.
Colorectal cancer; KLF5; Ultrahigh-throughput screen; Luciferase; Cell viability; Small-molecule compounds
Background & Aims
Krüppel-like factor 5 (KLF5) is transcription factor that is expressed by dividing epithelial cells of the intestinal epithelium. KLF5 promotes proliferation in vitro and in vivo and is induced by mitogens and various stress stimuli. To study the role of KLF5 in intestinal epithelial homeostasis, we examined the phenotype of mice with conditional deletion of Klf5 in the gut.
Mice were generated with intestinal-specific deletion of Klf5 (Vil-Cre;Klf5fl/fl).
Morphological changes in the small intestine and colon were examined by immunohistochemistry, immunoblotting, and real-time PCR.
Klf5 mutant mice were born at a normal Mendelian ratio but had high mortality compared to controls. Complete deletion of Klf5 from the intestinal mucosa resulted in neonatal lethality that corresponded with an absence of epithelial proliferation. Variegated intestinal-specific deletion of Klf5 in adult mice resulted in morphological changes that included a regenerative phenotype, impaired barrier function, and inflammation. Adult mutant mice exhibited defects in epithelial differentiation and migration. These changes were associated with reduced expression of Cdx 1, Cdx2, and Eph and ephrin signaling proteins. Concomitantly, Wnt signaling to β-catenin was reduced. Proliferation in regenerative crypts was associated with increased expression of the progenitor cell marker Sox9.
Deletion of Klf5 in the gut epithelium of mice demonstrated that KLF5 maintains epithelial proliferation, differentiation, and cell positioning along the crypt radial axis. Morphological changes that occur with deletion of Klf5 are associated with disruption of canonical Wnt signaling and increased expression of Sox9.
intestinal homeostasis; gastrointestinal development; genetics; GI tract
Recent advancement in understanding the role of both the genetics and molecular pathways in the formation and progression of colorectal cancer allowed the identification of factors that may be targeted for drug discovery. For the past decade various approaches have been developed to target specific steps or components of these pathways in order to prevent the development or progression of colorectal cancer. The innovation and optimization of high-throughput screening methods as well as the recent emphasis from the National Institutes of Health 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 towards pathways affected in colorectal cancer.
High-throughput screen; Colorectal cancer; Molecular pathways; Chemical probes
The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is expressed in the post-mitotic, differentiated epithelial cells lining the intestinal tract and exhibits a tumor suppressive effect on intestinal tumorigenesis. Here we report a role for KLF4 in maintaining homeostasis of intestinal epithelial cells. Mice with conditional ablation of the Klf4 gene from the intestinal epithelium were viable. However, both the rates of proliferation and migration of epithelial cells were increased in the small intestine of mutant mice. In addition, the brush-border alkaline phosphatase was reduced as was expression of ephrine-B1 in the small intestine, resulting in mispositioning of Paneth cells. In the colon of mutant mice, there was a reduction of the differentiation marker, carbonic anhydrase-1, and failure of differentiation of goblet cells. Mechanistically, deletion of Klf4 from the intestine resulted in a general activation of genes in the Wnt pathway and a global reduction in expression of genes encoding regulators of differentiation. Taken together, these data provide new insights into the function of KLF4 in regulating postnatal proliferation, migration, differentiation, and positioning of intestinal epithelial cells and demonstrate an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis in vivo.
KLF4; Wnt; Proliferation; Differentiation; Migration; Paneth Cells; Goblet Cells
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.
Levels of KLF4 were measured by immunohistochemical analysis of a tissue microarray containing 367 independent colon cancer sections. Univariate data analysis was performed 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).
Colon cancer tissues had significantly overall lower KLF4 levels compared to non-cancer tissues (P < 0.0001). Using logistic regression, a trend was noted for decreased odds of KLF4 expression in higher stages of tumors. In both univariate and multivariate analyses, KLF4 was a significant predictor of survival and recurrence.
KLF4 expression is significantly down-regulated in colon cancer and loss of KLF4 is an independent predictor of survival and recurrence.
These findings suggest that KLF4 may serve as a prognostic biomarker for colon cancer.
KLF4; Colon Cancer; Tissue microarray; Survival; Recurrence
The Krüppel-like factor (KLF) family of transcription factors regulates diverse biological processes that include proliferation, differentiation, growth, development, survival, and responses to external stress. Seventeen mammalian KLFs have been identified, and numerous studies have been published that describe their basic biology and contribution to human diseases. KLF proteins have received much attention because of their involvement in the development and homeostasis of numerous organ systems. KLFs are critical regulators of physiological systems that include the cardiovascular, digestive, respiratory, hematological, and immune systems and are involved in disorders such as obesity, cardiovascular disease, cancer, and inflammatory conditions. Furthermore, KLFs play an important role in reprogramming somatic cells into induced pluripotent stem (iPS) cells and maintaining the pluripotent state of embryonic stem cells. As research on KLF proteins progresses, additional KLF functions and associations with disease are likely to be discovered. Here, we review the current knowledge of KLF proteins and describe common attributes of their biochemical and physiological functions and their pathophysiological roles.
Krüppel-Like Factor; Proliferation; Differentiation; Inflammation; Cardiovascular Diseases; Tumorigenesis; Fat Metabolism; Induced Pluripotent Stem Cell
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4.
Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels.
One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data.
These data are not only consistent with previous functional studies of KLF4’s role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4’s functions.
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
The p53 tumor suppressor inhibits the proliferation of cells which undergo prolonged activation of the mitotic checkpoint. However, the function of this antiproliferative response is not well defined. Here we report that p53 suppresses structural chromosome instability following mitotic arrest in human cells. In both HCT116 colon cancer cells and normal human fibroblasts, DNA breaks occurred during mitotic arrest in a p53-independent manner, but p53 was required to suppress the proliferation and structural chromosome instability of the resulting polyploid cells. In contrast, cells made polyploid without mitotic arrest exhibited neither significant structural chromosome instability nor p53-dependent cell cycle arrest. We also observed that p53 suppressed both the frequency and structural chromosome instability of spontaneous polyploids in HCT116 cells. Furthermore, time-lapse videomicroscopy revealed that polyploidization of p53−/− HCT116 cells is frequently accompanied by mitotic arrest. These data suggest that a function of the p53-dependent postmitotic response is the prevention of structural chromosome instability following prolonged activation of the mitotic checkpoint. Accordingly, our study suggests a novel mechanism of tumor suppression for p53, as well as a potential role for p53 in the outcome of antimitotic chemotherapy.
p53; cell cycle arrest; chromosomal instability; DNA damage; mitotic checkpoint; polypoidization
IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca2+/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.
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 Krüppel-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.
Inactivation of the tumor suppressor adenomatous polyposis coli, with the resultant activation of β-catenin, is the initiating event in the development of a majority of colorectal cancers. Krüppel-like factor 5 (KLF5), a proproliferative transcription factor, is highly expressed in the proliferating intestinal crypt epithelial cells. To determine whether KLF5 contributes to intestinal adenoma formation, we examined tumor burdens in ApcMin/+ mice and ApcMin/+/Klf5+/− mice. Compared with ApcMin/+ mice, ApcMin/+/Klf5+/− mice had a 96% reduction in the number of intestinal adenomas. Reduced tumorigenicity in the ApcMin/+/Klf5+/− mice correlated with reduced levels and nuclear localization of β-catenin as well as reduced expression of two β-catenin targets, cyclin D1 and c-Myc. In vitro studies revealed a physical interaction between KLF5 and β-catenin that enhanced the nuclear localization and transcriptional activity of β-catenin. Thus, KLF5 is necessary for the tumor-initiating activity of β-catenin during intestinal adenoma formation in ApcMin/+ mice, and reduced expression of KLF5 offsets the tumor-initiating activity of the ApcMin mutation by reducing the nuclear localization and activity of β-catenin.
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.
Mitotic abnormalities are a common feature of human cancer cells, and recent studies have provided evidence that such abnormalities may play a causative, rather than merely incidental role, in tumorigenesis. One such abnormality is prolonged activation of the mitotic checkpoint, which can be provoked by a number of the gene changes which drive tumor formation. At the same time, antimitotic chemotherapeutics exert their clinical efficacy through the large-scale induction of prolonged mitotic checkpoint activation, indicating that mitotic arrest is influential in both the formation and treatment of human cancer. However, how this influence occurs is not well-understood. In this perspective, we will discuss the current evidence in support of the potential mechanisms by which prolonged activation of the mitotic checkpoint affects both tumorigenesis and antimitotic chemotherapy.
Aneuploidy; Antimitotic chemotherapy; Apoptosis; Cell cycle arrest; Centrosomes; Chromosomal instability; Checkpoint; DNA damage; Mitosis; Polypoidy; Tumorigenesis
The zinc finger-containing transcription factor, Krüppel-like factor 4 (KLF4), inhibits cell proliferation. An in vivo tumor suppressive role for KLF4 is demonstrated 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 (DBZ) to inhibit Notch or siRNA directed against Notch. KLF4 levels were increased in DBZ- or Notch siRNA-treated cells. Conversely, over-expression of Notch in HT29 cells reduced KLF4 levels, suppressed KLF4 promoter activity and increased proliferation rate. Treatment of ApcMin/+ mice with DBZ resulted in a 50% reduction in the number of intestinal adenomas compared to the vehicle-treated group (p < 0.001). Both the normal-appearing intestinal mucosa and adenomas obtained from DBZ-treated ApcMin/+ mice had increased goblet cell numbers and Klf4 staining accompanied by reduced cyclin D1 and Ki67 staining when compared to 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 anti-tumor effect of Notch inhibitors such as DBZ.
KLF4; goblet cells; γ-secretase inhibitor; ApcMin/+ mouse; adenomas
The potential for clinical application of pluripotent embryonic stem cells is immense but hampered by moral and ethical complications. Recent advances in the reprogramming of somatic cells by defined factors to a state that resemble embryonic stem cells have created tremendous excitement in the field. Four factors, Sox2, Oct4, Klf4 and c-Myc, when exogenously introduced into somatic cells, can lead to the formation of induced pluripotent stem (iPS) cells that have the capacity for self-renewal and differentiation into tissues of all three germ layers. In this review, we focus on the role of Krüppel-like factors (KLFs) in regulating somatic cell reprogramming. KLFs are zinc finger-containing transcription factors with diverse biological functions. We first provide an overview of the KLF family of regulatory proteins, paying special attention to the established biological and biochemical functions of KLF4 and KLF5. We then review the role of KLFs in somatic cell reprogramming and delineate the putative mechanism by which KLFs participates the establishment and self-renewal of iPS cells. Further research is likely to provide additional insight into the mechanisms of somatic cell reprogramming and refinement of the technique with which to generate clinically relevant iPS cells.
KLF; iPS cells; ES cells; Reprogramming; Somatic cells
Background & Aims
Krüppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in proliferating crypt cells of the intestinal epithelium. KLF5 has a pro-proliferative effect in vitro and is induced by mitogenic and stress stimuli. To determine whether KLF5 is involved in mediating proliferative responses to intestinal stressors in vivo, we examined its function in a mouse model of transmissible murine colonic hyperplasia (TMCH), which is triggered by colonization of the mouse colon by the bacterial pathogen, Citrobacter rodentium.
Heterozygous Klf5 knockout (Klf5+/−) mice were generated from embryonic stem cells carrying an insertional disruption of the Klf5 gene. Klf5+/− mice or wild-type (WT) littermates were infected with C. rodentium by oral gavage. At various time points post-infection (p.i.), mice were sacrificed and distal colons harvested. Colonic crypt heights were determined morphometrically from sections stained with hematoxylin and eosin. Frozen tissues were stained by immunofluorescence using antibodies against Klf5 and the proliferation marker, Ki67, to determine Klf5 expression and numbers of proliferating cells per crypt.
Infection of WT mice with C. rodentium resulted in a 2-fold increase in colonic crypt heights at 14 days p.i. and was accompanied by a 1.7-fold increase in Klf5 expression. Infection of Klf5+/− mice showed an attenuated induction of Klf5 expression, and hyperproliferative responses to C. rodentium were reduced in the Klf5+/− animals as compared to WT littermates.
Our study demonstrates that Klf5 is a key mediator of crypt cell proliferation in the colon in response to pathogenic bacterial infection.
Background & Aims
Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that regulates cell proliferation. Oncogenic KRAS mutations are commonly found in colorectal cancers. We aimed to determine whether KLF5 mediates KRAS functions during intestinal tumorigenesis.
The effects of KLF5 on proliferation and transformation were examined in IEC-6 intestinal epithelial cells stably transfected with an inducible KRASV12G. KLF5 expression was examined in intestinal tumors derived from transgenic mice expressing KRASV12G under a villin promoter and in human colorectal cancers with mutated KRAS.
Induction of KRASV12G in IEC-6 cells resulted in increased expression of KLF5, accompanied by an increased rate of proliferation and anchorage-independent growth. Inhibition of KLF5 expression by MEK inhibitors or KLF5-specific small interfering RNA (siRNA) reduced proliferation and anchorage-independent growth despite KRASV12G induction. Human colorectal cancer cell lines with mutated KRAS contained high levels of KLF5 and reduction of KLF5 by MEK inhibitors or KLF5 siRNA also led to reduced proliferation and transformation. In vivo, both intestinal tumors derived from mice transgenic for villin-KRASV12G and human primary colorectal cancers with mutated KRAS contained high levels of KLF5 and increased staining of the proliferative marker, Ki67.
Elevated levels of KLF5 protein are strongly correlated with activating KRAS mutations in intestinal tumors both in vitro and in vivo. Inhibition of KLF5 expression in these tumor cells resulted in significantly reduced rates of proliferation and transforming activities. We conclude that KLF5 is an important mediator of oncogenic KRAS transforming functions during intestinal tumorigenesis.
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.
Colorectal cancer (CRC) is a major cause of morbidity and mortality from cancers in the United States. Recent studies have revealed the paradigm in which sequential genetic changes (mutations) result in the progression from normal colonic tissues to frank carcinoma. In particular, the study of hereditary colorectal cancer and polyposis syndromes such as familial adenomatous polyposis and hereditary nonpolyposis colon cancer has contributed enormously to the understanding of the pathogenesis of CRC. Here we describe some of the common genetic pathways in CRC and the mechanisms of action for some of the key genes involved in the formation of CRC. The understanding of the genetic pathways and functions in CRC may lead to the development of novel therapeutic approaches for treating this deadly disease.
Mitosis is a crucial part of the cell cycle. A successful mitosis requires the proper execution of many complex cellular behaviors. Thus, there are many points at which mitosis may be disrupted. In cancer cells, chronic disruption of mitosis can lead to unequal segregation of chromosomes, a phenomenon known as chromosomal instability. A majority of colorectal tumors suffer from this instability, and recent studies have begun to reveal the specific ways in which mitotic defects promote chromosomal instability in colorectal cancer.
The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently down-regulated in colorectal cancer. Previous studies showed that the expression of KLF4 was activated by the colorectal cancer tumor suppressor adeno-matous polyposis coli (APC) and that KLF4 repressed the Wnt/β-catenin pathway. Here, we examined whether KLF4 plays a role in modulating intestinal tumorigenesis by comparing the tumor burdens in mice heterozygous for the ApcMin allele (ApcMin/+) and those heterozygous for both the ApcMin and Klf4 alleles (Klf4+/−/ApcMin/+). Between 10 and 20 weeks of age, Klf4+/−/ApcMin/+ mice developed, on average, 59% more intestinal adenomas than ApcMin/+ mice (P < 0.0001). Immunohistochemical staining showed that Klf4 protein levels were lower in the normal-appearing intestinal tissues of Klf4+/−/ApcMin/+ mice compared with wild-type, Klf4+/−, or ApcMin/+ mice. In contrast, the levels of β-catenin and cyclin D1 were higher in the normal-appearing intestinal tissues of Klf4+/−/ApcMin/+ mice compared with the other three genotypes. Klf4 levels were further decreased in adenomas from both ApcMin/+ and Klf4+/−/ApcMin/+ mice compared with their corresponding normal-appearing tissues. Reverse transcription-PCR showed an inverse correlation between adenoma size and Klf4 mRNA levels in both Klf4+/−/ApcMin/+ and ApcMin/+ mice. There was also a progressive loss of heterozygosity of the wild-type Apc allele in adenomas with increasing size from Klf4+/−/ApcMin/+ and ApcMin/+ mice. Results from this study show that KLF4 plays an important role in promoting the development of intestinal adenomas in the presence of ApcMin mutation.