Mandayam O. Nandan

Krüppel-like factors 4 and 5: The yin and yang regulators of cellular proliferation

ABSTRACTKrüppel-like factors (KLFs) are evolutionarily conserved zinc finger-containing transcription factors with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. KLF4 and KLF5 are two closely related members of the KLF family that have a similar tissue distribution in embryos and adults. However, the two KLFs often exhibit opposite effects on regulation of gene transcription, despite binding to similar, if not identical, cis-acting DNA sequences. In addition, KLF4 and 5 exert contrasting effects on cell proliferation in many instances; while KLF4 is an inhibitor of cell growth, KLF5 stimulates proliferation. Here we review the biological properties and biochemical mechanisms of action of the two KLFs in the context of growth regulation.

Krüppel-like factor 5 promotes mitosis by activating the cyclin B1/Cdc2 complex during oncogenic Ras-mediated transformation

We previously showed that the zinc finger‐containing transcription factor Krüppel‐like factor 5 (KLF5) is important in mediating transformation by oncogenic H‐Ras through induction of cyclin D1 expression and acceleration of the G1/S transition of the cell cycle. Here we present evidence of a role for KLF5 in accelerating mitotic entry in H‐Ras‐transformed NIH3T3 fibroblasts. When compared with non‐transformed parental NIH3T3 cells, H‐Ras‐transformed fibroblasts exhibit an increase in mitotic index, levels of cyclin B1 and Cdc2, and cyclin B1/Cdc2 kinase activity. Inhibition of KLF5 expression in H‐Ras‐transformed cells with KLF5‐specific small interfering RNA (siRNA) results in a decrease in each of the aforementioned parameters, with a concomitant reduction in the transforming potential of the cells. Conversely, over‐expression of KLF5 in NIH3T3 cells leads to an increase in the promoter activity of the genes encoding cyclin B1 and Cdc2. These results indicate that KLF5 accelerates mitotic entry in H‐Ras‐transformed cells by transcriptionally activating cyclin B1 and Cdc2, which leads to an increase in cyclin B1/Cdc2 kinase activity. Extending our previous observation that KLF5 activates cyclin D1 transcription to promote G1/S transition, our current results further support a crucial function for KLF5 in mediating cellular transformation caused by oncogenic H‐Ras.

Human Cancer Cells Commonly Acquire DNA Damage during Mitotic Arrest

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.

All-trans retinoic acid inhibits proliferation of intestinal epithelial cells by inhibiting expression of the gene encoding Krüppel-like factor 5

Retinoids are known inhibitors of epithelial cell proliferation. Previous studies indicate that Krüppel‐like factor 5 (KLF5) is a pro‐proliferative transcription factor. Here, we examined the effect of all‐trans retinoid acid (ATRA) on proliferation of the intestinal epithelial cell line, IEC6. Treatment of IEC6 cells with ATRA inhibited their proliferation due to G1 cell cycle arrest. This inhibition was correlated with a decrease in the levels of KLF5 mRNA and promoter activity. In contrast, constitutive expression of KLF5 in stably transfected IEC6 cells with a KLF5‐expressing plasmid driven by a viral promoter abrogated the growth inhibitory effect of ATRA. Moreover, ATRA inhibited proliferation of several human colon cancer cell lines with high levels of KLF5 expression but not those with low levels of KLF5 expression. Our results indicate that KLF5 is a potential mediator for the inhibitory effect of ATRA on intestinal epithelial cell proliferation.

Krüppel-like factor 4 prevents centrosome amplification following γ-irradiation-induced DNA damage

Centrosome duplication is a carefully controlled process in the cell cycle. Previous studies indicate that the tumor suppressor, p53, regulates centrosome duplication. Here, we present evidence for the involvement of the mammalian Krüppel-like transcription factor, KLF4, in preventing centrosome amplification following DNA damage caused by γ-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+/+), displayed stable centrosome numbers following γ-irradiation. In contrast, HCT116 cells null for the p53 alleles (HCT116 p53−/−) exhibited centrosome amplification after irradiation. In the latter cell line, KLF4 was not activated following γ-irradiation due to the absence of p53. However, centrosome amplification could be suppressed in irradiated HCT116 p53−/− cells by conditional induction of exogenous KLF4. Conversely, in a HCT116 p53+/+ cell line stably transfected with small hairpin RNA (shRNA) designed to specifically inhibit KLF4, γ-irradiation induced centrosome amplification. In these cells, the inability of KLF4 to become activated in response to DNA damage was directly associated with an increase in cyclin E level and Cdk2 activity, both essential for regulating centrosome duplication. Cotransfection experiments showed that KLF4 overexpression suppressed the promoter activity of the cyclin E gene. The results of this study demonstrated that KLF4 is both necessary and sufficient in preventing centrosome amplification following γ-radiation-induced DNA damage and does so by transcriptionally suppressing cyclin E expression.

Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined ApcMin and KRASV12 mutations

BackgroundBoth mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene and activation of the KRAS oncogene are implicated in the pathogenesis of colorectal cancer. Mice harboring a germline ApcMinmutation or intestine-specific expression of the KRASV 12gene have been developed. Both mouse strains develop spontaneous intestinal tumors, including adenoma and carcinoma, though at a different age. The zinc finger transcription factor Krüppel-like factor 5 (KLF5) has previously been shown to promote proliferation of intestinal epithelial cells and modulate intestinal tumorigenesis. Here we investigated the in vivo effect of Klf5 heterozygosity on the propensity of ApcMin/KRASV 12double transgenic mice to develop intestinal tumors.ResultsAt 12 weeks of age, ApcMin/KRASV 12mice had three times as many intestinal tumors as ApcMinmice. This increase in tumor number was reduced by 92% in triple transgenic ApcMin/KRASV 12/Klf5+/- mice. The reduction in tumor number in ApcMin/KRASV 12/Klf5+/- mice was also statistically significant compared to ApcMinmice alone, with a 75% decrease. Compared with ApcMin/KRASV 12, tumors from both ApcMin/KRASV 12/Klf5+/- and ApcMinmice were smaller. In addition, tumors from ApcMinmice were more distally distributed in the intestine as contrasted by the more proximal distribution in ApcMin/KRASV 12and ApcMin/KRASV 12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both ApcMinand ApcMin/KRASV 12mice but were attenuated in ApcMin/KRASV 12/Klf5+/- mice. The levels of β-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of ApcMin/KRASV 12/Klf5+/- mice when compared to ApcMin/KRASV 12mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of ApcMin/KRASV 12mice and modestly reduced in ApcMin/KRASV 12/Klf5+/- mice. Tumor tissues displayed higher levels of both Klf5 and β-catenin, irrespective of the mouse genotype from which tumors were derived.ConclusionsResults of the current study confirm the cumulative effect of Apc loss and oncogenic KRAS activation on intestinal tumorigenesis. The drastic reduction in tumor number and size due to Klf5 heterozygosity in ApcMin/KRASV 12mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.

Krüppel-like factor 5 mediates transmissible murine colonic hyperplasia caused by Citrobacter rodentium infection.

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 triggered by colonization of the mouse colon by the bacteria Citrobacter rodentium. METHODS 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 postinfection, mice were killed and distal colons were harvested. Colonic crypt heights were determined morphometrically from sections stained with H&E. 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. RESULTS Infection of WT mice with C rodentium resulted in a 2-fold increase in colonic crypt heights at 14 days postinfection 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 with WT littermates. CONCLUSION Our study shows that Klf5 is a key mediator of crypt cell proliferation in the colon in response to pathogenic bacterial infection.