• KLF5;
  • transcription factor;
  • tumour suppressor;
  • oncogene;
  • context dependent


  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular and Biological Roles of KLF5
  5. Molecular Mechanisms of KLF5 Target Gene Regulation
  6. Conflicting Roles of KLF5 in Carcinogenesis
  7. Acknowedgements
  8. References

The mechanisms by which cells control their growth and behavioral identities are complex and require adaptability to environmental changes. Transcription factors act as master controllers of many of these pivotal points through their ability to influence the expression of many thousands of downstream genes, and increasingly research is showing that transcription factor regulation of target genes can change in response to environmental stimuli and cell type such that their function is not prescribed but rather context-dependent. Krüppel like factor 5 (KLF5) is an example of such a transcription factor, where evidence of disparate effects on cell growth and differentiation in normal and transformed tissue are clear. Here we present and discuss the literature covering the differential roles of KLF5 in particular tissues and cancer states, and the mechanisms by which these differences are effected through the regulation of KLF5 protein function in response to different cellular states and the direct effect on target gene expression. © 2013 IUBMB Life, 65(12):999–1011, 2013


  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular and Biological Roles of KLF5
  5. Molecular Mechanisms of KLF5 Target Gene Regulation
  6. Conflicting Roles of KLF5 in Carcinogenesis
  7. Acknowedgements
  8. References

The Krüppel-Like Factor Family

Krüppel-like factor 5 (KLF5) belongs to the family of Krüppel-like transcription factors, of which 17 members have been identified to date. Members of this family have been implicated in an extensive array of biological processes including embryonic development, control of cellular proliferation and differentiation, stress response, and many others [1]. KLF family members are closely related to the Sp family of transcription factors but are distinguished by a unique pattern of three cysteine-2/histidine-2 zinc finger motifs separated by seven conserved amino acids at the carboxy terminal of the proteins [2, 3]. These zinc fingers comprise the DNA-binding domain of the KLF transcription factors, which all bind to similar elements within GC-rich promoter sequences of target genes. Outside of the DNA-binding domain, the KLF family members display relatively low conservation, although phylogenetic analysis has identified distinct subgroups within the KLF family [1]. Specificity of action for members of the KLF family is determined in part by differences in the amino-terminal transactivation and protein interaction domains and also by tissue-specific regulation of expression [2].

Krüppel-Like Factor 5

KLF5 was originally cloned as the Basic transcription element binding protein 2 (BTEB2) gene [4]. Prior to the reclassification of KLF family nomenclature KLF5 was known as Intestinal krüppel-like factor (IKLF) due to high levels of protein expression in intestinal epithelial cells [5]. Interest in this gene has greatly expanded in recent years as studies have revealed emerging functions in a variety of cell types and biological systems, with homologues identified in numerous vertebrate species. KLF5 is expressed in a range of epithelial cell types, as well as vascular smooth muscle cells (VSMCs), adipocytes, neural cells, and leukocytes [6-10]. Although the highest level of expression is found in the digestive system, KLF5 is also expressed in the reproductive organs, pancreas, prostate, skeletal muscle and lung [11]. Accordingly, in vivo studies have demonstrated essential roles for this protein in biological processes such as embryonic development, cardiovascular remodeling, adipogenesis, inflammatory stress responses, and intestinal development. KLF5 can function as a transcriptional activator or repressor, a promoter or inhibitor of cell growth and survival, and either an oncogene or tumor suppressor depending on the cellular and genetic context in which it operates [11].

Cellular and Biological Roles of KLF5

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular and Biological Roles of KLF5
  5. Molecular Mechanisms of KLF5 Target Gene Regulation
  6. Conflicting Roles of KLF5 in Carcinogenesis
  7. Acknowedgements
  8. References

Proliferation, Survival, and Oncogenic Function

Proliferation and Self-Renewal

Spatial and temporal expression patterns of KLF5 suggest a role in proliferation in many systems. Expression levels of KLF5 are high in actively dividing cell types such as embryonic stem cells (ESCs) or proliferating cells of the intestinal epithelium, and expression decreases on differentiation to terminally mature cells [12, 13]. Accordingly, serum or cytokine stimulation of fibroblasts and smooth muscle cells induces proliferation with a concordant increase in KLF5 expression [5, 14]. KLF5 expression is induced in rodent models of biological stress caused by physical injury, irradiation or bacterial pathogens, suggesting a proliferative and/or migratory role in the body's inflammatory and immune response systems [7, 15-19].

Ectopic expression of KLF5 in a variety of cell types provides supporting evidence for a role in positive regulation of cellular proliferation. KLF5 increases growth rates of intestinal and esophageal epithelial cells, keratinocytes, VSMCs, and NIH-3T3 fibroblasts [20-24]. RNA interference (RNAi) knock-down of Klf5 shows the expected opposite effect with reduced cellular proliferation of keratinocytes and VSMCs [14, 24, 25]. In vivo studies support a growth-promoting role for KLF5 in the intestine, with heterozygous knock-out mice displaying shorter intestinal crypts and villi and decreased thickening of arterial walls, whilst conditional deletion of KLF5 in intestinal epithelium abolishes epithelial cell proliferation and results in neonatal lethality [26]. Ablation or transgenic expression of KLF5 in mouse models of dextran sodium sulfate-induced colitis leads to increased or reduced disease severity respectively due to the essential role for KLF5 in epithelial cell proliferation and migration in the colon [27, 28]. KLF5 also contributes to vasculogenesis and angiogenesis via up-regulation of genes which increase VSMC proliferation and migration, and prevent differentiation, such as VEGFA, MYH10, TAGLN, SERPINE1, and EGR1 [7, 26, 29-31]. Accordingly, rat models of cardiovascular injury have shown that expression of KLF5 leads to enhanced neointima formation due to increased proliferation and migration of VSMCs [23, 25, 32]. In pulmonary arterial hypertension, a disease characterized by enhanced proliferation and suppressed apoptosis of pulmonary VSMCs, ablation of KLF5 in human explant cell models increased expression of the proliferative markers Ki67 and PCNA, and in rodent models delivery of a KLF5 siRNA to pulmonary VSMCs reduced disease severity [33]. In embryonic stem cells, KLF5 plays an essential role in self-renewal via direct regulation of the pluripotency genes OCT4, Nanog, TCL1, and BMP4 (reviewed by Bourillot and Savatier [34]). Ablation of KLF5 through gene targeting induces G1 cell cycle arrest accompanied by induction of spontaneous differentiation, and accordingly homozygous KLF5 null mice result in early embryonic lethality (before e8.5).

Cell Survival

As well as promoting proliferation and self-renewal, KLF5 may also modulate cell survival through inhibition of apoptotic pathways in certain cell types, such as in VSMCs. In vivo studies have demonstrated that Klf5+/− mice have increased apoptosis in vascular lesions, which is likely due to reduced interaction with the pro-apoptotic PARP1 protein whose function is usually directly inhibited by KLF5 binding [35]. Overexpression of KLF5 in a rat model has shown that VSMCs expressing KLF5 have decreased apoptosis on cardiovascular injury associated with reduced cleavage of Caspase-3 [23]. In human pulmonary VSMCs, KLF5 up-regulates expression of the anti-apoptotic Survivin gene [33]. A similar regulation is observed in Acute Lymphocytic Leukemia (ALL) cell lines, where KLF5 interacts with p53 to overcome p53-mediated repression of Survivin, and knock-down of KLF5 accordingly sensitizes cells to apoptosis induced by chemotherapeutic drugs [36]. It has been postulated that this process forms a positive regulatory loop as Survivin has been shown to increase KLF5 expression in VSMCs [37].

In intestinal epithelial cell lines, RNAi knock-down of KLF5 increases sensitivity to fluorouracil (5-FU) induced apoptosis through a p53-independent mechanism [38]. KLF5 contributes to survival in this system via activation of PIM1 kinase, which phosphorylates and inactivates the pro-apoptotic protein BAD. KLF5 conversely induces cell death in esophageal cancer cells through activation of JNK signaling and direct regulation of ASK1, MKK4, and the proapoptotic BAX protein [39], suggesting that KLF5 may have opposite effects on cell survival by mediating the activity of different members of the BCL-2 family.

KLF5 as an Oncogene

Consistent with a role as a promoter of proliferation and survival, KLF5 has been implicated as an oncogene in selected epithelial tissues, with a large number of studies particularly focusing on colorectal cancer. KLF5 acts to mediate responses downstream of the two most frequent classes of mutations found in human colorectal cancer, these being inactivating mutations in the APC gene (a component of the WNT signaling pathway which targets β-catenin for degradation) [40], and mutations in RAS genes (leading to aberrant activation of proliferative signaling pathways) [41]. It has been shown that KLF5 facilitates nuclear localization of β-catenin, which accumulates abnormally downstream of mutant APC, and the KLF5/β-catenin complex subsequently contributes to activation of proliferation-associated target genes such as CCND1 and MYC [42]. Accordingly, haploinsufficiency of KLF5 rescues the intestinal adenoma phenotype seen in Apcmin/+ mice [42]. The function of KLF5 in intestinal tumors is intimately linked with mutations in the KRAS proto-oncogene: in human intestinal cancers harboring KRAS mutations, KLF5 protein expression was found to be elevated in comparison to nontumorous tissues, consistent with oncogenic potential [43]. Accordingly, enforced expression or siRNA knock-down of KLF5 in intestinal cell lines positive for KRAS mutation enhanced or inhibited colony formation respectively [43, 44]. Interestingly, in double transgenic Apcmin/+ and KrasV12/+ mice, which demonstrate more aggressive tumor development than mice with Apcmin/+ alone, haploinsufficiency of KLF5 attenuated the cooperative effect of these mutations with a >90% reduction in tumor formation compared to control littermates [45]. KLF5 may also mediate signaling downstream of another member of the RAS family of proteins, HRAS, as knock-down of KLF5 in HRAS transformed fibroblast cells similarly leads to reduced proliferation and colony growth [46].

Differentiation and Tumor Suppressor Function

Differentiation and Growth Inhibition

Whilst the majority of data points to a growth-promoting and pro-survival role for KLF5 in a number of cell types, there is a growing body of evidence implicating KLF5 as an inducer of differentiation in selected systems. A well characterized example is in adipose tissue, where studies have shown that KLF5 expression is induced in the early stages of differentiation of pre-adipocyte cell lines. In the 3T3-L1 cell line, over-expression of KLF5 stimulates differentiation to adipocytes in a hormone-independent manner, whereas expression of a dominant-negative form of KLF5 inhibits insulin-induced differentiation [6]. In vivo studies support this observation, with heterozygous knock-out mice exhibiting decreased formation of mature adipose tissue due to reduced differentiation [6].

More recent studies have demonstrated essential roles for KLF5 in differentiation of selected epithelial cell systems using mouse models of conditional deletion. Knock-out in bladder epithelium leads to impaired bladder morphogenesis due to a block in terminal differentiation, with concordant microarray analyses implicating regulation of genes with essential roles in epithelial differentiation, such as PPARG and ELF3 [47]. Similarly, conditional ablation in ectodermal cells of the eye causes developmental defects, partly due to enhanced proliferation of ocular epithelial cells [47, 48]. Conditional knock-out of KLF5 in fetal respiratory cells results in neonatal lethality due to defective lung morphogenesis and an accumulation of immature respiratory epithelial cells. Microarray studies performed in lung tissues have demonstrated that KLF5 may contribute to this phenotype through regulation of genes involved in lung morphogenesis (vasculogenesis, smooth muscle cell differentiation, paracrine interactions), and also members of the TGFβ/BMP4 signaling pathways [29, 49]. Consistent with a differentiation-promoting role, functional evidence suggests that KLF5 may act as a tumor suppressor in lung cancer, with conditional deletion resulting in increased formation of cancerous lesions in a mouse model of KRAS-induced lung tumorigenesis [49]. In human non-small cell lung cancer, low KLF5 protein expression is correlated with poorer disease-specific survival, which may in part be due to increased expression of the efflux drug transporter ABCG2 [49].

In the hematopoietic system, we and others have recently demonstrated that KLF5 plays a role in terminal granulocyte differentiation [10, 50]. KLF5 is highly expressed in mature granulocyte populations in both mouse and human tissues, and accordingly siRNA knock-down of KLF5 attenuates granulocyte differentiation of the 32D myeloid cell line in response to G-CSF, and also of the acute promyelocytic leukemia cell line NB4 in response to the chemotherapeutic agent All-trans retinoic acid (ATRA). Consistent with this, in primary mouse hemopoietic cells, inducible knock-out of KLF5 leads to a decrease in peripheral blood myeloid cells. Interestingly loss of KLF5 also affects the homing and engraftment of the hematopoietic stem cell, through loss of Rab5-mediated β1/β2 integrin adhesion molecule expression [51]. KLF5 mRNA expression is reduced in acute myeloid leukemia (AML) samples relative to normal bone marrow controls and mature granulocyte populations, suggesting that down-regulation may contribute to the leukemic block in differentiation [10, 50]. We identified hypermethylation of the KLF5 locus as one mechanism contributing to reduced expression, which is correlated with inferior survival in AML [10, 52], and deletion of the chromosomal region containing KLF5 (13q21-22) has previously been reported in a range of hematopoietic malignancies [53, 54]. Humbert et al. demonstrated that bone marrow expression of KLF5 increases in patients with acute promyelocytic leukemia following treatment with ATRA, consistent with induction of differentiation and potential tumor suppressor function [50].

Other Examples of Tumor Suppressor Function

Some of the strongest evidence for KLF5 acting as a tumor suppressor gene is in prostate epithelium. KLF5 expression is commonly reduced in prostate cancer, with hemi- or homozygous deletion of 13q accounting for this in a large proportion of tumor samples and cell lines [55, 56]. Deletion mapping analysis using human tumor samples, xenograft models, and cancer cell lines identified KLF5 as the only complete gene to lie within the common region of deletion [55, 56]. Other studies have shown down-regulation of KLF5 expression or activity by excess protein ubiquitination and degradation, and increased cytoplasmic localization [57-61]. Enforced expression of KLF5 in prostate cancer cell lines, where the KLF5 protein is actively degraded, inhibits colony formation and enhances differentiation in vitro, and reduces tumor weight in vivo on subcutaneous injection [55, 61]. KLF5 also inhibits growth and promotes apoptosis of prostate cancer cells in response to treatment with estrogen antagonists via interaction with ERβ and CBP, and subsequent upregulation of the pro-apoptotic FOXO1 gene [61]. Consistent with this observation, patients displaying tumor samples positive for protein expression of KLF5, ERβ, or FOXO1 demonstrate improved disease-specific survival rates [61].

There is also substantial evidence for KLF5 acting as a tumor suppressor in breast cancer, with 70% of breast cancer cell lines tested showing reduced KLF5 mRNA levels compared to the BRF-97T non-neoplastic breast epithelial cell line [62]. Mechanisms for this down-regulation included hemizygous deletion and frequent copy number neutral loss of heterozygosity (LOH). One cell line tested also showed a point mutation in the coding sequence of KLF5 (resulting in a methionine to valine amino acid change at codon 294) in addition to hemizygous deletion. KLF5 function is aberrantly affected at the protein level in breast cancer, through excessive protein degradation or enhanced cytoplasmic localization [58, 63]. Loss of the 13q21-q22 genomic region, which includes KLF5 and seven other known genes, has been linked to hereditary breast cancer in families with BRCA1 and BRCA2-negative breast cancer [64]. Functional evidence for KLF5 acting as a tumor suppressor in breast cancer was initially provided by Chen et al., who demonstrated that re-expression of KLF5 in the T47d breast cancer cell line significantly inhibited colony formation of these cells [62]. More recently, it has been reported that KLF5 can inhibit proliferation of ERα-positive breast cancer cell lines in response to estrogen [65].

Molecular Mechanisms of KLF5 Target Gene Regulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular and Biological Roles of KLF5
  5. Molecular Mechanisms of KLF5 Target Gene Regulation
  6. Conflicting Roles of KLF5 in Carcinogenesis
  7. Acknowedgements
  8. References

A number of direct target genes of KLF5 have been identified with roles in cell proliferation, migration, survival, and differentiation (reviewed in Dong and Chen [11]). Earlier publications suggested that KLF5 mainly functions as a transcriptional activator, however, there have been a growing number of examples where it alternatively acts to repress transcription [11]. This is particularly evident in murine ESCs, where studies have demonstrated that 70% of direct target genes are repressed by KLF5 in this tissue [66].

Mechanisms for Target Gene Activation

KLF5 contains a proline-rich, hydrophobic transactivation domain within the N-terminal portion of the protein. Using a series of KLF5 deletion contructs fused to the DNA binding domain of GAL4 in a GAL4 reporter assay system, the transactivation domain was localized to amino acids 239 to 372 [67]. Mutation analysis using a similar reporter system has suggested that the region necessary for transcriptional activation can be further narrowed to 15 specific amino acids within this region (amino acids 324–338) [68]. The mechanism by which these amino acids act to induce transcription of target genes has not yet been elucidated. Direct interaction of KLF5 with other proteins has been shown by coimmunoprecipitation, yeast two-hybrid screening and mass spectrometry (Table 1). These interacting proteins include post-translational modifiers and coactivators or corepressors, which specify the transactivation function of KLF5. KLF5 can interact with basal transcriptional components including the transcription factors TFIIβ, TFIIEβ, TFIIFβ, and the TATA box-binding protein (TBP), however, these do not interact with the proline-rich activation domain [68]. A recent study by Mas et al. identified an atypical transactivation domain within the erythroid KLF1 protein, which shares homology with p53 and activates target gene expression via interaction with the TFIIH general transcription factor [89]. These amino acids are highly conserved in a subset of KLF family members, including amino acids 108 to 127 of KLF5, and it is interesting to speculate that this sequence is similarly important for transcriptional activity of KLF5.

Table 1. KLF5 interacting proteins
FunctionInteracting proteinsReferences
Transcriptional co-regulatorsCBP, CEBPα/β/δ, PIAS1, NF-κB, PPARδ, SREBP1, hhLIM, SMAD2/3/4, FOXO3, MYC, JUN, p53, RAR, NCoR1/2, ERβ, MIZ-1[6, 19, 25, 36, 61, 67, 69-78]
Post-translational modifiers affecting KLF5 functionPKC, HDAC1/2, SET, p300[67, 74, 79-81]
Post-translational modifiers influencing protein stabilityFBW7, WWP1, SMURF2, EFP, TAZ, YAP[58, 82-86]
OtherANP32B, β-catenin, ERα, HIF1α, TFIIB/Eβ/Fβ, KLF5, PARP1, TBP[35, 42, 65, 68, 71, 87, 88]

KLF5 interacts with a range of cofactor proteins to activate expression of target genes which induce specific biological effects in different tissues. In VSMCs, where KLF5 promotes proliferation, the KLF5 protein forms a heterodimer with RARα to activate expression of PDGFA [90]. PDGF-BB stimulation of VSMCs augments the interaction of KLF5 with the cofactor hhLIM, which recruits KLF5 to the promoter of CCNE1 and induces proliferation [25]. In COS-7 fibroblast cells, KLF5 induces cell cycle progression via interaction with the coactivator PIAS1, which enhances expression of the KLF5 target genes CCND1 and CDK1 [69].

Alternatively, in the pre-adipocyte 3T3-L1 cell line, C/EBPβ and C/EBPδ up-regulate expression of KLF5 which then forms complexes with these two C/EBP family members to activate expression of PPARG, promoting adipocyte differentiation [6]. KLF5 also interacts with another C/EBP family member, C/EBPα, in renal epithelial cells [19]. In resting renal epithelial collecting duct cells KLF5 directly activates expression of the cadherin CDH1, however on renal injury KLF5 instead binds to promoters of the genes for CEBPA and the chemoattractants S100a8 and S100a9. The C/EBPα protein then directly interacts with KLF5 to further increase expression of the chemoattractants, which induce an inflammatory response. These observations provide the first evidence that mechanical pressure may act as an external cue to cause a switch in target genes activated by KLF5.

KLF5 can also activate a number of lipid metabolism genes in concert with cotranscription factors; it interacts with SREBP1 to synergistically induce expression of the fatty acid enzyme FASN in prostate cancer cells [91], and it interacts with PPARδ and the coactivator CBP to induce transcription of the lipid oxidation genes CPT1B and UCP2/3 in skeletal muscle cells [72]. A key role for KLF5 in regulating expression of genes involved in energy metabolism, and in adipogenesis, is consistent with the observation that heterozygous knock-out mice are protected from obesity when fed a high-fat diet [72].

Protein Modifications and Transactivation Ability

The KLF5 protein undergoes numerous post-translational modifications (Fig. 1A), which can regulate protein levels by targeting KLF5 for degradation (reviewed in Chen [92]), or alternatively modulate the transactivation ability of KLF5 by altering binding affinity for different co-regulators (Fig. 1). Phosphorylation primarily enhances the transactivation ability of KLF5. Specific phosphorylation of serine 153 downstream of PKC signaling enhances the interaction of KLF5 with the cofactor CBP, leading to increased expression of a GAL4 reporter construct in the HEC-1B endometrial carcinoma cell line [67]. The interaction domain of CBP and KLF5 was mapped to amino acids 1 to 238 of the KLF5 protein, which is N-terminal to the KLF5 transactivation domain [67]. KLF5 can also be phosphorylated on serine 406 in the DNA-binding domain downstream of MEK/ERK and p38 signaling in VSMCs treated with the vasoconstrictive hormone Angiotensin II. This enhances the interaction of KLF5 with the AP-1 component c-JUN, and these proteins subsequently bind to the promoter of CCND1 to cooperatively induce expression and increase proliferation of VSMCs [76]. Interestingly, whilst the phospho-KLF5/c-JUN complex activates CCND1 in this context, this combination was found to have the opposite effect on transcription of p21 [77]. These observations suggest that there may be further interactions of KLF5 with as yet unknown cofactors which determine regulation of these genes in VSMCs. Phosphorylation of KLF5 on serine 406 also enhances interaction with unliganded RARα, which binds to the first zinc finger of the KLF5 DBD, to activate downstream target genes [90, 93]. RAR agonists are thought to act via two mechanisms to disrupt the interaction between KLF5 and RARα and subsequently inhibit VSMC proliferation. Firstly, treatment with the synthetic retinoid Am80 was found to inhibit p38 signaling which usually acts to phosphorylate KLF5, and to induce PI3K/AKT signaling which actively dephosphorylates KLF5, leading to reduced binding of KLF5 with RARα [93]. Secondly, ligand binding to RARα is presumed to effect a conformational change, as an RARα protein lacking the ligand binding domain still bound KLF5 in the presence of the agonist Acyclic retinoid and was able to activate expression of NFKB, whereas the full-length protein did not [90].


Figure 1. Post-translational modifications affecting transactivation function of KLF5. (A) The 457 amino acid (aa) protein contains three main functional domains—a nuclear export signal (NES), a transactivation domain (TAD), and a DNA-binding domain (DBD) consisting of three zinc finger motifs. KLF5 can be modified post-translationally by phosphorylation on S153, S303, T234, and T323 (Ph), SUMOylation on K162 and K209 (Su), acetylation on K369 (Ac), and ubiquitination on residues 323-348 (Ub). Note that Ph* indicates phosphorylation at these residues targets the KLF5 protein for ubiquitin-mediated degradation. Stimuli and signaling pathways which regulate (B) phosphorylation (Ph), (C) acetylation (Ac), and (D) sumoylation (Su) of the KLF5 protein. Dotted arrows represent how modified KLF5 subsequently activates or represses target genes through interaction with regulatory co-factors. For target genes marked with an asterisk, KLF5 has the opposite effect on transcription when unmodified.

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KLF5 can also be activated through acetylation of lysine 369 in its DBD. Although it has been shown that KLF5 directly interacts with the histone acetyltransferase CBP, CBP does not acetylate KLF5 [67]. Instead KLF5 is acetylated by the coactivator p300, and these two proteins then interact to induce expression of downstream target genes [74, 79, 80, 94]. In response to PMA stimulation of VSMCs, the acetyl-KLF5/p300 complex acts to increase expression of PDGFA and enhance cell proliferation, whereas in response to TGFβ stimulation of epithelial keratinocytes, these proteins subsequently recruit other coactivators such as SMADS2/3/4 and FOXO3 to form an activation complex on the promoter of CDKN2B (p15), which inhibits cell proliferation [74, 94]. As a negative regulatory mechanism, the oncogenic regulator SET and the histone deacetylase HDAC1 can bind to the DBD of KLF5 in competition with p300 to prevent acetylation and activation of KLF5 [79, 80]. These interactions also reduce binding of KLF5 to DNA in promoter regions of genes such as PDGFA and SMEMB (MYH10), which consequently inhibits VSMC proliferation. It is interesting to speculate that these proteins may in addition actively deacetylate KLF5, although there has been no experimental evidence provided to date to support this hypothesis.

Mechanisms for Target Gene Repression

Although a vast number of KLF5 target genes are activated by binding of KLF5 to their promoter regions, more recent studies have indicated that KLF5 can equally act as a repressor of transcription through altered binding to DNA control elements or recruitment of co-repressor proteins. A specific repression domain has not yet been identified for KLF5. Other KLF family members have empirically-determined repression domains, for example, KLFs 3, 8, and 12 have consensus sequences which bind the transcriptional repressor CtBP, and KLFs 9, 10, 11, 13, and 16 have hydrophobic consensus sequences for binding the repressor Sin3A [1]. Interestingly, the erythroid-associated KLF member KLF1 also binds Sin3A but it does not contain a similar conserved Sin-Interaction-Domain. Instead it has been shown that KLF1 can interact with Sin3A through its zinc finger DBD [95], and it will be of interest to determine whether the conserved DBD of KLF5 may also act in this manner to achieve repression of target genes.

Switching the Transactivation Function of KLF5

There is increasing evidence to demonstrate that KLF5 can switch from a transcriptional activator to a transcriptional repressor for the same sets of target genes. Parisi et al. used a combined microarray/ChIP-seq approach to demonstrate that a subset of KLF5 target genes were oppositely regulated in ESCs and keratinocytes on KLF5 knock-down [66], consistent with tissue-specific modulation of activity. Even within a given tissue type, such as VSMCs or keratinocytes, external signaling cues from growth factors or retinoids can switch the transactivation ability of KLF5 from an activator to a repressor for the same target genes, which can effectively convert the role KLF5 plays in cell growth and differentiation. This switch is likely modulated through post-translational modification of KLF5 downstream of external stimuli (Fig. 2). For example, whilst acetylated KLF5 recruits co-activators to induce expression of p15 and hence growth arrest of keratinocytes in response to TGFβ, under basal conditions KLF5 is not acetylated and instead interacts with co-repressors such as MYC to inhibit p15 expression [74]. Accordingly, knock-down of KLF5 by siRNA in the HaCaT keratinocyte cell line reduced proliferation in untreated cells, however, knock-down in TGFβ treated cells prevented differentiation of this cell line. Acetylation of KLF5 downstream of TGFβ signaling can alternatively cause KLF5 to act as a transcriptional repressor for selected target genes in keratinocytes. In unstimulated HaCaT cells, KLF5 normally activates expression of MYC by binding to a specific region of DNA containing two tandem CCCCACCC motifs designated the KLF5 binding element (KBE) [75]. Upon acetylation, KLF5 instead binds to another element in the promoter region designated the TGFβ inhibitory element (TIE). The MYC corepressor SMAD4 also binds the TIE in response to TGFβ, suggesting that these two proteins form part of a regulatory complex which inhibits expression of MYC and contributes to growth arrest. These studies may shed light on the contrasting observations that whilst most in vitro evidence supports a growth-promoting function for KLF5 in keratinocytes, epidermal-specific KLF5 over-expression in a transgenic mouse model results in loss of the epidermal stem cell population and consequently a reduction of keratinocyte regeneration potential [91].


Figure 2. Post-translational modifications which switch the transactivation function of KLF5. Acetylation of KLF5 downstream of TGFβ (top) alters the assembly of transcriptional co-regulators on the promoters of p15 and MYC, switching the transcriptional regulation of these genes in keratinocytes. Sumoylated KLF5 (bottom) forms a transcriptional repressor complex for lipid oxidation genes, and desumoylation in response to a PPARδ agonist alters affinity for co-regulators resulting in transcriptional activation.

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Acetylation also plays a role in determining KLF5 function in VSMCs, where studies have shown that KLF5 normally acts to enhance cell proliferation. Zheng et al. recently demonstrated that unliganded RARα interacts with the histone deacetylase HDAC2 and also with acetylated KLF5 to repress expression of the growth arrest gene p21 [81]. On treatment with an RAR agonist, liganded RARα instead binds more strongly to p300 which acetylates RARα, and these two proteins form a heterodimer to activate expression of p21. At the same time HDAC2 is phosphorylated, and subsequently binds to and deacetylates KLF5. This allows KLF5 to disassociate from the p21 promoter, and alleviates repression of p21 transcription. Recently, Xing et al. [96] provided the first evidence that the acetylation status of KLF5 may also impact function in prostate cancer, with acetylated KLF5 protein expressed in the more differentiated luminal cell subtype, and un-acetylated KLF5 acting to promote proliferation of prostate cancer cells.

Human KLF5 is SUMOylated at lysine residues 162 and 209, primarily by the sumoylating enzyme SUMO1 [44, 72]. SUMOylation of lysine 162 near the KLF5 nuclear export signal results in enhanced nuclear localization, however, SUMOylation also allows KLF5 to switch from an activator to a repressor of transcription. Whilst unmodified KLF5 activates expression of lipid metabolism genes (e.g. CPT1B and UCP2/3), SUMOylated KLF5 alternatively recruits the nuclear co-repressors NCoR1 and NCoR2 in a complex with unliganded PPARδ [72]. Treatment with a PPARδ agonist is required for deSUMOylation of KLF5 and subsequent activation of these genes.

Finally, KLF5 can also be ubiquitinated and targeted for proteasomal degradation via protein-protein interaction with the E3 ubiquitin ligases WWP1 and FBW7. FBW7 regulates ubiquitination of the KLF5 protein through binding to phosphorylated CDC4 phosphodegron (CPD) motifs, and it has been demonstrated that phosphorylation of serine 303 specifically occurs via direct interaction with the protein kinase GSK3β [82, 97]. Interestingly, it appears that KLF5 can also be degraded by the proteasome in a ubiquitin-independent manner via its N-terminal 19 amino acids; Chen et al. [98] generated a lysine-less mutant which could still be degraded without ubiquitination, however, deletion of the N-terminal 19 amino acids protected against this degradation. While such mechanisms do not affect the transactivation function of KLF5, they serve to control the level of KLF5 protein and may be important in modulating activity in many systems

Conflicting Roles of KLF5 in Carcinogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular and Biological Roles of KLF5
  5. Molecular Mechanisms of KLF5 Target Gene Regulation
  6. Conflicting Roles of KLF5 in Carcinogenesis
  7. Acknowedgements
  8. References

Opposite Regulation of Target Genes in Transformed Tissues

A large body of evidence exists to suggest that the biological role of KLF5 switches during oncogenic transformation of tissues such as breast, bladder, and esophageal epithelium (Table 2), and recent studies have highlighted opposite regulation of KLF5 target genes as a potential mechanism for these altered functional effects. In esophageal keratinocytes KLF5 normally promotes proliferation, however in esophageal cancer cells KLF5 displays tumor suppressor function through induction of cell death [39]. Accordingly, KLF5 expression is reduced in primary human squamous cell carcinoma samples relative to normal tissues [24, 105]. Yang et al. demonstrated that the status of p53 in human keratinocytes affects the transactivation ability of KLF5, specifically on expression of the cell cycle gene p21: KLF5 normally suppresses expression of p21, and indeed they found that in non-transformed keratinocytes with wild-type p53 this led to enhanced proliferation [104]. Conversely in transformed keratinocytes expressing the common p53 mutant protein R175H, KLF5 instead led to activation of p21 and inhibition of cell growth. Whilst the mechanism for the altered transactivation ability of KLF5 in this case has not been determined, the authors did show differential binding of p53 and KLF5 proteins to similar regions of the p21 promoter in each case. KLF5 and p53 also coordinately regulate the NOTCH1 tumor suppressor gene in esophageal keratinocytes through overlapping binding elements in the promoter region, and loss of KLF5 in the context of p53 mutation abolishes NOTCH1 expression leading to tumor growth in a xenograft mouse model [105].

Table 2. Conflicting functions of KLF5 in normal and transformed tissues
Tissue typeNormal functionCancerous tissues (oncogene)Cancerous tissues (tumor suppressor)Potential mechanisms
BladderNecessary for terminal differentiation in vivo [47]Enhances proliferation and cell cycle [99] Enhances tumorigenicity in vivo [99]No evidenceUnknown
BreastNecessary for proliferation and survival [65, 100, 101]Enhances and is necessary for proliferation [100, 102, 85] Necessary for proliferation in response to progesterone [103] Necessary for cell survival [101] Enhances tumorigenicity in vivo [85, 100]Reduces clonogenicity [62] Reduces proliferation of ER+ breast cancer cells in response to estrogen [65]Interaction with liganded ER Interaction with PR/AR?
EsophagusEnhances and is necessary for cell growth [25, 104] Enhances proliferation in vivo [20]No evidenceReduces proliferation [39] Reduces viability [39] Reduces proliferation of p53-mutant cells [104] Ablation enhances clonogenicity and in vivo tumorigenicity of p53-mutant cells [105]p53 status (wildtype or mutant) Alternate regulation of p21
IntestineEnhances proliferation and clonogenicity [21, 106] Necessary for proliferation in vivo [107]Enhances clonogenicity [46] Necessary for proliferation and clonogenicity of KRAS-mutant cells [43] Necessary for tumorigenesis in vivo [42, 45]Reduces clonogenicity [106] Reduces clonogenicity of HRAS-mutant cells [106]RAS/p53 status Alternate regulation of CCND1 Experimental variation
LungNecessary for terminal differentiation in vivo [29]Enhances clonogenicity and metastatic properties [49]Ablation may increase tumorigenesis in vivo [49]Different roles in tumor initiation and metastasis
ProstateNecessary for both survival and differentiation in vivo [96]Enhances migration of cancer cells [108]Reduces clonogenicity [55, 61] Reduces viability [55, 61] Induces differentiation [55] Reduces tumorigenicity in vivo [61]Dose and acetylation status Different roles in tumor initiation and metastasis

Whilst the majority of evidence supports an oncogenic role for KLF5 in intestinal tumorigenesis, Bateman, et al. initially reported that KLF5 mRNA expression is reduced in intestinal adenomas found in APCmin/+ mice and in human familial adenomatous polyposis adenomas when compared to healthy intestinal tissue [106], consistent with potential tumor suppressor function. The authors did not however assess KLF5 protein expression which has previously been reported to be increased in human colorectal cancer samples [43]. In direct contrast to the observations made by Nandan et al., where enforced expression of KLF5 enhanced colony formation of the intestinal cancer cell lines DLD-1 and HCT116 [43], Bateman et al. found that enforced expression in these cell lines reduced colony forming potential. While the reasons for these discrepancies are not clear, Nandan et al. suggest potential differences in the level of transfection of KLF5 or clonal variation. Bateman et al. proposed that KLF5 may have alternate functions in normal or cancerous intestinal tissue, and demonstrated opposite regulation of the CCND1 target gene as a mechanism for their observations. In non-transformed epithelial cells KLF5 activated expression of the positive cell cycle regulator CCND1 and enhanced proliferation, whereas in intestinal tumor derived cells the reverse effect was observed. It is interesting to observe that the colon cancer cell line used in these experiments, DLD1, harbors a p53 mutation [109], suggesting that p53 status may be a common mediator of KLF5 function in different types of epithelial tumors. The authors demonstrated a similar effect for KLF5 on CCND1 transcription in epithelial cell lines transformed with oncogenic HRAS. Aberrant activation of the RAS pathway cooperates with loss of p53 to synergistically influence tumor formation [110], and it is likely that the function of KLF5 will depend on the relative status of both of these pathways in epithelial tumorigenesis.

Steroid Hormone Modulation of Function

Whilst earlier publications collectively pointed to a tumor suppressor role for KLF5 in breast cancer, recent years have seen an increasing number of studies conversely demonstrating a pro-proliferative and oncogenic function for KLF5 in this tissue. Takagi et al. identified increased nuclear protein expression of KLF5 in cancerous breast epithelial cells relative to adjacent normal tissues [102], and accordingly, it has been shown that expression of KLF5 both at the mRNA level and nuclear protein expression is associated with poor disease-specific survival in breast cancer [102, 111]. Functional studies have demonstrated that siRNA knock-down of KLF5 can inhibit proliferation of non-transformed breast epithelial cell lines and breast cancer cells, and reduce tumor weight in xenograft models [65, 85, 86, 100, 102]. Accordingly, enforced expression of KLF5 in MCF7 breast cancer cells increased tumor weight when transplanted into the mammary pads of nude mice [100]. In breast cancer cell lines siRNA knock-down of KLF5 induces apoptosis through reduced expression of the FGF binding protein and subsequent inhibition of the pro-survival protein MKP1 [100, 101].

Although these discrepancies have not yet been fully explained, several studies have highlighted that KLF5 function may be altered according to expression of steroid hormone receptors in breast cancer. In estrogen receptor (ERα)-positive breast cancer cells, enforced expression of KLF5 inhibits proliferation in response to estrogen but does not have a significant effect in untreated cells or in ERα-negative cancer cells. It has been postulated that KLF5 and estrogen signaling pathways participate in a negative feedback loop in this system, as KLF5 can directly inhibit the activity of ERα through protein-protein interaction, whereas estrogen signaling results in proteasomal degradation of the KLF5 protein via up-regulation of the E3 ubiquitin ligase EFP [65, 84]. It is interesting to observe a parallel mechanism occurring in prostate cancer cells, where estrogen treatment induces KLF5 degradation through interaction with the WWP1 E3 ubiquitin ligase [61]. KLF5 appears to have the opposite effect in breast cancer cells positive for expression of the progesterone receptor, where KLF5 is necessary for up-regulation of cell cycle genes and concomitant proliferation in response to progesterone treatment [103]. These seemingly conflicting findings are nonetheless consistent with the observation that KLF5 expression is increased in ERα/PR/HER2 triple negative breast cancers [84, 97, 100, 111],), as we could postulate that aberrant KLF5 expression is necessary for cancer cell growth in the absence of PR, however it's proliferative function is only preserved with the additional absence of active ERα.

Transcription of KLF5 is also up-regulated in breast cancer cells in response to androgen receptor signaling, and in prostate cancer it has been shown that this effect is mediated by AR binding to a regulatory element within intron 1 [102, 108]. Accordingly, prostate-specific knock-out of KLF5 causes enhanced shrinkage of the prostate in response to androgen deprivation in mice (castration), consistent with a proliferative role for KLF5 in tumors positive for AR expression [96].

Metastatic Potential

It is likely that KLF5 may also play different roles in various stages of tumorigenesis. For example, although the majority of studies in prostate cancer collectively point to tumor suppressor function for KLF5 in this tissue, an independent investigation has suggested that KLF5 may alternatively have a role in promoting metastasis [108]. KLF5 was shown to up-regulate expression of the cell-surface chemokine receptor CXCR4, enabling migration of cancer cells to areas with high levels of the chemoattractant CXCL12 such as that found in the bone microenvironment. Similarly, whilst the majority of evidence points to a tumor suppressor role for KLF5 in lung tissue, enforced expression in KRAS-positive lung adenocarcinoma cell lines did not inhibit cell proliferation; rather, it resulted in reduced contact inhibition and increased anchorage-independent growth, both properties related to metastatic potential [49]. KLF5 may also play a pro-migratory role in other epithelial tissues such as in esophageal keratinocytes, where it stimulates expression of the promigratory protein ILK [112], or in skeletal muscle cells, where it up-regulates expression of MMP9, resulting in degradation of gelatine in skeletal muscle cell systems and enhanced migratory activity [113]. These observations collectively suggest that KLF5 may potentially act as a tumor suppressor which is down-regulated in initiation of tumorigenesis but later acts as an oncogene in metastatic transformation.

Concluding Remarks

Like other members of the Kruppel-like factor family, it is clear from accumulating evidence that KLF5 plays a lynchpin role in control of the growth, differentiation and motility of cells at different times and developmental stages. This has particular implications in the initiation, progression and metastatic potential of cancer cells where the influence of KLF5 may vary depending on the initial state of the cell and the ability of the cell to respond to environmental cues such as hormone stimulation and oxygen levels. The regulation of KLF5 transactivation ability at the protein level provides a therapeutic window through which we can now begin to design strategies to modify KLF5 activity appropriately in particular cancer types.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular and Biological Roles of KLF5
  5. Molecular Mechanisms of KLF5 Target Gene Regulation
  6. Conflicting Roles of KLF5 in Carcinogenesis
  7. Acknowedgements
  8. References

This work was supported by a grant from the National Health and Medical Research Council of Australia (NHMRC #626946).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular and Biological Roles of KLF5
  5. Molecular Mechanisms of KLF5 Target Gene Regulation
  6. Conflicting Roles of KLF5 in Carcinogenesis
  7. Acknowedgements
  8. References
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