Regulation of EMT by KLF4 in Gastrointestinal Cancer

Abstract

Gastrointestinal (GI) cancer is characterized by its aggressiveness, but the underlying mechanism is not fully understood. Studies reveal that epithelial to mesenchymal transition (EMT), which is regulated by a series of transcription factors and signaling pathways, is strongly associated with GI cancer cell proliferation, invasion and metastasis. In essential, EMT is a product of crosstalk between signaling pathways. Krüppel-like factor 4 (KLF4), a zinc finger-type transcription factor, is decreased or lost in most GI cancers. By transcriptional regulating its downstream target genes, KLF4 plays important roles of GI cancer tumorigenesis, proliferation and differentiation. In this review, we focus on the mechanism of KLF4 in GI cancer EMT, and demonstrate that through crosstalk with TGF-β, Notch, and Wnt signaling pathways, KLF4 negatively regulates EMT of GI cancers. Finally, we indicate the challenging new frontiers for KLF4 which contributes to better understanding of the mechanism of GI cancer aggressiveness.

1. Introduction

Gastrointestinal (GI) cancer, which refers to the cancers generated from esophagus, stomach, intestine, gallbladder, liver and pancreas, is a leading cause of cancer-related mortality worldwide [1]. In the United States, estimated 284,680 new GI cancer cases and 142,510 deaths in 2012, and is responsible for about one third of the cancer burden [2]. For GI cancers, surgical resection is the only potentially curative therapy, but most of the patients are diagnosed at the late stage and lose the opportunities of curative resection due to highly aggressive nature of GI cancers. Therefore, a better understanding of the mechanism which contributes to GI cancer aggressiveness is urgently needed.

Multiple lines of evidence have demonstrated the importance of epithelial to mesenchymal transition (EMT), a process of the transformation of epithelial cells to a mesenchymal phenotype and lose their cell-cell contacts, in GI cancer cells aggressiveness. In the 1980s, EMT was first described and later cancer researchers discovered that it was strongly associated with cancer cell proliferation, invasion and metastasis [3, 4]. The aggressive nature of cancer is associated to the abnormalities of a series molecular, including activation of tumor oncogenes, inactivation of tumor suppressor genes, reactivation of telomerase, and overexpression of growth factors and their receptors. Thereafter, effects of various genes, factors and molecular pathways which were identified as important mechanism of cancer development and progression have been extensively studied on EMT of GI cancer.

Krüppel-like factor 4 (KLF4), belonging to Krüppel-like factor family, is a zinc finger-type transcription factor highly expressed in various human tissues, including differentiated, post-mitotic epithelial cells of the gastrointestinal tract. Several lines of evidence have revealed that KLF4 acts as tumor suppressor in GI cancer, and it negatively regulates cell proliferation and promotes tissue differentiation, and loss of KLF4 expression is a predictor of poor survival [5]. Recent studies indicated that KLF4 was a key negative regulator of EMT and its expression was reduced during the EMT process [6]. This review will summarize what is known about KLF4 in GI cancer development and progression, and the role of KLF4 in regulating EMT, so as to get a better understanding of the mechanism that contributes to GI cancer aggressiveness.

2. EMT

EMT was discovered 30 years ago because of its critical roles in embryonic development, and in 1990s, cancer researchers found that EMT was strongly associated with tumor cell proliferation, invasion and metastasis [3, 4, 7]. It is a process typically characterized by epithelial cells losing cell-cell adhesions and apical-basal polarity, undergoing a remodeling of the cytoskeleton and developing a fibroblastoid motile phenotype, which activates the expression of a series of genes. By suppressing the expression of junctional complexes, such as E-cadherin, adenovirus receptor (CAR), Zona occludin-1 (ZO-1), occluding, claudin-1 and claudin-7, cells lose the cell-cell contacts and detach from each other, and by increasing the expression of mesenchymal markers, such as N-cadherin, vimentin, S100A4, α-smooth muscle actin, and extracellular matrix components, such as collagens 1 and 2, cells acquire the ability to migrate and invade the extracellular metrices [8, 9].

In cancer cells, essential step of EMT is the downregulation of E-cadherin which binds with its extracellular domain to another E-cadherin of the neighboring epithelial cells, and therefore disassembles the cell-cell contacts [10, 11]. It is well known that downregulation of E-cadherin is associated with the invasive and undifferentiated phenotype in many GI cancers, including esophageal cancer, gastric cancer, colorectal cancer, gallbladder cancer, liver cancer and pancreatic cancer [12–17]. Another adhesion molecule N-cadherin, which is typically expressed on mesenchymal cell surface, is correlated with the motility and invasive potential of cancer cells [18]. In our previous studies, we found that reduced E-cadherin expression and upregulated N-cadherin expression were associated with the EMT, invasion and metastasis potentials of pancreatic cancer cells [19]. In fact, multiple lines of researches have revealed that the cadherin-switch from E-cadherin to N-cadherin has critical function in cancer progression and is essential for increased motility and migration [20].

E-cadherin can be transcriptionally repressed by a number of transcription factors, such as ZEB (ZEB1 and ZEB2), the Snail (Snail1 and Snail2) and Twist [21]. These transcription factors are downstream of signaling pathways, including transforming growth factor-β (TGF-β), Wnt/β-catenin, Notch, fibroblast growth factor (FGF), signal transducer and activator of transcription-3 (STAT-3), epidermal growth factor (EGF) and nuclear factor (NF)-κB [20, 22]. Loss expression of E-cadherin leads to the release of its intracellular partner β-catenin which thereafter translocates into the nucleus and transcriptionally modulates a number of genes, such as cyclin D1, CD44, c-Myc, and vascular endothelial growth factor (VEGF), and promotes cancer development and progression [22]. Besides canonical Wnt signaling pathway, disassembly of the E-cadherin adhesion complex can also lead to the displacement of p120, which represses the small G protein RhoA and results in the activation of Cdc42 and Rac1 [20]. They together reorganize the actin cytoskeleton and promote the migration of cancer cells [23]. Therefore, the cadherin switch plays critical roles in EMT not only by changing the component of the cell-cell adhesion, but also by regulating various signaling pathways.

3. The Suppressor Role of KLF4 in GI Cancer

3.1 Krüppel-like factor family and KLF4

Krüppel-like factors (KLFs), which contain three C2H2-type zinc fingers at the C-terminal and transactivation domain at the N-terminal, are members of zinc-finger-containing transcription factors [24]. By binding to GC-rich or CACCC promoters with its co-activator p300/CBP, KLFs regulate the expression of a series of genes which play key roles in many biological processes, including cell proliferation, differentiation and stem cells reprogramming [25]. Till now, at least 17 members of KLFs have been identified in humans, and Klf4 was first cloned by two groups in 1996, and named gut-enriched Krüppel-like factor and epithelial zinc finger independently [26–28].

The gene of Klf4 has five exons and encodes a protein with 470 amino acids. Several functional domains have been identified in KLF4, including transcriptional activation and repression domains at N-terminus, DNA binding domain, and nuclear localization signal domain [29]. As a member of KLFs family, KLF4 binds to the CACCC element to regulate gene expression, yet KLF4 can also bind to other DNA sequences. The basic transcription element (BTE), which is often found in the promoter of a series highly conserved genes, is a high-affinity binding site of KLF4 [30, 31]. With both transcriptionally active and repressive domains, KLF4 alters positive and negative regulation of its downstream target genes. Recent studies showed that the expression of KLF4 was decreased or lost in most GI cancers, including esophageal cancer, gastric cancer, colon cancer and liver cancer and by transcriptionally regulating its target gene, KLF4 played essential roles in GI cancer tumorigensis, cell proliferation and differentiation (Fig. 1) [32–40].

Fig. 1. The Suppressor Role of KLF4 in GI Cancer.

The expression of KLF4 is inhibited by DNA methylation, microRNA and ubiquitin-proteasome pathway. In the case of DNA damage, p53 induces the expression of KLF4, and KLF4 can also be upregulated by HDAC inhibitors and CDX2. Overexpression of KLF4 leads to altered expression of a series of its downstream target genes which mediate the suppressor role of KLF4 in GI cancer tumorigenesis and proliferation, and the promoter role in cancer differentiation.

3.2 Roles of KLF4 in GI cancer tumorigenesis

Klf4 is one of a few genes which are proved to be downregulated in GI tumor early initiation. KLF4 is progressively lost as the tumor formation and progression. For example, in our previous studies, gastric cancer presented a significantly progressive loss of KLF4 expression as the stage advanced from I to IV [35]. Also, most human gastric cancer cell lines exhibited lost or decreased KLF4 expression at both mRNA and protein levels. In colon cancer development, KLF4 expression was significantly decreased in colonic adenomas as compared with adjacent normal mucosa [41, 42]. In a colon cancer tissue microarray, the expression of KLF4 was significantly decreased or lost in cancer tissues as compared with noncancer tissues and KLF4 was an independent predictor of survival [39]. Furthermore, Choi et al found that KLF4 was expressed in normal colonic mucosa and loss of KLF4 expression was observed in about a quarter of the colon cancers [38]. Decreased expression of KLF4 has also been found in esophageal cancer cell lines and tissues [32–34]. Besides these clinical evidence, by an established system in RKO colon cancer cells, which had an inducible promoter of Klf4, Dang et al demonstrated that overexpression of KLF4 reduced colony formation, in vivo tumrigenecity, and cell migration and invasion [43].

The tumor suppressor role of KLF4 in GI cancer is heightened by the fact that KLF4 is downregulated in the mouse models of gastrointestinal tumorigenesis. For example, in our previous studies, disruption of Klf4 in villin-positive antral mucosa cells (Villin-Cre(+);Klf4(fl/fl) mice) significantly increased the incidence of gastric tumors [44]. Further studies revealed that KLF4 suppressed the transcription of FOXM1, an important oncogenic transcription factor which were essential for tumor initiation and progression [45, 46]. In another study, Katz et al generated gastric epithelia-specific Klf4 ablation mouse models and found that Klf4 mutant mice presented with increased proliferation and altered differentiation in the gastric epithelia, and they further demonstrated that p21^WAF1/CIP1 was an in vivo target of Klf4 [47]. In colon, Klf4−/− mice presented with a 90% decrease in the number of goblet cells, and KLF4 was required for the terminal differentiation of the goblet cells [48]. The ApcMin/+ mice was an excellent model for studying intestinal tumorigenesis. As compared with the ApcMin/+ mice, Klf4+/−/ApcMin/+ mice developed, on average, 50% more intestinal adenomas. Further RT-PCR experiments showed an inverse correlation between KLF4 mRNA levels and adenoma size in both Klf4(+/−)/Apc(Min/+) and Apc(Min/+) mice [49].

All these clinical and experimental findings suggest a tumor suppressor role of KLF4 in GI cancer, and decreased or lost expression of KLF4 contributes to the GI cancer tumorigenesis. But the molecular basis of the KLF4 inactivation in GI cancer is not fully demonstrated. Some studies may give us a few clues. Elevated microRNA-10b directly repressed the expression of KLF4 in esophageal cancer [50]. In gastric cancer, we provided evidence that promoter hypermethylation and hemizygous deletion contributed to the low levels of KLF4 [35]. In colon cancer, Dang et al demonstrated that KLF4 was induced upon the activation of the adenomatous polyposis coli (APC) gene but not the DNA methylation [37]. Their further studies revealed that the decreased expression of KLF4 was the result of mutation of the putative tumor suppressor homeodomain protein, CDX2 [51]. In another study, Zhao et al provided evidence that the 5′-untranslated region of KLF4 was hypermethylated in colon cancer [52]. Furthermore, KLF4 is a direct target of ubiquitin-proteasome pathway [53]. These findings can’t fully reveal the mechanism of the KLF4 inactivation in GI cancer, and some even conflict with each other, so further detailed studies should be carried out.

3.3 Roles of KLF4 in GI cancer proliferation

The involvement of KLF4 in GI cancer tumorigenesis is largely dues to its essential roles in repressing cancer cell cycle, promoting apoptosis, and inhibiting proliferation. KLF4 has been reported to be overexpressed in growth-arrested cells and is rare detected in proliferation cells [27]. Decreased expression of KLF4 in esophageal cancer cells results in increased proliferation and decreased adhesion ability [34]. Overexpression of KLF4 activates DNA repair pathway and represses esophageal carcinogenesis through suppressing proliferation and inducing cell apoptosis [32]. Furthermore, KLF4 transcriptionally represses the expression of survivin, interferon induced transmembrane protein 3 (IFITM3), and B lymphoma Mo-MLV insertion region 1 (Bmi1), which play important roles in cancer development and progression, and results in significant decrease of cancer cell proliferation and increase of apoptosis [54–56].

KLF4 is a critical regulator for cell cycle, and cells with overexpressed KLF4 will arrest at G1/S checkpoint [57–59]. Cell cycle is controlled by cyclin-dependent kinases (CDKs) whose activity is regulated by activators (cyclins) and inhibitors (CKIs) [60]. Regulation of these CDKs, and their activators and inhibitors is the first indication of KLF4 contributing to cancer cell cycle. Constitutive expression of KLF4 transactivates the expression of p21, p27 and p53, which are the main CKI proteins, and inhibits the synthesis of DNA [27, 41, 52, 61–63]. In mouse models, Katz et al found that Klf4 mutant mice showed increased proliferation and altered differentiation of the gastric epithelia, and p21^WAF1/CIP1 was an in vivo target of KLF4 [47]. Our group identified 4 KLF4 isoforms in human pancreatic cancer cells, and KLF4α, which primarily localized to the cytoplasm, reduced the expression of KLF4 classical target genes p27^Kip and p21^WAF1/CIP1, and promoted cell cycle progression and tumor formation of pancreatic cancer [64]. Further studies revealed that enforced expression of KLF4 led to significant cytoplasmic distribution of KLF4 protein and repressed the transcriptional effect of KLF4 on p27^Kip gene. In another study, the authors demonstrated that KLF4 was an essential mediator of p53-dependent G1/S cell cycle arrest following DNA damage [57, 58]. Besides transactivating several CKIs, KLF4 also represses the expression of positive cell cycle regulators. By competitive binding Sp1-binding sites on cyclinD1 promoter, KLF4 suppresses mRNA and protein level of cyclinD1 [65]. With the same mechanism, KLF4 repressed the expression and enzyme activity ornithine decarboxylase (ODC), and led to cell cycle arrest at the G1/S checkpoint [66]. Besides KLF4’s essential role in G1/S checkpoint, in a DNA damage condition, KLF4 also played important roles in preventing centrosome amplification and maintaining the integrity of the G2/M checkpoint by transcriptionally repressing the cyclinB1 and cyclinE expression [59, 67].

All the above findings revealed an inhibitory role of KLF4 in GI cancer proliferation. In contrast with these studies, the expression of KLF4 was upregulated in other types of cancers, such as head and neck squamous cell cancer, breast cancer and oral squamous cancer, and functioned as an oncogene which enhanced tumor development and progression [68–70]. Apparently, Klf4 functioned as either a tumor suppressor gene or an oncogene, and had both negative and positive effects on cancer cell proliferation. Recent studies revealed that tumor suppressor or oncogenic function of KLF4 might dependent on the genetic background of the tumor type, especially the functional status of p21 [71]. Other factors or pathways, which contribute to switching between suppressor and oncogenic functions of KLF4 in cancer development and progression, should be further studied.

3.4 Roles of KLF4 of GI cancer differentiation

In addition to its well described role in GI cancer cell proliferation, KLF4 also has important regulating effect on differentiation. KLF4 was highly expressed in terminally differentiated layers of epidermis, and Klf4−/− mice died within 15 hours after birth because of the loss of skin barrier function and show disturbed late-stage differentiation of the epidermis [72]. In colon, Klf4 was a goblet cell-specific differentiation factor. Klf4−/− mice showed normal cell proliferation and death rates in colon, but had a 90% decrease in the number of goblet cells. By in situ hybridization, Klf4−/− mice exhibited abnormal expression of Muc2 which was the goblet cell-specific marker [48]. Therefore, KLF4 had an essential role in colonic epithelial cell differentiation. In gastric epithelia-specific Klf4 ablation mouse models, more than 50% decrease of mature zymogenic cells, but 4-fold increase in the number of TFF2/SP-positive mucue cells and 2-fold increase in the number of pit cells were found [47]. Besides mouse models, transcriptional profiling of KLF4 had showed that KLF4 regulated the expression of a series of epithelial-specific keratin [73]. Furthermore, in esophageal squamous epithelium, KLF4 transcriptionally upregulated the expression of differentiation genes Epstein-Barr virus ED-L2 and keratin 4, and meanwhile, in pancreatic ductal cells, KLF4 upregulated the expression of keratin 19 [74, 75].

Taken together, all the above studies reveal that KLF4 is critical in specific epithelial proliferation and differentiation. In GI cancers, KLF4 also functions as a promoter of cancer cell differentiation. For example, in esophageal cancer, overexpression of KLF4 transcriptionally elevated the expression of small proline-rich protein 1A (SPRR1A), small proline-rich protein 2A (SPRR2A) and keratin 4 (KRT4), which were squamous cell differentiation associated genes, and resulted in squamous cell differentiation [34]. In colon cancer cell lines, the expression level of KLF4 was increasing during the differentiation induced by butyrate, which was an inhibitor of histone deacetylases (HDAC) and an inducer of differentiation. KLF4 transcriptionally activated the expression of intestinal alkaline phosphatase (IAP), which was an enterocyte differentiation marker [76, 77].

The role of KLF4 in differentiation has been described for a long time, but recent studies of the induced pluripotent stem cells (iPS) have identified KLF4 as one of the four transcription factors which reprogram somatic cells into iPS through combination [78]. Furthermore, in cancer stem cells, Leng Z et al provided evidence that KLF4 was overexpressed only in spheroid cells and knockdown KLF4 in cancer stem cells led to significant decrease of cancer stem cell markers, resistance to chemicals, invasion, migration, and tumorigenesis both in vitro and in vivo [79]. These studies reveal another role of KLF4 in regulating dedifferentiation, which is inconsistent with previous studies, so further studies are still needed.

4. Roles of KLF4 in Regulating GI Cancer EMT

Over the past decades it has been demonstrated that EMT is a multistage process, during which dramatic changes in cell morphology and biology accompanied with substantial changes in gene expression profile take place. Ever-increasing evidences have revealed that a number of transcription factors play critical role in the initiation and execution of EMT, including Snail1 (Snail), Snail2 (Slug), ZEB1, ZEB2 (Sip1), Twist1, FoxC2, E47, KLF8, goosecoid, Sox9, NF-κB et al [80]. Recently, KLF4 has been suggested to function as a transcriptional activator of epithelial genes and as a suppressor of mesenchymal genes during the process of EMT [81–84]. In hepatocellular carcinoma, exogenous KLF4 expression repressed mesenchymal characteristics, and changed the cell morphology to a more epithelial phenotype, and then restrained cell migration and invasion activities, as well as tumor growth and lung colonization, whereas KLF4 knockdown enhanced mesenchymal features and cell migration. Further mechanism research demonstrated that KLF4 was able to bind and suppress the activity of the Slug promoter, and ectopic Slug expression partially revert the KLF4-mediated phenotypes [82]. There are limited literatures about the role of KLF4 in the GI cancer EMT, but other discoveries about KLF4 regulating EMT in non-GI cancer may bring inspiration to our understanding of KLF4 in GI cancer. Researches about transcriptional regulator of KLF4 in EMT mainly concentrated on the breast cancer. In a mouse model of breast cancer, KLF4 inhibited EMT through reducing the expression of Snail, a key mediator of EMT and metastasis [81]. The expression of E-cadherin, an epithelial marker, is controlled by a balance between ZEB2 and KLF4 in cancer cell lines. KLF4 binds to the E-cadherin promoter in a region overlapping with a known ZEB2 binding site, and these two transcription factors have opposite effects on the activity of E-cadherin promoter [84, 85]. A recent publication has shown that KLF4 was a transcriptional regulator of genes critical for EMT, and in addition, they revealed a series of key genes as direct transcriptional target of KLF4, including E-cadherin, N-cadherin, vimentin, β-catenin, VEGF-A, endothelin-1 and Jnk1 [83]. EMT is also regulated by other factors and it is a product of crosstalk of signaling pathways. In the following sections, we will discuss the crosstalk between KLF4 and selected signaling pathways, including TGF-β, Notch, and Wnt pathways which play critical roles in EMT (Fig. 2).

Fig. 2. The functions of KLF4 involved in the mechanism of EMT.

EMT is a product of crosstalk between signaling pathways, including TGF-β, Wnt/β-catenin, Notch, FGF, STAT3 and EGF. By regulating the transcription factors (ZEBs, Snails and Twist), these signaling pathways switch the epithelial phenotype to mesenchymal phenotype. In cancer cells, KLF4 represses the EMT through interacting with TGF-β, Notch and Wnt/β-catenin signaling pathways. The degradation of KLF4 through UPS is necessary to TGF-β induced transcriptional activation. In Notch signaling pathway, KLF4 negatively regulates the expression of Notch1 and functions as a negative modulator of Notch1 target genes’ transcription. KLF4 also interacts with Wnt/β-catenin by antagonizing the binding of β-catenin to TCF and represses the transcriptional activity of TCF.

4.1 Crosstalk between KLF4 and TGF-β

It is widely accepted that TGF-β has a dual role during carcinogenesis. Initially it suppresses tumor formation since it inhibits cell growth and stimulates apoptosis, however, in advanced cancer TGF-β is often overexpressed, and functions as a promoter through inducing EMT which strengthens invasiveness and metastasis [9]. TGF-β signaling pathway is initiated though interaction between TGF-β and a tetrameric cell surface complex of type I and type II transmembrane kinase receptors, in which type II receptor phosphorylates and activates the type I receptor. Then they phosphorylate receptor-activated Smads (Smad2 and Smad3) in their C-terminals, whereafter two activated Smad proteins form complex with the common-mediator Smad4. Smad complexes translocate into the nucleus and regulate target gene expression in cooperation with high-affinity DNA-binding transcription factors, co-activators and co-repressors [86]. During cancer progression, TGF-β is commonly overexpressed in various human cancers suggesting a pivotal role of TGF-β in EMT process. TGF-β/Smad signaling directly activates the expression of EMT transcription factors, including Snail, Slug, ZEB1, ZEB2 and Twist. Smad signaling also represses the expression of the microRNA-200 family through leading to the expression of ZEB transcription factors, afterwards, microRNA-200 family further increases the expression of ZEB at protein level and mRNA level in a feedback loop. In addition, TGF-β also promotes the expression of MMPs such as MMP2/9, as well as components of the ECM, most likely via activation of the EMT transcription factors [87].

Emerging studies have shown that KLF4 functionally contributed to the TGF-β induced EMT, in which the KLF4 mRNA and protein levels were found down-regulated already at first days of TGF-β treatment. Consistent with it, depletion of KLF4 expression could remarkable accelerated the process of TGF-β induced EMT [83]. Similar results were obtained from prostate cancer cell. In a model of TGF-β induced prostatic EMT, Slug is the dominant regulator of EMT initiation as demonstrated through the inhibition of EMT following Slug depletion, and TGF-β-stimulated KLF4 degradation is required for Slug induction [88]. Although it is common knowledge that KLF4 is regulated through various post-translational modifications, including phosphorylation, acetylation, emerging studies suggested that KLF4 alteration could be mediated by the Ubiquitin-Proteasome System (UPS). In response to TGF-β signaling, KLF4 is profoundly degraded through UPS, which is necessary to ensure TGF-β-induced transcriptional activation [89]. In the differentiation of vascular smooth muscle cells, KLF4 was found to transduce TGF-β signaling via phosphorylation-mediated activation of Smad2/3 and p38MAPK pathways. KLF4 could directly bind to the TGF-β promoter at KLF-4-binding sites 2 and 3, and recruit Smad2 to the Smad-responsive region [90]. TGF-β and bone morphogenetic proteins (BMPs), also belongs to TGF-β superfamily, down-regulated KLF4 via induction of microRNA-143 and microRNA-145 in modulation of vascular smooth muscle cell phenotype [91]. Together these findings provide convincing evidence that there was a tight crosstalk between KLF4 and TGF-β signaling pathways, and KLF4 may play a central role in tumor EMT through interaction with TGF-β signaling.

4.2 Crosstalk between KLF4 and Notch signaling

Notch signaling plays a key role in the control of cell fate determination, growth and differentiation, and also plays an instrumental role in the formation and progression of multiple human tumors, including GI cancer. The canonical pathway of Notch activation involves interaction between ligands and receptors of Notch, proteolytic cleavage and translocation of intracellular domain of the notch (ICN) into the nucleus, where it associated with DNA binding protein for transcriptional activation of Notch target genes. Recent experimental evidences have suggested that Notch signaling pathway was involved in the acquisition of EMT in both physiological conditions and pathological processes, especially in the field of malignancies [92, 93], and the interaction between KLF4 and Notch may suggest the role of KLF4 in EMT process. In normal intestine development, the regulation of differentiation of goblet cells in intestine by Notch signaling at least partially mediated via KLF4, which is essential for the repression of intestine cells proliferation and the terminal differentiation of enterocytes [94]. In specific cell types like keratinocytes, Notch plays a central pro-differentiation and tumor suppressing function with down-regulation of the Notch1 gene being associated with cancer development. Regulatory mechanisms is that KLF4 binds to the promoter of Notch1, together with Sp3, functions as a negative modulator of Notch1 target gene transcription [95]. KLF4 is highly expressed in more than 70% of breast cancers and functions as an oncogene, which is required for the maintenance of breast cancer stem cells. Knockdown of KLF4 inhibited migration and invasion of breast cancer cells. Further mechanistic studies revealed that the Notch signaling pathway was essential for KLF4 mediated cell migration and invasion [96]. Another research of human mammary epithelial cells suggested that KLF4 directly bound to the proximal Notch1 promoter, and siRNA-mediated suppression of KLF4 in human mammary cancer cells resulted in reduced expression of Notch1. Furthermore, the expression of KLF4 and notch1 are correlated in primary human breast tumors [96]. In intestine cancer, KLF4 is considered as a tumor suppressor gene, which 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. Overexpression of Notch in HT-29 colon cancer cells reduced KLF4 levels, suppressed KLF4 promoter activity, and increased proliferation rate [97]. Taken together, interaction between KLF4 and Notch signaling may be reciprocal, which possibly be responsible for the functions of KLF4 in EMT.

4.3 Crosstalk between KLF4 and Wnt signaling pathway

One major signal transduction pathway which is involved in the control of pathological process in human cancer is the canonical Wnt/β-catenin signaling. During development of human cancer, activated Wnt signaling promotes the nucleus translocation of β-catenin, leading to the consequent transcriptional activation of specific target genes via binding to the transcription factor T-cell factor/lymphocyte enhancer factor (TCF/LEF). Gene expression pattern induced by nuclear β-catenin has been found to favor tumor invasion and metastasis, and mounting evidences indicate that Wnt/β-catenin signaling is involved in the process of EMT in human cancer [98, 99]. In recent years, increasing evidences supported the existence of interaction between KLF4 and Wnt signaling, which may be correlated with EMT in human carcinoma. Sellak H et al recently revealed that KLF4 antagonized β-catenin/TCF binding in a series of normal and cancer cells, and the inhibition was concentration-dependent. Overexpression of KLF4 in cancer cells shows a concentration-dependent reduction of TCF-luciferase as well as the ability of TCF-binding [100]. When colorectal cancer tumor suppressor adenomatous polyposis coli (APC) is inactivated by mutation, Wnt signaling is unimpeded with the nuclear accumulation of β-catenin. Christian D et al found that APC could regulate the expression of KLF4 in a colon cancer cell line through transactivating KLF4 promoter. Overexpression of KLF4 resulted in decreases in β-catenin protein and mRNA levels, and down-regulation of KLF4 lead to increase in β-catenin concentration. Their findings indicated that the suppressive effect of KLF4 may be mediated via the APC/β-catenin signaling [101]. Zhang W et al also suggested that there was interaction between KLF4 and β-catenin, which played a critical role in homeostasis of the normal intestine as well as in carcinogenesis of colorectal cancers [102]. Further mechanical research revealed that KLF4 directly interacted with the C-terminal transaction domain of β-catenin and inhibited p300/CBP recruitment by β-catenin, additionally KLF4 also directly interacted with TCF4 independently of β-catenin, and then restrained the Wnt signaling pathway [103]. Taken together, there is a reciprocal interaction between KLF4 and Wnt signaling, which may contribute to the development of EMT in cancer.

5. Conclusions and Future Directions

EMT is associated with many processes of cancer development and progression, including proliferation, cell stemness, invasion and migration. Many transcription factors can trigger it, especially ZEB, Snail and Twist families. These transcription factors are downstream of signaling pathways, so in essential, EMT is a product of crosstalk between signaling pathways. KLF4, a transcription factor, functions as a tumor suppressor in GI cancers. Based on the association with main signaling pathways, including TGF-β, Wnt/β-catenin and Notch which play critical roles in regulating EMT, the negative role of KLF4 in GI cancer EMT has been established. This review has discussed the role of KLF4 in GI cancer tumorigenesis, proliferation, differentiation, and especially EMT in summarizing what is already known, but the exact mechanism of KLF4 in cancer early initiation, cancer stem cell reprogramming and maintaining are not clear, so more work is needed to provide deeper insight into them. Recent studies have suggested that KLF4 can also function as an oncogene and is one of the four transcription factors which reprogram somatic cells into iPS. In considering such a complicated transcription factor demonstrating the molecular basis of its switch between tumor suppressor gene and oncogene should be solved urgently. Recently, a series of studies have revealed that the functional status of p21 contributes to the switch but further studies are still needed [71].

Abbreviations

EMT

epithelial to mesenchymal transition

KLF4

Krüppel-like factor 4

CAR

adenovirus receptor

ZO-1

Zona occludin-1

TGF-β

transforming growth factor-β

FGF

fibroblast growth factor

STAT-3

signal transducer and activator of transcription-3

EGF

epidermal growth factor

NF-κB

nuclear factor -κB

VEGF

vascular endothelial growth factor

BTE

basic transcription element

APC

adenomatous polyposis coli

IFITM3

interferon induced transmembrane protein 3

Bmi1

B lymphoma Mo-MLV insertion region 1

CDK

cyclin-dependent kinase

ODC

ornithine decarboxylase

SPRR1A

small proline-rich protein 1A

SPRR2A

small proline-rich protein 2A

KRT4

keratin 4

HDAC

histone deacetylases

IAP

intestinal alkaline phosphatase

iPS

induced pluripotent stem cells