Emerging Evidence for Krüppel-Like Factor 4 (KLF4) as a Tumor Suppressor in Neuroblastoma

Abstract

Currently, 17 transcription factors are known to form the Krüppel-like factor (KLF) family. All transcription factors of the KLF family resemble the members of the specificity protein (Sp) family; however, all members of the KLF family are distinctly characterized by three Cys ₂/His ₂zinc finger motifs at their C-terminal ends. The Cys ₂/His ₂zinc finger motifs of KLF family members are used for efficient binding to the GC/GT box or CACCC element of the enhancer or promoter of the target genes. For the regulation of tumor cell growth or death, KLF4 is an important player. The precise localization of the KLF4 gene is known to be chromosome 9q31. As a transcription regulator, KLF4 is capable of controlling tumor cell proliferation, differentiation, apoptosis, migration, angiogenesis, and metastasis emphasizing its significance in tumor diagnosis, treatment, and prognosis. Various studies suggest that KLF4 plays important roles in tumor progression as well as in tumor suppression depending on the tumor types and contexts. Malignant neuroblastoma is a deadly tumor that mostly occurs in children. Emerging evidence strongly suggests that KLF4 inhibits the cell cycle and activates cell differentiation and death pathways in human malignant neuroblastoma, thereby behaving as a tumor suppressor. Our increasing understanding of the molecular mechanism of expression and activity of KLF4 in human malignant neuroblastoma will be highly useful for better diagnosis, therapy, and prognosis of this deadly pediatric tumor in the near future.

Keywords

Expression and activity, KLF4, Neuroblastoma, Transcription factor, Tumor suppressor

Acknowledgments

This work was supported in part by an award from Soy Health Research Program (SHRP, United Soybean Board, Chesterfield, MO, USA), University of South Carolina School of Medicine Research Development Fund (USC SOM RDF, Columbia, SC, USA), South Carolina Spinal Cord Injury Research Fund (SC SCIRF-2015-I-01, Columbia, SC, USA), and earlier grants (R01 CA91460 and R01 NS057811) from the National Institutes of Health (Bethesda, MD, USA).

Introduction

The Krüppel-like factor (KLF) family in humans includes 17 members, which are transcription factors and have homology with their founding member Krüppel (“crippled” in German), the deletion of which is associated with a crippled-like phenotype in Drosophila melanogaster . It is now well recognized that some of the members of the KLF family have very important roles in human health and diseases including tumor progression or suppression . As such, there is a great interest in the deeper understanding of their structural features, regulation of expression, and molecular mechanism of action for controlling the growth or death of malignant neuroblastoma, which is a deadly solid tumor occurring mostly in children. Among all the members of the KLF family, now KLF4 is of special interest because its transcription regulatory roles (as transactivator or transrepressor) may result in oncogenic or tumor suppressive effects ( Fig. 16.1 ). Dual roles of KLF4 as oncogene and tumor suppressor are due to its interaction with the binding elements on promoters of the target genes in different cellular contexts . Results from recent studies strongly suggested that indirect upregulation of KLF4 was associated with suppression of growth, invasion, and metastasis in human malignant neuroblastoma cells in vitro and in vivo . A thorough understanding of the level of expression and function of KLF4 may help in the diagnosis, treatment, and prognosis of malignant neuroblastoma.

The transcription factors of both KLF family and specificity protein (Sp) family have key roles in critical biological processes such as stem cell maintenance, proliferation, differentiation, development, and metabolism, and thereby, impairment in their regulation is associated with many diseases including cancers in humans . Results from the phylogenic studies show that members of the KLF family share a common structural feature with the members of the Sp family. All members of these two protein families have zinc finger (ZnF) motifs at their C-terminal ends . Nevertheless, the proteins of the KLF family are structurally different as they have three highly conserved and distinct ZnF motifs with additional conserved residues between each ZnF . To act as transcription factors, proteins of the KLF family employ their conserved ZnF motifs to recognize and bind to the nearly identical GC/GT box or CACCC element consensus sequences: even thereafter, the specificity of their activities is imposed by their varying N-terminals and/or by their expression in distinctive tissues .

Because of abundant expression of KLF4 in the gut, it is every so often called the gut-enriched KLF . Moreover, KLF4 has been highly found to be expressed in the epithelial tissues. Conspicuously, KLF4 is highly expressed in terminally differentiated epithelial cells of the intestinal mucosa. Considering its customary roles and ample expression in epithelial cells of the gut, KLF4 certainly has key roles to play in maintaining cellular homeostasis in the intestinal epithelium. Studies show that colon cancer has a notable alteration in expression of KLF4 . Many other solid tumors also exhibit alterations in the expression of KLF4 . Because it can function as an oncoprotein or a tumor suppressor protein depending on the cellular, tissue, and genetic contexts, the pathobiology of KLF4 is very complicated . Results accumulated so far from various studies almost convincingly show that the transcription regulatory roles of KLF4 make it a tumor suppressor in human malignant neuroblastoma.

Neuroblastoma in Need for New and More Reliable Prognostic Biomarkers

Human malignant neuroblastoma is one of the deadliest pediatric solid tumors, as it currently accounts for more than 15% of all cancer deaths in children . Although the pathogenesis of neuroblastoma involves upregulation of many oncogenes and downregulation of some tumor suppressors, many other key factors remain unknown. Proper diagnosis of neuroblastoma is problematic. Neuroblastoma follows an unpredictable clinical course. Some of these tumors undergo regression spontaneously without any treatment or respond quickly to the right treatment strategies . But some other tumors grow relentlessly due to drug resistance, resulting in poor therapeutic outcomes and eventually leading to the death of many pediatric patients . It is increasingly becoming evident that new and reliable prognostic biomarkers must be identified by the basic scientists for helping clinical scientists devise the most appropriate treatment strategies for saving many children who are currently under the dark spell of malignant neuroblastoma.

To predict the clinical outcomes of neuroblastoma patients, currently, there exist several prognostic markers such as the age of the patient at diagnosis, amplification of N-Myc, and level of expression of TrkA . Studies suggest that the more than 1 year age of the patient is associated with poor prognosis and amplification of N-Myc in neuroblastoma is also an indication of poor prognosis . There are no doubts that the age of the patient and amplification of N-Myc in neuroblastoma are currently useful in determining the stages of a progressive disease and predicting the gloomy therapeutic outcomes . On the other hand, the functional TrkA and EphB signaling pathways have been associated with a good prognosis of neuroblastoma patients . The functional TrkA and EphB signaling pathways are known to block the growth of neuroblastoma, respectively, by induction of terminal differentiation and inhibition of proliferation, angiogenesis, and metastasis in neuroblastoma cells . A later study shows that recruitment of histone deacetylase 1 (HDAC1) by a potent repression complex containing N-Myc at the TrkA promoter represses TrkA expression directly and thereby neuroblastoma cell response to the nerve growth factor, resulting in inhibition of terminal differentiation and promotion of aggressive growth of neuroblastoma .

A relatively recent study showed that expression of KLF4 at a low level in primary neuroblastoma in patients was a harbinger of unfavorable outcomes, while expression of KLF4 at a high level in human malignant neuroblastoma SH-SY5Y cells profoundly prevented cell proliferation due to direct upregulation of the cell cycle inhibitor protein p21Waf1/Cip1 and promoted loss of their neuroblastic phenotypes making them resemble epithelial cells, ardently adherent to the substrate, express smooth muscle marker, and non-tumorigenic cells, suggesting that the expression of KLF4 at a high level triggered terminal differentiation of neuroblastoma cells paving their path to spontaneous death . This study in neuroblastoma patients as well as in preclinical models of human neuroblastoma revealed the signaling pathways for strongly supporting the contention that KLF4 acts as a tumor suppressor in human malignant neuroblastoma. Expression of KLF4 at a high level in neuroblastoma patients seems to be a new and reliable biomarker for prediction of favorable outcomes.

Members of KLF Family and Subfamilies

All the 17 transcription factors in the KLF family harbor a highly conserved C-terminal DNA binding domain (DBD) that contains three Cys ₂/His ₂ZnF motifs for recognition and binding to the GC/GT rich sequences of the target genes in the genome . All members of the KLF4 family display sequence similarity with the DBD of the members of the Sp family . The C-terminal ends of the members of the KLF family are highly preserved, but their N-terminal ends are highly variable for making contributions to different biological functions. Structural homologies and functional similarities of the members of the KLF family are correlated, and this correlation seems to be due to their homologous protein interaction motifs at their N-terminal ends. All the currently known 17 mammalian KLF members can be grouped into three major subfamilies based on their structural similarities in the N-terminal ends .

Group one subfamily members (KLF3, 8, and 12) are generally known to act as transcription repressors (transrepressors) as they recruit the chromatin-modifying enzymes to augment the repressive signals to the histones . Group two subfamily members (KLF1, 2, 4, 5, 6, and 7) harbor acidic activation domains that usually portray them as transcription activators (transactivators). Group three subfamily members (KLF9, 10, 11, 13, 14, and 16) are transrepressors of the target genes, but they may also act as transactivators . It needs to be noted here that KLF15 and KLF17 are imprecisely related as revealed from the phylogenetic analysis, but these two members of the KLF family have no defined protein interaction motifs, and hence, they are not included in any of the three subfamilies.

Molecular Structure of KLF4 to Account for Its Transcription Regulatory Roles

Because of the dual functions of KLF4 as oncogene and tumor suppressor gene depending on cancers and conditions, there is a great interest in understanding its molecular structure at the DNA, RNA, and protein levels . The gene locus for human KLF4 is made up of a 6.3-kb section and located on chromosome 9q31; it contains five exons for generating around 3.5 kb mRNA of KLF4 as identified by Northern blotting of total RNA from the human umbilical vein endothelial and other cells . Molecular biology of not only human KLF4 but also mouse KLF4 is well studied. Both human and mouse KLF4 proteins consist of nearly 91% resemblance in their sequences of amino acids. The human cDNA that codes for human KLF4 protein has been forecasted to contain a sequence of 513 amino acids with a molecular weight of 54 kDa . Several alternatively spliced isoforms of human KLF4 gene are currently known to exist in normal and cancer cells . Various studies over the years have well established the molecular structure of the human KLF4 protein and its distinct functional domains ( Fig. 16.2 ). The whole KLF4 protein can be divided roughly into four discrete functional domains: (1) an N-terminal activation domain contains 1–157 amino acids , (2) a middle repression domain has 158–385 amino acids , (3) then a nucleus localization signal (NLS) sequence is comprised of the hexapeptide PKRGRR (386–401 amino acids), and (4) lastly a C-terminal DBD has 81 highly preserved 402–483 amino acids .