Original ArticlesTAp73 inhibits cell invasion and migration by directly activating KAI1 expression in colorectal carcinoma
Introduction
p53 is a sequence-specific transcription factor that plays a central role as a tumor suppressor by regulating the transcription of target genes in response to intracellular and extracellular stimuli, but it is frequently mutated in a variety of human cancers [1]. p73 is a member of the p53 family and shares significant similarity at the amino acid level with p53 and p63. Specifically, the highest similarity is found in the DNA-binding domain (DBD), in which p73 has more than 60% similarity with p53 and p63. Thus, p73 binds to canonical p53-responsive elements to transactivate many p53 target genes [2]. However, unlike p53, p73 is rarely mutated in human tumors [3]. Furthermore, altered expression levels of various p73 isoforms, which primarily include transactivation (TA) isoforms and transactivation-deficient N-terminally truncated dominant negative (DN) isoforms, are frequently detected in different types of cancer [3]. TA isoforms of p73 form homo- or heteromeric complexes with p53 and/or p63 and directly bind to their response elements to transactivate target gene promoters, while DN isoforms of p73 are known to act as dominant-negative inhibitors of p53 family proteins by either directly blocking their binding to target gene promoters and/or by forming inactive heteromeric complexes with TA isoforms [4]. However, the physiological roles of p73 in tumor cells are not yet fully understood. Previous studies have demonstrated that TAp73 acts as a tumor suppressor in cell proliferation, cell cycle, apoptosis, invasion, migration and tumor metastasis through direct regulation of its unique or shared target genes with p53, whereas DNp73 can serve as an antagonist of p53, TAp63 and TAp73 [5], [6], [7], [8], [9]. On the other hand, previous studies also showed that TAp73 and DNp73 can act as oncogenes or tumor suppressors in cell proliferation and invasion [10], [11].
Tetraspanins are a family of membrane proteins with four transmembrane domains, and they interact with other tetraspanins and several different types of proteins, including integrins, membrane receptors, intracellular cytoskeletal proteins and signaling molecules [12]. Due to their multiple interactions with a diverse range of molecules, tetraspanins have been implicated in a wide range of biological processes, including cell motility, invasion, morphology, metastasis, proliferation and differentiation. KAI1/CD82 (Cluster of Differentiation 82) is a member of the tetraspanin superfamily and acts as a metastasis suppressor during the progression of a variety of solid tumors [13]. KAI1 also interacts with several cell surface receptors, such as EGFR, c-Met and Integrin β1, to inhibit their signaling transduction, resulting in suppression of invasion, migration and metastasis of cancer cells [14], [15], [16], [17]. Down-regulation of KAI1 has been observed during the progression of many types of human tumors and is correlated with poor survival in patients with those cancers. However, the underlying causes of the down-regulation of KAI1 in advanced or metastatic cancers remain unknown. The available evidence does not support mechanisms that involve mutations within the coding or promoter regions of KAI1, loss of heterozygosity, or CpG hypermethylation of the KAI1 promoter [18], [19], [20], [21], [22]. Rather, down-regulation of KAI1 expression is likely due to negative and/or positive alterations of its transcriptional activity.
In this study, we found that TAp73 and KAI1 were down-regulated in metastatic cancer tissues, which inhibited invasion, migration and EMT of colorectal cancer cells. We identified TAp73 as a positive transcriptional regulator that directly regulates the expression of KAI1 and found that KAI1 is indispensable for TAp73-mediated inhibition of cancer cell invasion, migration and metastasis.
Section snippets
Experimental procedures
Plasmid construction, western blot analysis, luciferase reporter assay, chromatin immunoprecipitation (ChIP) assay, electrophoretic mobility shift assay (EMSA), hepatic metastasis model, immunofluorescence, immunohistochemistry, RNA-seq analysis and ChIP-seq analysis are described in Supplementary Materials and Methods.
Tissue samples, cell culture and transfection
The biospecimens and data used for this study were provided by the Biobank of Chonnam National University Hwasun Hospital, a member of the Korea Biobank Network. HEK293T, HT29,
TAp73 inhibits invasion and migration of colorectal cancer cells
To analyze the role of TAp73 in cancer progression, a tetracycline-inducible TAp73 expression plasmid (pTet-TAp73) was constructed and transfected into HCT116 (p53-/-) and HT29 colorectal cancer cell lines. An empty vector (pTet) was transfected as a control. The TAp73 protein levels in pTet-TAp73-transfected cells were significantly increased by treatment with the tetracycline analog doxycycline (Dox) at 100 ng/ml compared with control cells (Fig. 1A). Conversely, application of gene-specific
Discussion
TAp73 is known to function as a tumor suppressor and regulate genomic integrity, cellular proliferation, and apoptosis. Recently, it has been reported and reviewed that TAp73 and its multiple isoforms control cell invasion, migration, metastasis and EMT-mediated cancer cell stemness by regulation of EPLIN, p57Kip2, FOXF1, POSTN, miR-885-5p, MIR34A and MIR3158 in a variety of cancer cells [8], [9], [11], [29], [30], [31], [32], [33]. However, its role in colorectal cancer invasion and metastasis
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the National Research Foundation of Korea grant (MRC, 2011-0030132) funded by the Korea government (MSIP). We thank Myung-Sook Park and Ji-Na Choi for conducting the experimental studies.
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These authors contributed equally in this work.