Hakai reduces cell-substratum adhesion and increases epithelial cell invasion

Translational Cancer Research Group, Instituto de Investigación Biomédica A Coruña (INIBIC), Complejo Hospitalario Universitario A Coruña (CHUAC), SERGAS, A Coruña, Spain.
BMC Cancer (Impact Factor: 3.36). 11/2011; 11(1):474. DOI: 10.1186/1471-2407-11-474
Source: PubMed


The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells.
Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed.
Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The expression of Paxillin was found to be regulated by a proteasome-independent mechanism, possibly due to the decreased abundance of E-cadherin.
Taken together, these results suggest that Hakai may be involved in two hallmark aspects of tumour progression, the lowering cell-substratum adhesion and the enhancement of cell invasion.

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    • "Since then, novel proteins substrates for Hakai have been identified, such as Cortactin, a protein critically involved in the reorganization of actin cytoskeleton in cell protrusions, and DOK1, which binds to p120-rasGAP, a potent inhibitor of Ras oncogene [10]. Besides influencing cell adhesion, Hakai has also been implicated in controlling cell migration and embryogenesis [11]–[13], and it can control cell proliferation in an E-cadherin-independent manner, further supporting a role for Hakai in early stages of tumorigenesis [14], [15]. Accordingly, Hakai is highly up-regulated in human colon adenocarcinomas compared to normal tissues. "
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    ABSTRACT: Gene expression is potently regulated through the action of microRNAs (miRNAs). Here, we present evidence of a miRNA regulating Hakai protein. Hakai was discovered as an E3 ubiquitin-ligase that mediates the posttranslational downregulation of E-cadherin, a major component of adherens junctions in epithelial cells and a potent tumour suppressor. Recent data have provided evidence that Hakai affects cell proliferation in an E-cadherin-independent manner, thus revealing a role for Hakai in the early stages of tumour progression. Furthermore, Hakai is highly up-regulated in human colon adenocarcinomas compared to normal tissues. However, the molecular mechanisms that regulate Hakai abundance are unknown. We identified two putative sites of miR-203 interaction on the Hakai mRNA, in its 3'-untranslated region (UTR). In several human carcinoma cell lines tested, overexpression of a miR-203 precursor (Pre-miR-203) reduced Hakai abundance, while inhibiting miR-203 by using an antisense RNA (Anti-miR-203) elevated Hakai levels. The repressive influence of miR-203 on the Hakai 3'-UTR was confirmed using heterologous reporter constructs. In keeping with Hakai's proliferative influence, Anti-miR-203 significantly increased cell number and BrdU incorporation, while Pre-miR-203 reduced these parameters. Importantly, the growth-promoting effects of anti-miR-203 required the presence of Hakai, because downregulation of Hakai by siRNA suppressed its proliferative action. Finally, in situ hybridization showed that miR-203 expression is attenuated in colon tumour tissues compared to normal colon tissues, suggesting that miR-203 could be a potential new prognostic marker and therapeutic target to explore in colon cancer. In conclusion, our findings reveal, for the first time, a post-transcriptional regulator of Hakai expression. Furthermore, by lowering Hakai abundance, miR-203 also reduces Hakai-regulated-cell division.
    Full-text · Article · Dec 2012 · PLoS ONE
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    • "Moreover, sequence inspection suggests that the Numb-associated E3 ligase LNX may contain an HYB domain [96]. Thus, the HYB domain appears to be a recurring feature in certain E3 ligases and its presence in cell adhesion regulatory proteins such as Hakai suggests a role for this domain in cell-cell adhesion and cancer metastasis [129]. The HYB domain is likely conserved through evolution as a sequence search identifies HYB-like domain sequences in different animals and plants with complete conservation of the zinc-coordinating residues (Figure 5B). "
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    ABSTRACT: SH2 domains are long known prominent players in the field of phosphotyrosine recognition within signaling protein networks. However, over the years they have been joined by an increasing number of other protein domain families that can, at least with some of their members, also recognise pTyr residues in a sequence-specific context. This superfamily of pTyr recognition modules, which includes substantial fractions of the PTB domains, as well as much smaller, or even single member fractions like the HYB domain, the PKCdelta and PKCtheta C2 domains and RKIP, represents a fascinating, medically relevant and hence intensely studied part of the cellular signaling architecture of metazoans. Protein tyrosine phosphorylation clearly serves a plethora of functions and pTyr recognition domains are used in a similarly wide range of interaction modes, which encompass, for example, partner protein switching, tandem recognition functionalities and the interaction with catalytically active protein domains. If looked upon closely enough, virtually no pTyr recognition and regulation event is an exact mirror image of another one in the same cell. Thus, the more we learn about the biology and ultrastructural details of pTyr recognition domains, the more does it become apparent that nature cleverly combines and varies a few basic principles to generate a sheer endless number of sophisticated and highly effective recognition/regulation events that are, under normal conditions, elegantly orchestrated in time and space. This knowledge is also valuable when exploring pTyr reader domains as diagnostic tools, drug targets or therapeutic reagents to combat human diseases.
    Full-text · Article · Nov 2012 · Cell Communication and Signaling
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    • "Hakai’s influence on cell attachment to the substrate and invasion capacity of epithelial MDCK cells was addressed [89]. In this system, Hakai overexpression leads to a reduction in cell adhesion to the substrate with impact on decreasing protein levels of Paxillin, a key focal adhesion-associated protein, although its downregulation was controlled by a proteasome-independent mechanism [89]. Nevertheless, further investigations of Hakai in in vitro and in vivo model systems would lead us to validate its role during tumorigenesis. "
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    ABSTRACT: In order to metastasize, cancer cells must first detach from the primary tumor, migrate, invade through tissues, and attach to a second site. Hakai was discovered as an E3 ubiquitin-ligase that mediates the posttranslational downregulation of E-cadherin, a major component of adherens junctions in epithelial cells that is characterized as a potent tumor suppressor and is modulated during various processes including epithelial-mesenchymal transition. Recent data have provided evidences for novel biological functional role of Hakai during tumor progression and other diseases. Here, we will review the knowledge that has been accumulated since Hakai discovery 10 years ago and its implication in human cancer disease. We will highlight the different signaling pathways leading to the influence on Hakai and suggest its potential usefulness as therapeutic target for cancer.
    Full-text · Article · Feb 2012 · CANCER AND METASTASIS REVIEW
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