Hakai is a 491 amino acid E3 ubiquitin-protein ligase that forms a dimer if zinc is available. This allows Src tyrosine kinase dependant interaction with the transmembrane adhesion protein E-cadherin. This interaction results in ubiquitination and degradation of E-cadherin, a key protein that normally interlocks epithelial cells. The re-targeting of E-cadherin away from the cell surface, resulting in its eventual degradation, is a key feature of cancers of an epithelial origin. Preventing the addition of ubiquitin to E-cadherin is potentially a novel therapeutic intervention.
Angélica Figueroa of Universidade da Coruña, Spain, and colleagues, generated a small molecule inhibitor of hakai called Hakin-1. The effect of Hakin-1 on the enzymatic activity of hakai was determined in a panel of banked tumour and Src transformed cell lines. The results were published in the journal Cancers.
Upon Hakin-1 treatment ubiquitination was reduced in a dose dependent manner, but hakai protein levels remained unaltered. This effect was not observed if hakai was not overexpressed. Reduced ubiquitination was also observed on immunoprecipitated E-cadherin in the presence of Hakin-1. In cell lines Hakin-1 treatment increased the total protein expression of E-cadherin.
Hakin-1 reduced colorectal tumour cell viability in a dose dependent manner. Some cell lines showed evidence of an altered morphology upon treatment. At concentrations above 100 µM Hakin-1 significantly decreased proliferation in some tumour cell lines.
Hakin-1 prevented tumour cell lines from forming colonies in soft agar and reduced cell migration. Treatment of mice with xenografts of hakai expressing MDCK cells reduced the tumour volume significantly and reduced expression of N-cadherin, a mesenchymal marker.
The study is limited by the fact that Hakin-1 may not be completely specific for hakai. The authors noted that Hakin-1 could potentially bind and inhibit other E3 ubiquitin-ligases containing a HYB domain, such as the testis-specific E3 ubiquitin-ligase ZNF645, also known as CBLL2. This possibility was not verified or refuted experimentally.
“Future investigations will help to elucidate the beneficial effect of Hakin-1 in human colon cancer treatment. In conclusion, Hakin-1 emerged as an effective therapeutic agent for [epithelial to mesenchymal transition] inhibition with therapeutic potential and our results constitute the first preclinical proof-of-concept that Hakai inhibitors could be useful as anticancer agents,” concluded the authors.