Abstract
Some highly metastatic types of breast cancer show decreased intracellular levels of the tumor suppressor protein NME1, also known as nm23-H1 or nucleoside diphosphate kinase A (NDPK-A), which decreases cancer cell motility and metastasis. Since its activity is directly correlated with the overall outcome in patients, increasing the cytosolic levels of NDPK-A/NME1 in such cancer cells should represent an attractive starting point for novel therapeutic approaches to reduce tumor cell motility and decrease metastasis. Here, we established the Bacillus anthracis protein toxins’ transport component PA63 as transporter for the delivery of His-tagged human NDPK-A into the cytosol of cultured cells including human MDA-MB-231 breast cancer cells. The specifically delivered His6-tagged NDPK-A was detected in MDA-MB-231 cells via Western blotting and immunofluorescence microscopy. The PA63-mediated delivery of His6-NDPK-A resulted in reduced migration of MDA-MB-231 cells, as determined by a wound-healing assay. In conclusion, PA63 serves for the transport of the tumor metastasis suppressor NDPK-A/NME1 into the cytosol of human breast cancer cells In Vitro, which reduced the migratory activity of these cells. This approach might lead to development of novel therapeutic options.
Highlights
Metastasis is the leading cause of mortality in cancer patients, even if the primary tumor has been removed
In In Vitro experiments performed in a variety of tumor cells, it was shown that nucleoside diphosphate kinase A (NDPK-A)/NME1 re-expression reduced the migration in Boyden chamber as well as wound healing assays stimulated with multiple attractants, which suggest a central role in the regulation of tumor cell motility [8,9,10]
His6-NDPK-A was recombinantly expressed in E. coli BL21 and purified via affinity chromato2g.1r.aPpuhriyfi.caTtihoneainddeCnhtaitryactoerfizthateionpuofrRifieceodmbHiniasn6tl-yNEDxpPreKss-eAd Hpirs6o-NteDinPKw-Aas confirmed by Western blotting with a specificHaisn6-tNibDoPdKy-Adirweacsterdecaogmabininsatntthlye aexmpirnesoseadcidinreEs.idcuoliesB1L3241-1a5n2dofphuruifmiedanvNiaDaPffKin-iAty (Figure 1B)
Summary
Metastasis is the leading cause of mortality in cancer patients, even if the primary tumor has been removed. Metastasis suppressors are proteins which exert no inhibitory effect on primary tumor growth, but significantly reduce metastasis. Increasing the amount of such a protein present in highly metastatic tumor cells might offer an opportunity to interfere with these processes [3,4]. The human nm23-H1 gene, more generally named nme, was the first metastasis suppressor identified [5]. Overexpression of, for example, NDPK-A/NME1 in metastatic tumor cell lines significantly reduced In Vivo metastasis with no effect on primary tumor size [7]. Recent data showed that dynamin 2 oligomerization is promoted by NDPK-A/NME1 in breast cancer cells. As dynamin oligomerization is required for endocytosis of, e.g., chemotactic EGF receptors and others, the enhancement of the internalization of such receptors by NDPK-A/NME1 might be part of the underlying mechanism [11]
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