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  • gap-27 Here we report for the

    2020-02-05

    Here, we report for the first time that EphB4 contains two NLS sequences and localises to the nucleus. This is mediated via an importin-α pathway and we show that EphB4 could potentially have a direct role in gene regulation. These data provide new insight into Eph receptor localisation and action and provides a basis for further study of novel functions for this cancer-associated receptor tyrosine kinase.
    Materials and methods
    Results
    Discussion Abundant evidence shows that both full-length and/or proteolytic gap-27 fragments of some RTKs may be translocated to the nucleus where they play important roles in oncogenesis [33], [34], [35]. To date, of the 58 reported human receptor tyrosine kinases, 18 have been found within the nucleus [36]. This includes another member of the Eph family, EphA4, which was found in the nuclei of the SaOS-2 osteoblastic cancer cell line, however the mechanism by which EphA4 translocates to the nucleus remains to be determined [37]. Here we provide evidence that the receptor tyrosine kinase EphB4 is located in the nucleus of cancer gap-27 that endogenously over-express this receptor and in non-tumorigenic breast cells that have been transformed by exogenous EphB4 over-expression. The most common mechanism of nuclear translocation of any protein is via an encoded NLS to which binds a member of the dedicated nuclear transport receptor family of β-karyopherins which includes the importins [23]. Our results show that EphB4 has two NLS sequences that are both capable of directing protein sequences into the nucleus and given that immunoprecipitation of EphB4 under native conditions also pulls-down importin-α, we suggest that nuclear translocation of EphB4 can occur by a classical nuclear transport process. The intracellular portion of ephrin-B1 has been suggested to translocate to the nucleus after γ-secretase-dependent cleavage in neuronal cells in a classic nuclear import manner [38]. We suggest that full-length EphB4 is located to the nucleus of cancer cells as demonstrated by sub-cellular fractionation. For the full-length protein to translocate from its membrane spanning location into the nucleus, it is hypothesised that it needs to ‘escape’ from the lipid bilayer [39]. Based on the research exploring trafficking of EGFR from early endosomes to the nucleus, several mechanisms have been proposed such as retrograde transport to the Golgi and the ER and lateral diffusion from the ER all of which depend on importin-α/β-mediated nuclear transport to get EGFR into the nucleus via the importins binding to a NLS [18], [40], [41]. Further research needs to address the precise trafficking mechanisms of EphB4 to the nucleus which could involve blocking importin-α [42] and generating full-length EphB4 NLS mutants. Several possible nuclear functions for EphB4 may be implied from studies of other RTKs that translocate to the nucleus. In prostate cancer, nuclear ERBB3 has been shown to discriminate normal from malignant tissues and between tumours from hormone-sensitive versus hormone-refractory prostate cancer [43]. Nuclear EGFR has been implicated as a prognostic factor in breast cancer [44], [45], as a driver in chemo- and radiation-resistance [46], [47] and as a predictor of poorer outcomes in various cancer types [33], [48]. It will be important to determine whether nuclear EphB4 also correlates with aggressive and/or resistant prostate cancers. Nuclear EGFR is reported to act as a co-activator for transcription of genes such as cyclin-D1 [22], [49] in regulating DNA replication and repair [50], [51]. In our study we have identified several genomic sequences to which EphB4 may bind and these include sequences upstream of, and within, coding sequences of genes that have been linked to prostate cancer including GPR103, a G-protein coupled receptor that shows increased expression with increased severity of disease and is linked to neuroendocrine differentiation [52] and Lef1, whose over-expression in LNCaP cells resulted in increased androgen receptor expression and enhanced growth and invasion [32]. Lef1 was validated as being differentially regulated when EphB4 levels were manipulated. Further studies need to determine the relationship between those two molecules as EphB4 could potentially drive Lef1 expression.