The Anti-Migratory Effects of FKBPL and Its Peptide Derivative, AD-01: Regulation of CD44 and the Cytoskeletal Pathway

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity in vitro and in vivo and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development in vivo suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks

FKBPL: A marker of good prognosis in breast cancer

FK506-binding protein-like (FKBPL) has established roles as an anti-tumor protein, with a therapeutic peptide based on this protein, ALM201, shortly entering phase I/II clinical trials. Here, we evaluated FKBPL's prognostic ability in primary breast cancer tissue, represented on tissue microarrays (TMA) from 3277 women recruited into five independent retrospective studies, using immunohistochemistry (IHC). In a meta-analysis, FKBPL levels were a significant predictor of BCSS; low FKBPL levels indicated poorer breast cancer specific survival (BCSS) (hazard ratio (HR) = 1.30, 95% confidence interval (CI) 1.14-1.49, p < 0.001). The prognostic impact of FKBPL remained significant after adjusting for other known prognostic factors (HR = 1.25, 95% CI 1.07-1.45, p = 0.004). For the sub-groups of 2365 estrogen receptor (ER) positive patients and 1649 tamoxifen treated patients, FKBPL was significantly associated with BCSS (HR = 1.34, 95% CI 1.13-1.58, p < 0.001, and HR = 1.25, 95% CI 1.04-1.49, p = 0.02, respectively). A univariate analysis revealed that FKBPL was also a significant predictor of relapse free interval (RFI) within the ER positive patient group, but it was only borderline significant within the smaller tamoxifen treated patient group (HR = 1.32 95% CI 1.05-1.65, p = 0.02 and HR = 1.23 95% CI 0.99-1.54, p = 0.06, respectively). The data suggests a role for FKBPL as a prognostic factor for BCSS, with the potential to be routinely evaluated within the clinic

Functions of the Hsp90-binding FKBP Immunophilins.

Hsp90 functionally interacts with a broad array of client proteins, but in every case examined Hsp90 is accompanied by one or more co-chaperones. One class of co-chaperone contains a tetratricopeptide repeat domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is now clear that the client protein influences, and is influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members

FKBP (FK506 Binding Protein)

In the 70s, after a decade from the purification of cyclosporine, a selective immunosuppressant agent and potent tool in transplantation medicine, a novel molecule was purified from bacteria Streptomyces tsukubaensis. This molecule, called FK506, showed the same selective immunosuppressant action as cyclosporine but was 10 to 100 fold more potent. In an attempt to clarify the molecular mechanism through which the new drug exerted such a selective effect on T-cells activation, two laboratories identified the cytosolic receptor for FK506. This so-called FK506 binding protein (FKBP) was purified from bovine thymus, human spleen, and Jurkat T-cell line. The isolated FKBP had an approximate molecular mass of 14 kDa and showed an isomerase activity similar to the recently purified cyclosporine-binding protein, cyclophilin, but, it was inhibited by FK506 and rapamycin but not cyclosporine. The subsequent cloning of FKBP gene revealed that FKBP and cyclophilin had dissimilar sequences in spite of their common enzymatic activity. The identified FKBP gene encoded for a protein of 108 aminoacids with a relative molecular mass of 11,819. For this reason, the progenitor of this nascent class of proteins was later known as FKBP12. The subsequent studies showed that FKBP12 was just a member of a ubiquitous and evolutionarily conserved sub-family of proteins which differ from each other in their molecular weight and structure. All FKBPs share a highly conserved domain, termed “FK-12 like domain”, capable of binding to FK506 and exerting isomerase properties, i.e. interconversion from cis-to-trans and trans-to-cis of peptide bonds involving proline, on protein substrates. A schematic historical background of the 17 FKBPs so far identified is shown. A general overview of FKBP structure, function and eventually associated disease is given in this monograph, with the order of proteins following the chronology of discovery

Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore

In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and MR (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR)-domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor “transformation”) is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore