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

RALA-mediated delivery of FKBPL nucleic acid therapeutics

AIMS: RALA is a novel 30 mer bioinspired amphipathic peptide that is showing promise for gene delivery. Here, we used RALA to deliver the FK506-binding protein like – FKBPL gene (pFKBPL) – a novel member of the immunophilin protein family. FKBPL is a secreted protein, with overexpression shown to inhibit angiogenesis, tumor growth and stemness, through a variety of intra- and extracellular signaling mechanisms. We also elucidated proangiogenic activity and stemness after utilizing RALA to deliver siRNA (siFKBPL). MATERIALS & METHODS: The RALA/pFKBPL and RALA/siFKBPL nanoparticles were characterized in terms of size, charge, stability and toxicity. Overexpression and knockdown of FKBPL was assessed in vitro and in vivo. RESULTS: RALA delivered both pFKBPL and siFKBPL with less cytotoxicity than commercially available counterparts. In vivo, RALA/pFKBPL delivery retarded tumor growth, and prolonged survival with an associated decrease in angiogenesis, while RALA/siFKBPL had no effect on tumor growth rate or survival, but resulted in an increase in angiogenesis and stemness. CONCLUSION: RALA is an effective delivery system for both FKBPL DNA and RNAi and highlights an alternative therapeutic approach to harnessing FKBPL's antiangiogenic and antistemness activity

FKBPL regulates estrogen receptor signaling and determines response to endocrine therapy

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor alpha (ER alpha) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERa phosphorylation on Ser(118) in response to 17 beta-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome. Cancer Res; 70(3); 1090-100. (C)2010 AACR

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

The emerging role of FK506-binding proteins as cancer biomarkers: a focus on FKBPL

FKBPs (FK506-binding proteins) have long been recognized as key regulators of the response to immunosuppressant drugs and as co-chaperones of steroid receptor complexes. More recently, evidence has emerged suggesting that this diverse protein family may also represent cancer biomarkers owing to their roles in cancer progression and response to treatment. FKBPL (FKBP-like) is a novel FKBP with roles in GR (glucocorticoid receptor), AR (androgen receptor) and ER (oestrogen receptor) signalling. FKBPL binds Hsp90 (heat-shock protein 90) and modulates translocation, transcriptional activation and phosphorylation of these steroid receptors. It has been proposed as a novel prognostic and predictive biomarker, where high levels predict for increased recurrence-free survival in breast cancer patients and enhanced sensitivity to endocrine therapy. Since this protein family has roles in a plethora of signalling pathways, its members represent novel prognostic markers and therapeutic targets for cancer diagnosis and treatment

Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01, via the CD44 pathway

PURPOSE: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC). EXPERIMENTAL DESIGN: Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples, and xenografts. Delays in tumor initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays, quantitative PCR (qPCR), and immunofluorescence. RESULTS: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA(+)/CD44(+)/CD24(-) or aldehyde dehydrogenase (ALDH)(+) cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01-mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4, and Sox2, were also significantly reduced. Furthermore, we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signaling. CONCLUSIONS: AD-01 has dual antiangiogenic and anti-BCSC activity, which will be advantageous as this agent enters clinical trial

FKBPL and its peptide derivative, AD-01, bind CD44.

<p>(<b>A</b>) Representative western blot showing FKBPL co-immunoprecipitated with CD44 in HMEC-1 cells; immuno-blotted with anti-CD44 antibody; n = 3; Cdc42 was used as a positive control for the CD44 interaction and rabbit and murine IgGs were used as negative controls. (<b>B</b>) Schematic diagram of the Biacore assay using AD-01 immobilised on CM5 chip surface. Binding of anti-AD-01 antibody to AD-01-CM5 surface was inhibited by AD-01 in solution in a dose dependent manner, with excellent sensitivity in lower concentration range of peptide; 1–500 nM. Scrambled AD-01, used as a negative control, did not demonstrate any binding to anti-AD-01 up to 200 µM. Competition of the anti-AD-01 antibody interaction with its cellular partner/s results in increased binding of anti-AD-01 antibody on chip surface. (<b>C</b>) Representative graph demonstrating that AD-01 specifically binds to CD44 immunoprecipitated from MDA-MB-231 cells using the assay described. CD44 was immuno-purified from cell lysate and analysed using Biacore Q. Isotype control mIgG antibody was used as control. Bar charts show the relative binding of anti-AD-01 antibody in presence of AD-01, calculated as the percentage of the maximum resonance binding units in the presence of 0.001 and 0.01 µM AD-01. Data points show means ± SEM of 5 independent experiments (p-value was determined by one way ANOVA). (<b>D</b>) No competition of anti-AD-01 antibody binding to immobilised AD-01 was obtained in the presence of various concentrations of rEGFR indicating a specificity of AD-01-CD44 interaction.</p

AD-01 and FKBPL mediate cytoskeletal changes

<p><b>and AD-01 disrupts the RhoA-Rac1 dynamics.</b> (<b>A</b>) Confocal images (60x) representing the changes in phalloidin/F-actin dynamics in MDA-MB-231 upon wounding and treatment with AD-01 (10⁻⁹M) for 24 h; n = 3. Treated monolayers were fixed and stained with TRITC-phallodin (red) and DAPI (blue). Intense actin staining was accompanied by the loss in cell direction and communication. (<b>B</b>) Images (40x) representing disruption in tubulin distribution in HMEC-1 cell monolayers, wounded and treated with rFKBPL 750 ng/ml for 5 h. Fixed monolayers were stained for tubulin, followed by FITC conjugated secondary antibody (green) and nucleus with PI (red). (<b>C</b>) RhoA expression was increased after wounding and treatment with AD-01 for 24 h, resulting in a concomitant increase in the downstream actin binding proteins vinculin and profilin. Cells monolayers were treated with AD-01 (± wounding) for 24 h and total cell lysates were subjected to immuno blotting as indicated. (<b>D</b>) Treatment with AD-01 (10⁻⁹M) for 10/60 min inhibited fMLP induction (30 sec) of GTP-Rac-1 in HMEC-1 cells. Cell lysates of treated monolayers were subjected to Rac GTPase pull down assay; n = 3. (<b>E</b>) Representative western blots and quantitative densitometric analysis demonstrating an inhibition of cofilin phosphorylation after treatment with AD-01. HA treatment up-regulated cofilin phosphorylation maintaining its inactive state. HMEC-1 cell monolayers were treated with AD-01 for 3 h or HA for 10 min, and the extracted membrane fractions were subjected to immunoblotting with cofilin/p-cofilin; n = 3.</p

FKBPL is present in various cell compartments and regulates cell migration and tumour vasculature.

<p>(<b>A</b>) Representative blot demonstrating that FKBPL is present predominantly in the cytosol and membrane compartments of both HMEC-1 (H) and MDA-MB-231 (M) cells and in the nuclear fraction of MDA-MB-231 cells<b>.</b> Protein extracts from each subcellular compartment probed with specific compartmental markers, vimentin, calpain and histone-H1 were used as loading controls. (<b>B</b>) Representative confocal images (60x) of MDA-MB-231 and HMEC-1 cells fixed, permeabilised and stained with DAPI (blue) and with anti-AD-01 primary antibody and Alexa-488 tagged secondary antibody demonstrating vesicular staining for FKBPL (green); n = 3. (<b>C</b>) Anti-AD-01 antibody targets the active domain of FKBPL and accelerates HMEC-1 cell migration in comparison to cells treated with an isotype control. Data points show means ± SEM; n = 3 (<b>D</b>) FKBPL knockdown with siRNA accelerated migration of HMEC-1 cells in comparison to un-transfected and NT-siRNA-transfected cells. Data points show means ± SEM; n = 3. Cell migration was assessed using scratch wound assay. Wound size is normalised to that of T₀. p-value was determined using two-way ANOVA. (<b>E</b>) Intravital microscopy images (20x) representing disruption of tumour vasculature <i>in vivo</i> in FKBPL-overexpressing MDA-MB-231 xenografts in comparison to those derived from parental MDA-MB-231 cells. Tumours (21 days) were imaged using Epi-fluoresence microscopy following injection of mice with FITC-Dextran. Quantification of vessel dynamics was carried out on 3D images using ImageJ software. n = 5 mice per treatment group (p-value was determined using two-tailed T –test).</p