Lana McClements

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. Clin Cancer Res; 19(14); 3881–93. ©2013 AACR.

Identification of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identified a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-β-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-α (ERα). Furthermore, FKBPL and RBCK1 associate with ERα at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

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 FKBPLs antiangiogenic and antistemness activity.

FKBPL Is a Critical Antiangiogenic Regulator of Developmental and Pathological Angiogenesis

Objective— The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models. Approach and Results— FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL’s critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl +/− mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish. Conclusions— FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes.

Risk of pre-eclampsia in women taking metformin: a systematic review and meta-analysis

To perform meta‐analyses of studies evaluating the risk of pre‐eclampsia in high‐risk insulin‐resistant women taking metformin prior to, or during pregnancy.

Elucidating the Pathogenesis of Pre-eclampsia Using In Vitro Models of Spiral Uterine Artery Remodelling

Purpose of ReviewThe aim of the study is to perform a critical assessment of in vitro models of pre-eclampsia using complementary human and cell line-based studies. Molecular mechanisms involved in spiral uterine artery (SUA) remodelling and trophoblast functionality will also be discussed.Recent FindingsA number of proteins and microRNAs have been implicated as key in SUA remodelling, which could be explored as early biomarkers or therapeutic targets for prevention of pre-eclampsia.SummaryVarious 2D and 3D in vitro models involving trophoblast cells, endothelial cells, immune cells and placental tissue were discussed to elucidate the pathogenesis of pre-eclampsia. Nevertheless, pre-eclampsia is a multifactorial disease, and the mechanisms involved in its pathogenesis are complex and still largely unknown. Further studies are required to provide better understanding of the key processes leading to inappropriate placental development which is the root cause of pre-eclampsia. This new knowledge could identify novel biomarkers and treatment strategies.

The Role of Peptidyl Prolyl Isomerases in Aging and Vascular Diseases

Peptidyl prolyl isomerases (PPIases) are proteins belonging to the immunophilin family and are characterised by their cis-trans isomerization activity at the X-Pro peptide bond, in addition to their tetratricopeptide repeat (TPR) domain, important for interaction with the molecular chaperone, Hsp90. Due to this unique structure these proteins are able to facilitate protein-protein interactions which can impact significantly on a range of cellular processes such as cell signalling, differentiation, cell cycle progression, metabolic activity and apoptosis. Malfunction and/or dysregulation of most members of this class of proteins promotes cellular damage and tissue/organ failure, predisposing to ageing and age-related diseases. Many individual genes within the PPIase family are associated with several age-related diseases including cardiovascular diseases (CVDs), atherosclerosis, type II diabetes mellitus (T2D), chronic kidney disease (CDK), neurodegeneration, cancer and age-related macular degeneration (AMD), in addition to the ageing process itself. This review will focus on the different roles of PPIases, and their therapeutic/ biomarker potential in these age-related vascular diseases.

MESENCHYMAL STEM CELLS INFLUENCE TROPHOBLAST AND ENDOTHELIAL CELL FUNCTIONALITY IMPORTANT FOR PREVENTION OF PRE-ECLAMPSIA VIA A NOVEL ANTI-ANGIOGENIC PROTEIN, FKBPL

Objective: Pre-eclampsia is a disorder affecting 5–6% of all pregnancies globally. It is characterised by the new onset of hypertension and proteinuria post 20 weeks gestation. Pre-eclampsia is a leading cause of morbidity and mortality in both mothers and children. Although the pathogenesis of pre-eclampsia is poorly understood, aberrant angiogenesis and inadequate trophoblast cell function have both been implicated. Mesenchymal Stem Cell (MSC)-based therapies have shown benefits in animal models of pre-eclampsia however, the underlying mechanisms are not well understood. FKBPL is a novel anti-angiogenic protein which has a critical role in developmental, physiological and pathological angiogenesis. In this study, our objective was to evaluate the effects of MSC–conditioned medium (MSC-CM) on migration and differentiation of trophoblast and endothelial cell lines under both normoxic and hypoxic conditions. The role of FKBPL signalling in these processes was also investigated. Design and method: Human trophoblast (BeWo and Jar) and endothelial cells (HUVEC) were incubated in the presence of human bone marrow- derived MSC-CM, normal or serum free medium under normoxia (21% oxygen) or hypoxia (1% oxygen). The confluent cell monolayer was wounded and the percentage of wound closure assessed at 24 h. HUVEC were stained with Calcein prior to 6-hour incubation in the presence of MSC-CM, normal or serum free medium under normoxia or hypoxia. Tubule formation was assessed using Image J. FKBPL protein expression was evaluated using western blot analysis where cells were lysed in RIPA buffer before being subjected to western blotting and probed for FKBPL and GAPDH. Results: MSC-CM promoted cell migration significantly in all three cell lines under both normoxia and hypoxia compared to normal medium (n = 6; p < 0.001). MSC-CM also significantly increased the formation of HUVEC tubule networks under both normoxia and hypoxia compared to normal medium. Furthermore, concomitant reduction in FKBPL protein expression was observed, demonstrating potential FKBPLs involvement in MSC-driven effects on trophoblast and endothelial cell functionality. Conclusions: Our findings suggest that MSCs could be explored as potential preventative therapy for pre-eclampsia by ameliorating trophoblast and endothelial cell functionality, and that the mechanism is likely to involve a novel anti-angiogenic protein, FKBPL.

6 The role of a novel angiogenesis related protein, FKBPL, in spiral uterine artery remodelling important for the pathogenesis of preeclampsia

Introduction Preeclampsia is a complication which occurs in 5%–6% of pregnancies, characterised by high blood pressure and/or other organ dysfunction in the third trimester of pregnancy. Preeclampsia has short-term risks for the mother and child, but is also associated with remote cardiovascular disease and/or type 2 diabetes mellitus in both. The pathogenesis of preeclampsia is unclear but it appears to be attributed to inappropriate remodelling of spiral uterine artery as a result of dysregulated trophoblast function. We investigated the involvement of novel regulator of developmental and pathological angiogenesis, FKBPL, and its role in the pathophysiology of preeclampsia. Methods Trophoblast cells (HTR8.SV.neo, BeWo and JAR) were exposed to hypoxic (1%) or normoxic (21%) conditions before wound scratch migration assays were performed, and FKBPL protein levels measured. BeWo cells were treated with the HIF-1α activator, DMOG, for 24 hour before protein lysates were extracted for western blotting analysis. Colony forming efficiency and the number of holoclones, meroclones and paraclones of both HTR8.SV.neo and JAR trophoblast cells were determined in the presence of hypoxia or normoxia via clonogenic assay. Results BeWo and JAR migration increased by approximately 40% following 24 hour exposure to hypoxia (n=6; BeWo, p<0.05; JAR, p<0.01), and FKBPL protein expression was downregulated (n=3; HTR8.SV.neo, p<0.01; BeWo, p<0.05; JAR, p<0.01), when compared to normoxia. DMOG treatment downregulated FKBPL protein levels in BeWo cells (n=3, p<0.01). JAR colony formation was reduced by approximately 70% in hypoxia (n=3, p<0.01); all colonies appeared to be holoclones. No change in colony formation was observed in HTR8.SV.neo cells; however, there was over two-fold reduction of holoclones, and an increase in differentiated colonies, meroclones plus paraclones (n=3, p<0.05). Conclusion Our in vitro data suggest that FKBPL plays an important role in trophoblast functionality, which may extend to spiral uterine artery remodelling underlying the pathogenesis of pre-eclampsia.