Changes in dietary fat intake and associations with mental health in a UK public sample during the COVID-19 pandemic.

BACKGROUND: Consumption of unhealthy foods may have changed during the COVID-19 pandemic. This study explored how dietary fat intake was impacted in a sample of the UK public who were social distancing during the COVID-19 pandemic. METHODS: Data were collected from a UK COVID-19 online survey. Fat intake was measured using the Dietary Instrument for Nutrition Education questionnaire. Anxiety and depressive symptoms were assessed using Becks' Anxiety and Depression Inventories, while the short-form Warwick-Edinburgh Mental Well-being Scale assessed mental well-being. Differences between individuals who increased versus decreased fat intake were explored using chi-square or independent sample t-tests. Association between fat intake and mental health was explored using adjusted linear regression models. RESULTS: Eight hundred and eighty-seven adults were included. Approximately, 34% recorded medium-to-high levels of fat consumption during social distancing. Around 48% reported decreased fat intake during social distancing compared to usual levels, while 41.3% documented increased fat intake. Fat intake was not significantly associated (P > 0.05) with any measures of mental health. CONCLUSIONS: A higher proportion of a sample of UK adults social distancing during the COVID-19 pandemic recorded decreased fat intake when compared to levels prior to social distancing. There appeared to be no associations between fat intake and mental health.

FKBPL-based peptide, ALM201, targets angiogenesis and cancer stem cells in ovarian cancer.

BACKGROUND: ALM201 is a therapeutic peptide derived from FKBPL that has previously undergone preclinical and clinical development for oncology indications and has completed a Phase 1a clinical trial in ovarian cancer patients and other advanced solid tumours. METHODS: In vitro, cancer stem cell (CSC) assays in a range of HGSOC cell lines and patient samples, and in vivo tumour initiation, growth delay and limiting dilution assays, were utilised. Mechanisms were determined by using immunohistochemistry, ELISA, qRT-PCR, RNAseq and western blotting. Endogenous FKBPL protein levels were evaluated using tissue microarrays (TMA). RESULTS: ALM201 reduced CSCs in cell lines and primary samples by inducing differentiation. ALM201 treatment of highly vascularised Kuramochi xenografts resulted in tumour growth delay by disruption of angiogenesis and a ten-fold decrease in the CSC population. In contrast, ALM201 failed to elicit a strong antitumour response in non-vascularised OVCAR3 xenografts, due to high levels of IL-6 and vasculogenic mimicry. High endogenous tumour expression of FKBPL was associated with an increased progression-free interval, supporting the protective role of FKBPL in HGSOC. CONCLUSION: FKBPL-based therapy can (i) dually target angiogenesis and CSCs, (ii) target the CD44/STAT3 pathway in tumours and (iii) is effective in highly vascularised HGSOC tumours with low levels of IL-6.

Challenges in the Practice of Sexual Medicine in the Time of COVID-19 in the United Kingdom.

BACKGROUND: On 23rd March 2020, the UK government released self-isolation/social distancing guidance to reduce the risk of transmission of SARS-CoV-2. The influence such guidance has on sexual activity is not known. AIM: To investigate levels and correlates of sexual activity during COVID-19 self-isolation/social distancing in a sample of the UK public. METHODS: This paper presents preplanned interim analyses of data from a cross-sectional epidemiological study, administered through an online survey. OUTCOMES: Sexual activity was measured using the following question: "On average after self-isolating how many times have you engaged in sexual activity weekly?" Demographic and clinical data were collected, including sex, age, marital status, employment, annual household income, region, current smoking status, current alcohol consumption, number of chronic physical conditions, number of chronic psychiatric conditions, any physical symptom experienced during self-isolation, and number of days of self-isolation/social distancing. The association between several factors (independent variables) and sexual activity (dependent variable) was studied using a multivariable logistic regression model. RESULTS: 868 individuals were included in this study. There were 63.1% of women, and 21.8% of adults who were aged between 25 and 34 years. During self-isolation/social distancing, 39.9% of the population reported engaging in sexual activity at least once per week. Variables significantly associated with sexual activity (dependent variable) were being male, a younger age, being married or in a domestic partnership, consuming alcohol, and a higher number of days of self-isolation/social distancing. CLINICAL IMPLICATIONS: In this sample of 868 UK adults self-isolating owing to the COVID-19 pandemic, the prevalence of sexual activity was lower than 40%. Those reporting particularly low levels of sexual activity included females, older adults, those not married, and those who abstain from alcohol consumption. STRENGTH AND LIMITATIONS: This is the first study to investigate sexual activity during the UK COVID-19 self-isolation/social distancing. Participants were asked to self-report their sexual activity potentially introducing self-reporting bias into the findings. Second, analyses were cross-sectional and thus it is not possible to determine trajectories of sexual activity during the current pandemic. CONCLUSION: Interventions to promote health and well-being during the COVID-19 pandemic should consider positive sexual health messages in mitigating the detrimental health consequences in relation to self-isolation/social distancing and should target those with the lowest levels of sexual activity. Jacob L, Smith L, Butler L, et al. Challenges in the Practice of Sexual Medicine in the Time of COVID-19 in the United Kingdom. J Sex Med 2020;17:1229-1236.

FKBPL and its peptide derivatives inhibit endocrine therapy resistant cancer stem cells and breast cancer metastasis by downregulating DLL4 and Notch4.

BACKGROUND: Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease. METHODS: In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett's multiple comparison test. RESULTS: We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4. CONCLUSION: This study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.

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.

Heart rate variability biofeedback to reduce anxiety in autism spectrum disorder - a mini review.

There is a reported high prevalence of anxiety in people with autism spectrum disorder. This mini review appraises existing research investigating heart rate variability biofeedback to help manage symptoms of anxiety in people with autism spectrum disorder. A thorough search of electronic databases was conducted to find relevant literature. Consultation with experts and a librarian helped develop search terms following the PICO framework. Five databases were searched, and screening was undertaken using Covidence software, with the process outlined in a PRISMA flowchart. The latest review showed positive short-term effects but there is a need for long-term follow-up. Future investigations should consider device type, training settings, and control interventions. Accurate heart rate variability assessment independent of biofeedback devices is crucial. Additional measures like cortisol assessment and user feedback are recommended for comprehensive evaluation. The findings highlight progress in the evidence base and offer insight to future directions.

Changes in Health-Related Behaviours and Mental Health in a UK Public Sample during the First Set of COVID-19 Public Health Restrictions.

Public health restrictions, in response to the COVID-19 pandemic, have had potentially wide-ranging, unintended effects on health-related behaviours such as diet and physical activity and also affected mental health due to reduced social interactions. This study explored how health-related behaviours and mental health were impacted in a sample of the UK public during the first set of COVID-19 public health restrictions. Two online surveys were administered in the UK, one within the first three months of the restrictions (Timepoints 1 (T1­involving pre-pandemic recall) and 2/T2) and another ten weeks later (Timepoint 3/T3). Moderate−vigorous physical activity (MVPA), outdoor time, sitting time, screen time and sexual activity were self-reported. Diet was assessed using the Dietary Instrument for Nutrition Education questionnaire. Mental health was measured using the short-form Warwick−Edinburgh Mental Wellbeing Scale and Becks' Anxiety and Depression Inventories. Differences between timepoints were explored using the Friedman, Wilcoxon signed-rank, McNemar and McNemar−Bowker tests. Two hundred and ninety-six adults (74% under 65 years old; 65% female) provided data across all timepoints. Between T1 and T2, MVPA, time outdoors and sexual activity decreased while sitting, and screen time increased (p < 0.05). Between T2 and T3, saturated fat intake, MVPA, time outdoors, and mental wellbeing increased while sitting, screen time and anxiety symptoms decreased (p < 0.05). This study found that depending on the level of COVID-19 public health restrictions in place, there appeared to be a varying impact on different health-related behaviours and mental health. As countries emerge from restrictions, it is prudent to direct necessary resources to address these important public health issues.

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.

Alcohol use and mental health during COVID-19 lockdown: A cross-sectional study in a sample of UK adults.

BACKGROUND: The aim was to examine the correlates of increased alcohol consumption during the COVID-19 pandemic-related restrictions that were implemented in a sample of UK adults. METHODS: This paper presents analyses of data from a cross-sectional study. Adults aged 18 years and over, residing in the UK and self-isolating from others outside their own household were eligible to participate. Participants reported increase or no increase in their level of alcohol consumption from before to during lockdown, as well as symptoms of anxiety, depression and mental wellbeing. Socio-demographic characteristics were compared between adults with and without reported increased alcohol consumption. The associations between reported increased alcohol consumption and mental health outcomes were investigated using logistic and linear regression analyses. RESULTS: 691 adults (61.1 % women; 48.8 % aged 35-64 years) were included in the analysis. Of these, 17 % reported increased alcohol consumption after lockdown. A higher proportion of 18-34-year olds reported increased alcohol consumption compared to older groups. The prevalence of poor overall mental health was significantly higher in individuals with increased alcohol consumption (vs. no increase) (45.4 % versus 32.7 %; p-value = 0.01). There was a significant association between increased alcohol consumption and poor overall mental health (OR = 1.64; 95 % CI = 1.01, 2.66), depressive symptoms (unstandardized beta = 2.93; 95 % CI = 0.91, 4.95) and mental wellbeing (unstandardized beta=-1.38; 95 % CI=-2.38, -0.39). CONCLUSIONS: More than one in six UK adults increased their alcohol consumption during lockdown and a higher proportion of these were younger adults. Increased alcohol consumption was independently associated with poor overall mental health, increased depressive symptoms and lower mental wellbeing. These findings highlight the importance of planning targeted support as we emerge from lockdown and plan for potential second and subsequent waves.

Prevalence and correlates of physical activity in a sample of UK adults observing social distancing during the COVID-19 pandemic.

OBJECTIVE: To investigate the levels and correlates of physical activity during COVID-19 social distancing in a sample of the UK public. METHODS: This paper presents analyses of data from a cross-sectional study. Levels of physical activity during COVID-19 social distancing were self-reported. Participants also reported on sociodemographic and clinical data. The association between several factors and physical activity was studied using regression models. RESULTS: Nine hundred and eleven adults were included (64.0% were women and 50.4% of the participants were aged 35-64 years). 75.0% of the participants met the physical activity guidelines during social distancing. Meeting these guidelines during social distancing was significantly associated with sex (reference: male; female: OR=1.60, 95% CI 1.10 to 2.33), age (reference: 18-34 years; ≥65 years: OR=4.11, 95% CI 2.01 to 8.92), annual household income (reference: <£15 000; £15 000-<£25 000: OR=2.03, 95% CI 1.11 to 3.76; £25 000-<£40 000: OR=3.16, 95% CI 1.68 to 6.04; £40 000-<£60 000: OR=2.27, 95% CI 1.19 to 4.34; ≥£60 000: OR=2.11, 95% CI 1.09 to 4.09), level of physical activity per day when not observing social distancing (OR=1.00 (per 1 min increase), 95% CI 1.00 to 1.01), and any physical symptom experienced during social distancing (reference: no; yes: OR=0.31, 95% CI 0.21 to 0.46). CONCLUSION: During COVID-19, social distancing interventions should focus on increasing physical activity levels among younger adults, men and those with low annual household income. It should be noted in the present sample that women and younger adults are over-represented.

Correlates of symptoms of anxiety and depression and mental wellbeing associated with COVID-19: a cross-sectional study of UK-based respondents.

BACKGROUND: The aim was to assess the impact of COVID-19 self-isolation/social distancing on mental health, and potential correlates, among a sample of the UK population. METHODS: A cross-sectional study. Mental health was measured using the Beck Anxiety and Depression Inventory. Mental wellbeing was measured using The Short Warwick-Edinburgh Mental Well-being Scale. Data collected on predictors included sex, age, marital status, employment, annual income, region, current smoking, current alcohol consumption, physical multimorbidity, any physical symptoms experienced during self-isolation/social distancing, and the number of days of self-isolation/social distancing. The association between potential predictors and poor mental health was studied using a multivariable logistic regression. RESULTS: 932 participants were included. Factors associated with poor mental health were sex (reference: male; female: OR=1.89, 95%CI=1.34-2.68), age (18-24 years: reference;45-54 years: OR=0.27, 95%CI=0.14-0.53; 55-64 years: OR=0.24, 95%CI=0.12-0.47; 65-74years: OR=0.10, 95% CI=0.05-0.22; and ≥75years: OR=0.08,95% CI=0.03-0.24),annual income (<£15,000: reference; £25,000-<£40,000: OR=0.54, 95% CI=0.31-0.93; £40,000-<£60,000: OR=0.39, 95% CI=0.22-0.69; and ≥£60,000: OR=0.38, 95% CI=0.21-0.67), current smoking (yes: OR=2.59, 95%CI=1.62-4.20), and physical multimorbidity (OR=2.35, 95%CI=1.61-3.46). CONCLUSIONS: In this sample of UK adults self-isolating/social distancing females, younger age groups, those with a lower annual income, current smokers and those with physical multimorbidity were associated with higher levels of poor mental health.

The relationship between physical activity and mental health in a sample of the UK public: A cross-sectional study during the implementation of COVID-19 social distancing measures.

AIM: The aim of the present study was to investigate the cross-sectional association between physical activity levels with depressive symptoms, anxiety symptoms, and positive mental well-being in a sample of the UK public social distancing owing to COVID-19. METHOD: This paper presents pre-planned interim analyses of data from a cross-sectional epidemiological study. Levels of physical activity during COVID-I9 social distancing were self-reported. Mental health was measured using the Beck Anxiety and Depression Inventory. Mental wellbeing was measured using The Short Warwick-Edinburgh Mental Well-being Scale. Participants also reported on sociodemographic and clinical data. The association between physical activity and mental health was studied using regression models. RESULTS: 902 adults were included in this study (63.8% of women and 50.1% of people aged 35-64 years). After adjusting for covariates, there was a negative association between moderate-to-vigorous physical activity per day in hours and poor mental health (OR = 0.88, 95% CI = 0.80-0.97). Similar findings were obtained for moderate-to-severe anxiety symptoms, moderate-to-severe depressive symptoms and poor mental wellbeing. CONCLUSIONS: In the present sample of UK adults social distancing owing to COVID-19 those who were physically active have better overall mental health. Owing, to the cross-sectional design of the present study the direction of the association cannot be inferred.

User Centered Reading Intervention for Individuals with Autism and Intellectual Disability.

Individuals with autism and intellectual disability (ID) have complex learning needs and often have difficulty in acquiring reading comprehension skills using conventional teaching tools. Evidence based reading interventions for these learners and the use of assistive technology and application of behaviour analysis to develop user-centered teaching is discussed in this paper.

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.

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: 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.

Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N.

The bioreductive drug, AQ4N, is metabolized under hypoxic conditions and has been shown to enhance the antitumor effects of radiation and chemotherapy drugs. We have investigated the role of cytochrome P450 3A4 (CYP3A4) in increasing the metabolism of AQ4N using a gene-directed enzyme prodrug therapy (GDEPT) strategy. RIF-1 murine tumor cells were transfected with a mammalian expression vector containing CYP3A4 cDNA. In vitro AQ4N metabolism, DNA damage, and clonogenic cell kill were assessed following exposure of transfected and parental control cells to AQ4N. The presence of exogenous CYP3A4 increased the metabolism of AQ4N and significantly enhanced the ability of the drug to cause DNA strand breaks and clonogenic cell death. Cotransfection of CYP reductase with CYP3A4 showed a small enhancement of the effect in the DNA damage assay only. A single injection of CYP3A4 into established RIF-1 murine tumors increased the metabolism of AQ4N, and when used in combination with radiation, three of nine tumors were locally controlled for >60 days. This is the first demonstration that CYPs alone can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only CYP-activated bioreductive agent in clinical trials. Combination with a GDEPT strategy may offer a further opportunity for targeting radiation-resistant and chemo-resistant hypoxic tumor cells.

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.

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.