Exploring assessment of balance using virtual reality in patients at risk of chemotherapy-induced peripheral neuropathy.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting toxicity for people treated for cancer. Impaired balance and falls are functional consequences of CIPN. Virtual reality (VR) technology may be able to assess balance and identify patients at risk of falls. AIMS: To assess the impact of potentially neurotoxic chemotherapy on balance using VR, and explore associations between VR balance assessment, falls and CIPN. METHODS: This prospective, repeated measures longitudinal study was conducted at two Australian cancer centres. Eligible participants were commencing adjuvant chemotherapy containing a taxane for breast cancer, or oxaliplatin for colorectal cancer (CRC), per institutional guidelines. Balance assessments using VR were conducted at baseline, end of chemotherapy and 3 and 6 months after completion of chemotherapy. Participants also completed a comprehensive CIPN assessment comprising clinical and patient-reported outcomes, and recorded falls or near falls. RESULTS: Out of 34 participants consented, 24 (71%) had breast cancer and 10 (29%) had CRC. Compared to baseline, balance threshold was reduced in 10/28 (36%) evaluable participants assessed at the end of chemotherapy, and persistent in 7/22 (32%) at 6 months. CIPN was identified in 86% at end of chemotherapy and persisted to 6 months after chemotherapy completion in 73%. Falls or near falls were reported by 12/34 (35%) participants, and were associated with impaired VR balance threshold (P = 0.002). CONCLUSIONS: While VR balance assessment was no better at identifying CIPN than existing measures, it is a potential surrogate method to assess patients at risk of falls from CIPN.

Patterns of Patient-Reported Chemotherapy-Induced Peripheral Neuropathy in Colorectal Cancer Survivors.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) can be a debilitating toxicity of oxaliplatin used for treatment of colorectal cancer (CRC). We aimed to assess CIPN symptoms and associations in our colorectal survivorship population and review the impact of neurotoxicity on dose delivery of oxaliplatin. PATIENTS AND METHODS: Patients attending their first visit to the Sydney Cancer Survivorship Centre following completion of adjuvant treatment for CRC completed comprehensive patient-reported outcome measures, including symptoms, quality of life (QoL), alcohol intake, and exercise habits. Participants scored symptoms of "numbness or pins and needles" in hands or feet from 0 (no trouble at all) to 10 (worst I can imagine). Diagnosis, treatment, and comorbidity details were obtained from medical records. A subset of patients completed serial assessments of PN symptoms at follow-up visits. RESULTS: Data were analyzed from 233 patients (52% male; mean age, 63 years) with CRC attending their first visit at the Sydney Cancer Survivorship Centre. A subset of 104 patients were included in the longitudinal analysis. The odds of patient-reported numbness were significantly higher in patients receiving oxaliplatin (odds ratio, 5.6; 95% CI, 3.2-9.8), with 72.4% of oxaliplatin-treated CRC survivors reporting numbness an average of 5.9 months after chemotherapy. Mean patient-reported numbness was significantly higher in those who received oxaliplatin-containing chemotherapy (mean, 3.31) compared with fluoropyrimidines alone (mean, 1.37) and no chemotherapy (mean, 0.66). Of the patients receiving oxaliplatin, 80% required dose reduction or early cessation, with PN the most common reason reported. QoL in physical, emotional, and functional well-being domains was lower in patients with numbness. We found a weak negative association between numbness score and age, and between (1) numbness and cardiovascular disease and (2) numbers and pain score. CONCLUSIONS: CIPN symptoms are common in CRC survivors who have received oxaliplatin and are associated with lower QoL. Neurotoxicity is underreported in clinical trials compared with real-world populations and is a major barrier to oxaliplatin treatment delivery.

Evaluating laser photobiomodulation for chemotherapy-induced peripheral neuropathy: a randomised phase II trial.

PURPOSE: This study aims to evaluate the efficacy and safety of laser photobiomodulation (PBM) for treatment of established chemotherapy-induced peripheral neuropathy (CIPN) in cancer survivors. METHODS: We conducted a randomised phase II, non-comparative, sham-controlled, single-blinded clinical trial in 44 cancer survivors reporting CIPN symptoms at least 3 months following completion of neurotoxic chemotherapy. Participants were randomised 2:1 to either PBM laser or sham control delivered twice weekly for 12 sessions. Assessments were conducted at baseline, the end of intervention (6 weeks), and 6 weeks post intervention (12 weeks). Participants completed neuropathy, quality of life and function questionnaires, and a clinical neurological assessment. The primary outcome was proportion of participants with CIPN response, defined as either symptom resolution or reduction of minimally clinically important difference. RESULTS: In the laser and control groups, CIPN response rates were - 48% and 53% at 6 weeks and 45% and 33% at 12 weeks, respectively. The null hypothesis that the true response rate is 5% in the laser arm was rejected at both 6 and 12 weeks (p < 0.001 for both). Compared to baseline, patient-reported CIPN improved in both laser and control groups after the intervention. At 12 weeks, improvement was sustained in the laser group and approaching baseline in the control group. Clinical signs, quality of life, and function remained stable in both groups. Low-grade "side-effects" were observed in both arms. CONCLUSION: PBM may offer clinically meaningful symptom benefit in cancer survivors with established CIPN with improvement potentially continuing beyond completion of the intervention. A larger study is warranted to evaluate this further.

Systematic review of long-term chemotherapy-induced peripheral neuropathy (CIPN) following adjuvant oxaliplatin for colorectal cancer.

PURPOSE: Chemotherapy-induced peripheral neuropathy (CIPN) is the most common dose-limiting side effect of oxaliplatin. It often persists and can adversely affect quality of life of colorectal cancer (CRC) survivors. This systematic review explored the proportions of patients with persistent CIPN and the reporting methods used. METHODS: MEDLINE, EMBASE, Web of Science and CINAHL were searched up to March 2021 for publications reporting CIPN outcomes following adjuvant oxaliplatin-containing chemotherapy at prespecified timepoints in participants with CRC. Secondary outcomes assessed the tools used to measure CIPN. Two authors reviewed full text publications for eligibility, data extraction and appraisal. Meta-analysis was performed where Common Terminology Criteria for Adverse Events (any grade) was reported at the most frequent timepoints. RESULTS: From 7895 citations identified, 27 studies met the eligibility criteria: six were randomised control trials, and 21 were non-randomised studies. Pooled prevalence of CIPN at 6, 12, 24 and 36 months after chemotherapy were 58%, 45%, 32% and 24% respectively. The average prevalence of CIPN decreased by 26% per year after chemotherapy (pooled RR = 0.74; 95% CI 0.72-0.75). Across all studies, ten separate tools were used as the primary measure of CIPN. Quality appraisal identified open-label design and inadequate reporting of participants lost to follow-up as the main methodological limitations. CONCLUSION: Our summary of reported rates of persistent CIPN indicates substantial long-term toxicity affecting CRC survivors, and will help clinicians estimate CIPN risk and its change over time. The heterogeneity of CIPN measures identified in the review highlights the need for a standardised CIPN assessment.

Identification of Compounds That Inhibit Estrogen-Related Receptor Alpha Signaling Using High-Throughput Screening Assays.

The nuclear receptor, estrogen-related receptor alpha (ERRα; NR3B1), plays a pivotal role in energy homeostasis. Its expression fluctuates with the demands of energy production in various tissues. When paired with the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), the PGC/ERR pathway regulates a host of genes that participate in metabolic signaling networks and in mitochondrial oxidative respiration. Unregulated overexpression of ERRα is found in many cancer cells, implicating a role in cancer progression and other metabolism-related diseases. Using high throughput screening assays, we screened the Tox21 10K compound library in stably transfected HEK293 cells containing either the ERRα-reporter or the reporter plus PGC-1α expression plasmid. We identified two groups of antagonists that were potent inhibitors of ERRα activity and/or the PGC/ERR pathway: nine antineoplastic agents and thirteen pesticides. Results were confirmed using gene expression studies. These findings suggest a novel mechanism of action on bioenergetics for five of the nine antineoplastic drugs. Nine of the thirteen pesticides, which have not been investigated previously for ERRα disrupting activity, were classified as such. In conclusion, we demonstrated that high-throughput screening assays can be used to reveal new biological properties of therapeutic and environmental chemicals, broadening our understanding of their modes of action.

Computational prediction of multidisciplinary team decision-making for adjuvant breast cancer drug therapies: a machine learning approach.

BACKGROUND: Multidisciplinary team (MDT) meetings are used to optimise expert decision-making about treatment options, but such expertise is not digitally transferable between centres. To help standardise medical decision-making, we developed a machine learning model designed to predict MDT decisions about adjuvant breast cancer treatments. METHODS: We analysed MDT decisions regarding adjuvant systemic therapy for 1065 breast cancer cases over eight years. Machine learning classifiers with and without bootstrap aggregation were correlated with MDT decisions (recommended, not recommended, or discussable) regarding adjuvant cytotoxic, endocrine and biologic/targeted therapies, then tested for predictability using stratified ten-fold cross-validations. The predictions so derived were duly compared with those based on published (ESMO and NCCN) cancer guidelines. RESULTS: Machine learning more accurately predicted adjuvant chemotherapy MDT decisions than did simple application of guidelines. No differences were found between MDT- vs. ESMO/NCCN- based decisions to prescribe either adjuvant endocrine (97%, p = 0.44/0.74) or biologic/targeted therapies (98%, p = 0.82/0.59). In contrast, significant discrepancies were evident between MDT- and guideline-based decisions to prescribe chemotherapy (87%, p < 0.01, representing 43% and 53% variations from ESMO/NCCN guidelines, respectively). Using ten-fold cross-validation, the best classifiers achieved areas under the receiver operating characteristic curve (AUC) of 0.940 for chemotherapy (95% C.I., 0.922-0.958), 0.899 for the endocrine therapy (95% C.I., 0.880-0.918), and 0.977 for trastuzumab therapy (95% C.I., 0.955-0.999) respectively. Overall, bootstrap aggregated classifiers performed better among all evaluated machine learning models. CONCLUSIONS: A machine learning approach based on clinicopathologic characteristics can predict MDT decisions about adjuvant breast cancer drug therapies. The discrepancy between MDT- and guideline-based decisions regarding adjuvant chemotherapy implies that certain non-clincopathologic criteria, such as patient preference and resource availability, are factored into clinical decision-making by local experts but not captured by guidelines.

Development of a stable cell line with an intact PGC-1α/ERRα axis for screening environmental chemicals.

The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway.

Lactoferrin: the path from protein to gene.

This review focuses on the basic research that was performed on the lactoferrin protein and gene that was conducted in my laboratory over the past 25 years. This manuscript will outline how we discovered that lactoferrin is a target gene for estrogen, and how the first mouse lactoferrin cDNA, promoter and gene was cloned. Additionally, study was further extended to investigating the human lactoferrin protein and gene. Lastly the expression of lactoferrin in various tissues of both human and rodent under various physiological conditions were examined.

Ibudilast for prevention of oxaliplatin-induced acute neurotoxicity: a pilot study assessing preliminary efficacy, tolerability and pharmacokinetic interactions in patients with metastatic gastrointestinal cancer.

PURPOSE: This prospective, open-label, sequential 'before vs. after' pilot study was conducted to provide preliminary efficacy and tolerability data for ibudilast in the prevention of oxaliplatin-induced neurotoxicity in patients with metastatic upper gastrointestinal or colorectal cancer. Any potential impact of ibudilast on oxaliplatin and 5-fluorouracil pharmacokinetics was also explored. METHODS: Participants were administered a chemotherapy cycle (FOLFOX or CapeOx), followed by a chemotherapy cycle with co-administration of ibudilast 30 mg b.i.d. p.o. Efficacy was assessed on Day 3 and end of cycle using the Oxaliplatin-Specific Neurotoxicity Scale (OSNS) and additional clinical/patient-reported neurotoxicity measures. A population pharmacokinetic approach was used to determine oxaliplatin and 5-fluorouracil pharmacokinetics with and without ibudilast. RESULTS: Sixteen participants consented; 14 completed both chemotherapy cycles. Across all measures, the majority of participants experienced either an improvement or no worsening of neurotoxicity with ibudilast treatment. Based on OSNS assessments, acute neurotoxicity was unchanged in 12/14 participants and improved in 2/14 participants. The 90% confidence interval (CI) of the dose-normalised ratio of oxaliplatin AUC (90% CI 95.0-109%) and 5-fluorouracil AUC (90% CI 66.5-173%) indicated no significant impact of ibudilast on systemic exposure. CONCLUSION: This pilot study indicated ibudilast co-administration may improve or stabilise oxaliplatin-induced neurotoxicity. Given the expected worsening of symptoms in patients with continued chemotherapy, this represents a signal of effect that warrants further investigation. Pharmacokinetic analysis indicates ibudilast has no significant effect on oxaliplatin pharmacokinetics, and is unlikely to influence pharmacokinetics of 5-fluorouracil. CLINICAL TRIAL REGISTRATION: Trial registration number: UTN U1111-1209-0075 and ANZCTRN12618000232235 (registered 13/02/2018).

PGC-1alpha induces dynamic protein interactions on the ERRalpha gene multi-hormone response element nucleosome in kidney cells.

ERR (oestrogen-related receptor)-alpha modulates the oestrogen signalling pathway and regulates genes participating in the physiological energy balance programme. Oestrogen and PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator-1alpha), the master regulator of the energy homoeostasis programme, both regulate the expression of ERRalpha through the MHRE (multi-hormone response element) of the ERRalpha gene. Although the molecular mechanism of oestrogen action on ERRalpha regulation is well characterized, the mechanism of PGC-1alpha induction is unclear. In this study, we examine chromatin structural changes and protein interactions at the MHRE nucleosome in response to PGC-1alpha expression in HK2 human kidney cells. We mapped the nucleosome positions of the ERRalpha gene promoter and examined the changes of histone acetylation in response to PGC-1alpha expression. The interactions of DNA-binding proteins, ERRalpha and ERRgamma, co-activators {CBP [CREB (cAMP-response-element-binding protein)-binding protein], p300, PCAF (p300/CBP-associated factor)}, co-repressor [RIP140 (receptor-interacting protein of 140 kDa)] and RNA polymerase II at the MHRE nucleosome region were investigated over time before and after PGC-1alpha expression in the HK2 cells. We found a dynamic cyclic interaction of these proteins shortly after PGC-1alpha expression and a slower cycling interaction, with fewer proteins involved, 20 h later. By using the siRNA (small interfering RNA) knockdown approach, we discovered that ERRgamma was involved in the initial phase, but not in the later phase, of PGC-1alpha-induced ERRalpha expression.

Differential in Vitro Biological Action, Coregulator Interactions, and Molecular Dynamic Analysis of Bisphenol A (BPA), BPAF, and BPS Ligand-ERα Complexes.

BACKGROUND: Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) that might be harmful to human health. Recently, there has been widespread usage of bisphenol chemicals (BPs), such as bisphenol AF (BPAF) and bisphenol S (BPS), as replacements for BPA. However, the potential biological actions, toxicity, and the molecular mechanism of these compounds are still poorly understood. OBJECTIVES: Our objective was to examine the estrogenic effects of BPA, BPAF, and BPS and the molecular mechanisms of action in the estrogen receptor alpha (ERα) complex. METHODS: In vitro cell models were used to compare the estrogenic effects of BPA, BPAF, and BPS to estrogen. Microarray Assay for Real-Time Coregulator-Nuclear receptor Interaction (MARCoNI) analysis was used to identify coregulators of BPA, BPAF, and BPS, and molecular dynamic (MD) simulations were used to determine the compounds binding in the ERα complex. RESULTS: We demonstrated that BPA and BPAF have agonistic activity for both ERα and ERß, but BPS has ERα-selective specificity. We concluded that coregulators were differentially recruited in the presence of BPA, BPAF, or BPS. Interestingly, BPS recruited more corepressors when compared to BPA and BPAF. From a series of MD analysis, we concluded that BPA, BPAF, and BPS can bind to the ER-ligand-binding domain with differing energetics and conformations. In addition, the binding surface of coregulator interactions on ERα was characterized for the BPA, BPAF, and BPS complexes. CONCLUSION: These findings further our understanding of the molecular mechanisms of EDCs, such as BPs, in ER-mediated transcriptional activation, biological activity, and their effects on physiological functions in human health. https://doi.org/10.1289/EHP2505.

Identification of Estrogen-Related Receptor α Agonists in the Tox21 Compound Library.

The estrogen-related receptor α (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has also recently been shown to regulate an enzyme in the ß-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., small interfering RNA knockdown), we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. To our knowledge, this study is the first comprehensive analysis in discovering potential endocrine disrupters that affect the ERRα signaling pathway.

Interplay between estrogen-related receptor alpha (ERRalpha) and gamma (ERRgamma) on the regulation of ERRalpha gene expression.

Estrogen-related receptor alpha (ERRalpha) modulates estrogen receptor (ER)-mediated activity and is participating in the energy homeostasis by regulation of downstream target genes. The ERRalpha gene itself is proposed to be regulated by peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) through an autoregulatory loop under physiological stimulation. We have previously shown that the close family member ERRgamma is a positive regulator of ERRalpha gene expression. ERRalpha and ERRgamma are coexpressed in metabolically active tissues such as heart, kidney and muscle, yet the physiological role of ERRgamma and its relationship with ERRalpha in gene regulation are currently unknown. The present study examined the interplay of ERRgamma and ERRalpha in regulation of ERRalpha gene expression. Using real-time PCR analyses we found that ERRgamma, like the ERRalpha and PGC-1alpha is induced in mouse liver during fasting. Overexpression of ERRgamma in the HEC-1B cells robustly stimulated the multi-hormone response element (MHRE) of the ERRalpha gene promoter and this activity was repressed by increasing expression of ERRalpha. The two ERRs bind MHRE simultaneously in electrophoretic mobility shift assay (EMSA) and they were detected as multimeric complexes in cells by coimmunoprecipitation. Although ERRalpha and ERRgamma share high sequence identity, they differ in biochemical and molecular characteristics as examined by trypsin digestion, reporter activation and coactivator interaction and utilization. Using chromatin immunoprecipitation (ChIP) assay, we showed that ectopic expression of both ERRalpha and ERRgamma modifies chromatin structure at the MHRE region while ectopic expression of PGC-1alpha in HEC-1B cells promotes ERRgamma but not ERRalpha occupancy at the MHRE region of the ERRalpha gene promoter and enhances the recruitment of coactivator SRC1. These data suggested that ERRalpha and ERRgamma regulate ERRalpha gene expression with different molecular mechanisms.

Estrogen-related receptors-stimulated monoamine oxidase B promoter activity is down-regulated by estrogen receptors.

Although there are studies published about the neuroprotective effect of estrogen, little is known about the mechanisms and cellular targets of the hormone. Recent reports demonstrate that estrogen down-regulates the expression of monoamine oxidase A and B (MAO-A and MAO-B) in the hypothalamus of the Macaques monkey, both of which are key isoenzymes in the neurotransmitter degradation pathway. Additionally, estrogen-related receptor alpha (ERRalpha) up-regulates MAO-B gene expression in breast cancer cells. ERRalpha recognizes a variety of estrogen response elements and shares many target genes and coactivators with estrogen receptor alpha (ERalpha). In this study, we investigate the interplay of ERs and ERRs in the regulation of MAO-B promoter activity. We demonstrate that ERRalpha and ERRgamma up-regulate MAO-B gene activity, whereas ERalpha and ERbeta decrease stimulation in both a ligand-dependent and -independent manner. Ectopically expressed ERRalpha and ERRgamma stimulate the expression of MAO-B mRNA and protein as well as increase the MAO-B enzymatic activity in ER-negative HeLa cells. The ability of ERRs to stimulate MAO-B promoter activity was reduced in ER-positive MCF-7 and T47D cells. Several AGGTCA motifs of the MAO-B promoter are responsible for up-regulation by ERRs. Interestingly, ERalpha or ERbeta alone have no effect on MAO-B promoter activity but can down-regulate the activation function of ERRs, whereas glucocorticoid receptor does not. By using chromatin immunoprecipitation assay, we demonstrate that ERs compete with ERRs for binding to the MAO-B promoter at selective AGGTCA motifs, thereby changing the chromatin status and cofactor recruitment to a repressed state. These studies provide new insight into the relationship between ERalpha, ERbeta, ERRalpha, and ERRgamma in modulation of MAO-B gene activity.

TEPAPA: a novel in silico feature learning pipeline for mining prognostic and associative factors from text-based electronic medical records.

Vast amounts of clinically relevant text-based variables lie undiscovered and unexploited in electronic medical records (EMR). To exploit this untapped resource, and thus facilitate the discovery of informative covariates from unstructured clinical narratives, we have built a novel computational pipeline termed Text-based Exploratory Pattern Analyser for Prognosticator and Associator discovery (TEPAPA). This pipeline combines semantic-free natural language processing (NLP), regular expression induction, and statistical association testing to identify conserved text patterns associated with outcome variables of clinical interest. When we applied TEPAPA to a cohort of head and neck squamous cell carcinoma patients, plausible concepts known to be correlated with human papilloma virus (HPV) status were identified from the EMR text, including site of primary disease, tumour stage, pathologic characteristics, and treatment modalities. Similarly, correlates of other variables (including gender, nodal status, recurrent disease, smoking and alcohol status) were also reliably recovered. Using highly-associated patterns as covariates, a patient's HPV status was classifiable using a bootstrap analysis with a mean area under the ROC curve of 0.861, suggesting its predictive utility in supporting EMR-based phenotyping tasks. These data support using this integrative approach to efficiently identify disease-associated factors from unstructured EMR narratives, and thus to efficiently generate testable hypotheses.

Binding of bisphenol A, bisphenol AF, and bisphenol S on the androgen receptor: Coregulator recruitment and stimulation of potential interaction sites.

Bisphenol A (BPA), bisphenol AF (BPAF), and bisphenol S (BPS) are well known endocrine disruptors. Previous in vitro studies showed that these compounds antagonize androgen receptor (AR) transcriptional activity; however, the mechanisms of action are unclear. In the present study, we investigated interactions of coregulator peptides with BPA, BPAF, or BPS at the AR complexes using Micro Array for Real-time Coregulator Nuclear Receptor Interaction (MARCoNI) assays and assessed the binding of these compounds on AR by molecular dynamics (MD) simulations. The set of coregulator peptides that are recruited by BPA-bound AR, either positively/or negatively, are different from those recruited by the agonist R1881-bound AR. Therefore, the data indicates that BPA shows no similarities to R1881 and suggests that it may recruit other coregulators to the AR complex. BPAF-bound AR recruits about 70-80% of the same coregulator peptides as BPA-bound AR. Meanwhile, BPS-bound AR interacts with only few peptides compared to BPA or BPAF-bound AR. MD results show that multiple binding sites with varying binding affinities are available on AR for BPA, BPAF, and BPS, indicating the availability of modified binding surfaces on AR for coregulator interactions. These findings help explain some of the distinct AR-related toxicities observed with bisphenol chemicals and raise concern for the use of substitutes for BPA in commercial products.

Development of Novel Cell Lines for High-Throughput Screening to Detect Estrogen-Related Receptor Alpha Modulators.

Estrogen-related receptor alpha (ERRα), the first orphan nuclear receptor discovered, is crucial for the control of cellular energy metabolism. ERRα and its coactivator, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), are required for rapid energy production in response to environmental challenges. They have been implicated in the etiology of metabolic disorders such as type 2 diabetes and metabolic syndrome. ERRα also plays a role in the pathogenesis of breast cancer. Identification of compounds that modulate ERRα signaling may elucidate environmental factors associated with these diseases. Therefore, we developed stable cell lines containing an intact ERRα signaling pathway, with and without the coactivator PGC-1α, to use as high-throughput screening tools to detect ERRα modulators. The lentiviral PGC-1α expression constructs and ERRα multiple hormone response element (MHRE) reporters were introduced into HEK293T cells that express endogenous ERRα. A cell line expressing the reporter alone was designated "ERR." A second cell line expressing both reporter and PGC-1α was named "PGC/ERR." Initial screenings of the Library of Pharmacologically Active Compounds (LOPAC) identified 33 ERR and 22 PGC/ERR agonists, and 54 ERR and 15 PGC/ERR antagonists. Several potent ERRα agonists were dietary plant compounds (e.g., genistein). In conclusion, these cell lines are suitable for high-throughput screens to identify environmental chemicals affecting metabolic pathways and breast cancer progression.

Phosphorylation of Kruppel-like factor 5 (KLF5/IKLF) at the CBP interaction region enhances its transactivation function.

The Kruppel-like factor 5 (KLF5/IKLF) belongs to the Kruppel family of genes which bind GC-rich DNA elements and activate or repress their target genes in a promoter context and/or cellular environment-dependent manner. In the present study, we used the Gal4 fusion assay system to characterize the mechanism of transactivation by KLF5. We demonstrated that the transactivation function of KLF5 was enhanced by CREB-binding protein (CBP) and blocked by wild-type but not mutant E1A. Over expression of CBP reversed the inhibition effect of E1A. With various lengths of KLF5 fusion protein, the transactivation functions were localized to 156 amino acid residues at the N-terminal region and 133 amino acid residues adjacent to the Zn finger motif. We mapped the CBP and KLF5 interaction domain to the N-terminal region of CBP (amino acids 1-232) and the N-terminal region of KLF5 (amino acids 1-238) where one of the activation functions resides. The histone acetyltransferase (HAT) activity of CBP does not play a role in the transactivation function of KLF5 nor does it acetylate KLF5 in vitro. However, phosphorylation is important in KLF5 transactivation activity. Inhibition of protein kinase activity by H7 or calphostin C blocked both full-length and N-terminal fragment (amino acids 1-238) KLF5 activities. Mutation at a potential protein kinase C phosphorylation site within the CBP interaction domain of KLF5 reduces its transactivation function. Furthermore, using the GST pull-down approach, we showed that phosphorylation of KLF5 enhances its interaction with CBP. The results of the present study provide a mechanism for KLF5 transactivation function.

Full length and delta lactoferrin display differential cell localization dynamics, but do not act as tumor markers or significantly affect the expression of other genes.

Lactoferrin is a secreted protein related to transferrin. Lactoferrin indirectly protects host cells against foreign insults by killing bacteria, scavenging free iron, and binding to receptors required for viral invasion. However, lactoferrin is also proposed to act directly on cells as a transcription factor and tumor suppressor gene. In addition to full length lactoferrin, a truncated form, called delta lactoferrin, can also be produced by alternative splicing. We show here that transformed and nontransformed cells are equally able to express both full length and delta lactoferrin. Moreover, both forms of lactoferrin failed to substantially modulate the expression of other genes. Thus, lactoferrin does not seem to directly control gene expression or inhibit tumor cell growth.