Lasofoxifene as a potential treatment for aromatase inhibitor-resistant ER-positive breast cancer.

BACKGROUND: Breast cancers treated with aromatase inhibitors (AIs) can develop AI resistance, which is often driven by estrogen receptor-alpha (ERα/ESR1) activating mutations, as well as by ER-independent signaling pathways. The breast ER antagonist lasofoxifene, alone or combined with palbociclib, elicited antitumor activities in a xenograft model of ER + metastatic breast cancer (mBC) harboring ESR1 mutations. The current study investigated the activity of LAS in a letrozole-resistant breast tumor model that does not have ESR1 mutations. METHODS: Letrozole-resistant, MCF7 LTLT cells tagged with luciferase-GFP were injected into the mammary duct inguinal glands of NSG mice (MIND model; 6 mice/group). Mice were randomized to vehicle, lasofoxifene ± palbociclib, fulvestrant ± palbociclib, or palbociclib alone 2-3 weeks after cell injections. Tumor growth and metastases were monitored with in vivo and ex vivo luminescence imaging, terminal tumor weight measurements, and histological analysis. The experiment was repeated with the same design and 8-9 mice in each treatment group. RESULTS: Western blot analysis showed that the MCF7 LTLT cells had lower ERα and higher HER2 expressions compared with normal MCF7 cells. Lasofoxifene ± palbociclib, but not fulvestrant, significantly reduced primary tumor growth versus vehicle as assessed by in vivo imaging of tumors at study ends. Percent tumor area in excised mammary glands was significantly lower for lasofoxifene plus palbociclib versus vehicle. Ki67 staining showed decreased overall tumor cell proliferation with lasofoxifene ± palbociclib. The lasofoxifene + palbociclib combination was also associated with significantly fewer bone metastases compared with vehicle. Similar results were observed in the repeat experiment. CONCLUSIONS: In a mouse model of letrozole-resistant breast cancer with no ESR1 mutations, reduced levels of ERα, and overexpression of HER2, lasofoxifene alone or combined with palbociclib inhibited primary tumor growth more effectively than fulvestrant. Lasofoxifene plus palbociclib also reduced bone metastases. These results suggest that lasofoxifene alone or combined with a CDK4/6 inhibitor may offer benefits to patients who have ER-low and HER2-positive, AI-resistant breast cancer, independent of ESR1 mutations.

The relationship between fat distribution and diabetes in US adults by race/ethnicity.

Introduction: This study examined the relationship between fat distribution and diabetes by sex-specific racial/ethnic groups. Methods: A secondary data analysis of National Health and Nutrition Examination Survey 2011-2018 data (n = 11,972) was completed. Key variables examined were visceral adipose tissue area (VATA), subcutaneous fat area (SFA), diabetes prevalence, and race/ethnicity. The association of VATA and SFA and diabetes prevalence was examined separately and simultaneously using multiple logistic regression. Bonferroni corrections were applied to all multiple comparisons between racial/ethnic groups. All analyses were adjusted for demographics and muscle mass. Results: VATA was positively associated with diabetes in both sexes (p < 0.001) and across all racial/ethnic groups (p < 0.05) except Black females. No statistically significant relationships were observed between SFA and diabetes while accounting for VATA with the exception of White females (p = 0.032). When comparing racial/ethnic groups, the relationship between VATA and diabetes was stronger in White and Hispanic females than in Black females (p < 0.005) while the relationship between SFA and diabetes did not differ between any racial/ethnic groups. Conclusion: This study found that VATA is associated with diabetes for both sexes across almost all racial/ethnic groups independent of SFA whereas the only significant relationship between SFA and diabetes, independent of VATA, was observed in White females. The findings indicated that visceral fat was more strongly associated with diabetes than subcutaneous. Additionally, there are health disparities in sex-specific racial/ethnic groups thus further study is warranted.

Effect of a Technology-Integrated Curriculum on Sugary Drink and Snack Intake of Elementary-Aged Youth Experiencing Low Income.

OBJECTIVE: To describe the results of a technology-integrated intervention on sugar-sweetened beverage (SSB) and energy-dense snack intake with third graders experiencing low income. DESIGN: A 2 × 2 quasi-randomized cluster-block, parallel-group experimental research design. SETTING: Low-income schools in Rhode Island. PARTICIPANTS: Two-hundred seventeen intervention and 242 control third-grade students in low-income (89.6% and 88.2% free/reduced meals, respectively), ethnically and racially diverse (63% Hispanic/20% Black and 62% Hispanic/18% Black, respectively) schools. INTERVENTION(S): A 13-week in-school program held once per week for 1 hour. The hands-on, technology-integrated program used a modified version of the Body Quest: Food of the Warrior curriculum. MAIN OUTCOME MEASURE(S): Intake of SSB and energy-dense snacks, both salty and sweet snacks, using baseline (week 1) and postassessment (week 13) previous day self-recall. ANALYSIS: Generalized mixed modeling with nesting. RESULTS: Intervention students significantly reduced their SSB intake by 38% (0.5 times/d; F[1, 540] = 4.26; P = 0.04) and salty snack intake by 58% (0.8 times/d; F[1, 534] = 6.58, P < 0.01) from baseline to postassessment as compared with the control students. CONCLUSIONS AND IMPLICATIONS: Findings suggest a technology-integrated curriculum is effective in decreasing SSB and salty snacks in elementary-aged students of low-income, minoritized populations. Improved dietary habits can potentially influence other facets of students' lives.

ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation.

The constitutively active ESR1 Y537S mutation is associated with endocrine therapy (ET) resistance and progression of metastatic breast cancer through its effects on estrogen receptor (ERα) gene regulatory functions. However, the complex relationship between ERα and the progesterone receptor (PR), known as ERα/PR crosstalk, has yet to be characterized in the context of the ERα Y537S mutation. Using proximity ligation assays, we identify an increased physical interaction of ERα and PR in the context of the ERα Y537S mutation, including in the nucleus where this interaction may translate to altered gene expression. As such, more than 30 genes were differentially expressed in both patient tumor and cell line data (MCF7 and/or T47D cells) in the context of the ERα Y537S mutation compared to ERα WT. Of these, IRS1 stood out as a gene of interest, and ERα and PR occupancy at chromatin binding sites along IRS1 were uniquely altered in the context of ERα Y537S. Furthermore, siRNA knockdown of IRS1 or treatment with the IRS1 inhibitor NT-157 had a significant anti-proliferative effect in ERα Y537S cell lines, implicating IRS1 as a potential therapeutic target for restoring treatment sensitivity to patients with breast cancers harboring ERα Y537S mutations.

Glucocorticoid Receptor Activation in Lobular Breast Cancer Is Associated with Reduced Cell Proliferation and Promotion of Metastases.

Estrogen receptor-positive (ER+) invasive lobular breast cancer (ILC) comprises about ~15% of breast cancer. ILC's unique genotypic (loss of wild type E-cadherin expression) and phenotypic (small individual round cancer cells that grow in discontinuous nests) are thought to contribute to a distinctive pattern of metastases to serosal membranes. Unlike invasive ductal carcinoma (IDC), ILC metastases often intercalate into the mesothelial layer of the peritoneum and other serosal surfaces. While ER activity is a known driver of ILC proliferation, very little is known about how additional nuclear receptors contribute to ILC's distinctive biology. In ER+ IDC, we showed previously that glucocorticoid receptor (GR) activity inhibits pro-proliferative gene expression and cell proliferation. Here we examined ER+ ILC models and found that GR activation similarly reduces S-phase entry gene expression and ILC proliferation. While slowing tumor growth rate, our data also suggest that GR activation results in an enhanced metastatic phenotype through increasing integrin-encoding gene expression, extracellular matrix protein adhesion, and mesothelial cell clearance. Moreover, in an intraductal mouse mammary gland model of ILC, we found that GR expression is associated with increased bone metastases despite slowed primary mammary tumor growth. Taken together, our findings suggest GR-mediated gene expression may contribute to the unusual characteristics of ILC biology.

Reinvestigating the Synthesis and Characterization of Ethyl 3-[5-(2-Ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]-but-2-enoate.

We reinvestigated the reported method for the synthesis of ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]-but-2-enoate (MIBE), which was obtained by the reaction of 5-hydroxy-1-methyl-1H-indole with excess ethyl acetoacetate catalyzed by indium(III) chloride. Based on the NMR and MS data, we assigned the structure of the isolated product as (3E)-3-(2-ethoxy-2-oxoethylidene)-1,2,3,4-tetrahydro-7-hydroxy-1,4-dimethylcyclopent[b]indole-1-acetate (2a) rather than the reported MIBE.

Evaluating steroid hormone receptor interactions using the live-cell NanoBRET proximity assay.

Steroid hormone receptors play a crucial role in the development and characterization of the majority of breast cancers. These receptors canonically function through homodimerization, but physical interactions between different hormone receptors play a key role in cell functions as well. The estrogen receptor (ERα) and progesterone receptor (PR), for example, are involved in a complex set of interactions known as ERα/PR crosstalk. Here, we developed a valuable panel of nuclear receptor expression plasmids specifically for use in NanoBRET assays to assess nuclear receptor homo- and heterodimerization. We demonstrate the utility of this assay system by assessing ERα/PR physical interaction in the context of the endocrine therapy resistance-associated ERα Y537S mutation. We identify a role of the ERα Y537S mutation beyond that of constitutive activity of the receptor; it also increases ERα/PR crosstalk. In total, the NanoBRET assay provides a novel avenue for investigating hormone receptor crosstalk. Future research may use this system to assess the effects of other clinically significant hormone receptor mutations on hormone receptor crosstalk.

Sex and race/ethnicity specific reference predictive equations for abdominal adiposity indices using anthropometry in US adults.

BACKGROUND AND AIMS: Abdominal adiposity indices have stronger associations with cardiometabolic risk factors compared to anthropometric measures but are rarely used in large scale studies due to the cost and efficiency. The aim of this study is to establish sex and race/ethnicity specific reference equations using anthropometric measures. METHODS AND RESULTS: A secondary data analysis (n = 6589) of healthy adults was conducted using data from National Health and Nutrition Examination Survey 2011-2018. Variables included in the analyses were anthropometric measures (height; weight; waist circumference, WC) and abdominal adiposity indices (android percent fat; android to gynoid ratio, A/G ratio; visceral adipose tissue area, VATA; visceral to subcutaneous adipose area ratio, VSR). Multivariable prediction models were developed using quantile regression. Bland-Altman was used for external validation of prediction models. Reference equations to estimate android percent fat, A/G ratio, VATA and VSR from anthropometric measurements were developed using a randomly selected subsample of 4613. These reference equations for four abdominal adiposity indices were then cross-validated in the remaining subsample of 1976. The measured and predicted android percent fat, A/G ratio, VATA and VSR were not statistically different (p > 0.05) except for the A/G ratio in Asian males and VSR in White females. The results of Bland-Altman further revealed that ≥93% of predicted abdominal adiposity indices fell within the limits of agreement (±1.96 standard deviation). CONCLUSION: The sex and race/ethnicity specific reference equations for abdominal adiposity indices established using anthropometrics in the present study have strong predictive ability in US healthy adults.

Formative evaluation of a STEAM and nutrition education summer program for low-income youth.

In-depth formative evaluations are vital for curriculum development and program planning but are often not conducted before a program pilots. A formative evaluation of Project stRIde was conducted to gain insight from experts and identify revisions to the curriculum. Project stRIde is a science, technology, engineering, arts, and mathematics (STEAM) and nutrition-based curriculum developed for 4th and 5th grade students from low-income and diverse families. Nine experts spanning the fields of nutrition education, cultural competency, elementary education, summer programs, and STEAM outreach were recruited to participate in an expert content review (ECR) survey and virtual interviews. Seven core themes were identified: effectively promoting student engagement, increased guidance or support needed, activity too difficult for age, time, confidence in teaching lessons, cultural appropriateness, and strengths of curriculum in promoting STEAM education and innovation. Across the lessons, all reviewers agreed that the lessons were accurate, incorporated STEAM concepts, and were culturally appropriate for this population. Future major edits to the curriculum include creating supplemental videos, modifying some activities for age level, and incorporating more opportunities for participant engagement. Overall, an ECR is an effective way to examine a program's strengths and limitations and should be included in the beginning stages of program planning.

Development and validation of the ASKFV-SE tool to measure children's self-efficacy for requesting fruits and vegetables.

The aim of the present study was to develop the ASKFV-SE tool to measure self-efficacy (SE) towards requesting fruits and vegetables (FV) in the home and school environment with school-age children (grades 4-5) from urban, ethnically diverse, low-income households. Cognitive interviews reduced the tool from eleven items to seven. The 7-item questionnaire was tested with 444 children. The items loaded on two factors: home SE (four items) and school SE (two items) with one item was excluded (<0⋅40). The reduced 6-item, 2-factor structure was the best fit for the data (χ 2 = 45⋅09; df = 9; CFI = 0⋅835; RMSEA = 0⋅147). Confirmatory factory analysis revealed that the 4-item home SE had high reliability (α = 0⋅73) and marginally acceptable reliability for the 2-item school SE (α = 0⋅53). The pre-COVID intra-class correlation coefficient (ICC) was 0⋅584 (P < 0⋅001; fair; n = 57) compared to 0⋅736 during-COVID (P < 0⋅001; good; n 50). The ASKFV-SE tool measures children's SE for asking for FVs with strong psychometric properties and low participant burden.

Eating pace instruction is effective in slowing eating rate in women with overweight and obesity.

Slow eating is associated with lower body mass index (BMI), enhanced satiety, and reduced food intake in laboratory settings. This study developed and tested a 5-week slow-eating intervention, delivered either through individual or small group weekly meetings, in women with overweight and obesity. Women (n = 65; 20.5 ± 3.6 years; BMI 31.3 ± 2.7 kg/m2) were assigned to experimental or parallel non-treatment control. Main outcomes, measured pre- and post-intervention, included eating rate, meal duration, and energy intake during a standardized meal served on a universal eating monitor. Exploratory outcomes included Weight Related Eating Questionnaire (WREQ), Intuitive Eating Scale (IES), and Mindful Eating Questionnaire (MEQ) scores. All women in the experimental group underwent the same slow-eating intervention, but half had individual sessions while the other half had small group sessions. No differences were seen for any outcomes between session modalities, so experimental data were pooled (n = 25) and compared to control data (n = 25). Time-by-group interactions showed reduced eating rate (F(1,48df) = 13.04, η2 = 0.214, p = .001) and increased meal duration (F(1,48df) = 7.949, η2 = 0.142, p = .007) in the experimental group compared to the control group but change in energy intake was not significant (F(1,48df) = 3.298, η2 = 0.064, p = .076). Experimental within-group changes for WREQ subscale scores External Cues (t(23) = 3.779, p = .001) and Emotional Eating (t(23) = 2.282, p = .032) decreased over time, along with increased total and summary IES (t(23) = 2.6330, p = .015) and MEQ (t(23) = 2.663, p = .014) scores. Promising findings of reduced eating rate, increased meal duration, and improved WREQ, IES, and MEQ scores should be followed up in larger more diverse samples for longer durations.

Demethylation of dimethyl phosphonate esters with sodium ethanethiolate: Improved synthesis of 5'-methylene substituted 2',5'-deoxyribonucleotides.

We describe a new demethylation method for dimethyl phosphonate esters using sodium ethanethiolate. The new procedure allows demethylation of nucleoside dimethyl phosphonate esters without 1'-α-anomerization, providing an improved synthesis of 5'-methylene substituted 2',5'-deoxynucleotides.

Targeting MYC with modular synthetic transcriptional repressors derived from bHLH DNA-binding domains.

Despite unequivocal roles in disease, transcription factors (TFs) remain largely untapped as pharmacologic targets due to the challenges in targeting protein-protein and protein-DNA interactions. Here we report a chemical strategy to generate modular synthetic transcriptional repressors (STRs) derived from the bHLH domain of MAX. Our synthetic approach yields chemically stabilized tertiary domain mimetics that cooperatively bind the MYC/MAX consensus E-box motif with nanomolar affinity, exhibit specificity that is equivalent to or beyond that of full-length TFs and directly compete with MYC/MAX protein for DNA binding. A lead STR directly inhibits MYC binding in cells, downregulates MYC-dependent expression programs at the proteome level and inhibits MYC-dependent cell proliferation. Co-crystallization and structure determination of a STR:E-box DNA complex confirms retention of DNA recognition in a near identical manner as full-length bHLH TFs. We additionally demonstrate structure-blind design of STRs derived from alternative bHLH-TFs, confirming that STRs can be used to develop highly specific mimetics of TFs targeting other gene regulatory elements.

The Sex and Race/Ethnicity-Specific Relationships of Abdominal Fat Distribution and Anthropometric Indices in US Adults.

The purpose of this study was to examine demographic-specific relationships between direct abdominal fat measures and anthropometric indices. A cross-sectional study was conducted utilizing abdominal fat measures (visceral fat area, VFA; visceral to subcutaneous adipose area ratio, VSR) and anthropometrics (body mass index, BMI; waist circumference, WC) data from the 2011-2018 National Health and Nutrition Examination Survey. Linear or polynomial linear regression models were used to examine the relationships of abdominal fat measures to anthropometrics with adjustment for demographics. The results revealed that while VFA was linearly related to BMI and WC across all demographics (p < 0.001), the relationships between VSR and both BMI and WC were concave in men and convex in women. The relationships between VFA, VSR, and BMI, WC varied by sex and race/ethnicity. In conclusion, increasing BMI and WC were linearly associated with increased VFA, but their relationships with VSR were nonlinear and differed by sex.

The Demographic Specific Abdominal Fat Composition and Distribution Trends in US Adults from 2011 to 2018.

Despite the rising awareness of abdominal adiposity associated health problems and demographic health disparities, research is lacking about abdominal fat trends using a national representative sample of US adults. Our purpose was to examine national demographic specific abdominal fat composition and distribution trends from 2011 to 2018. This trend analysis was using National Health and Nutrition Examination Survey data (n = 13,163). Visceral adipose percent (VAT%), visceral adipose tissue area (VAA) and visceral to subcutaneous adipose area ratio (VSR) were utilized in data analyses. Multiple polynomial linear regression was utilized with adjustment for confounding variables. Our findings revealed that VAT%, VAA and VSR trends were concave among all demographic groups. The VAT%, VAA and/or VSR changes were observed in most demographic groups (p < 0.05) except younger, White and Black respondents. The pattern was consistent with biennial increases up to 2014 or 2016 followed by decreases in 2017-2018. There were demographic disparities, with middle-aged respondents and Hispanics having the most evident VAT%, VSR and/or VAA changes biennially when compared to their counterparts (p < 0.05). In conclusion, abdominal fat composition and distribution increased before 2014 or 2016 but decreased afterwards with variations by age and/or race/ethnicity. Further research is needed to explore the possible causes of abdominal fat changes overtime.

The Relationship of Physical Activity and Dietary Quality and Diabetes Prevalence in US Adults: Findings from NHANES 2011-2018.

This study aimed to examine the relationship of physical activity and/or dietary quality and diabetes prevalence in the general population and within specific age groups. It was a cross-sectional study using 2011−2018 National Health and Nutrition Examination Survey and the US Department of Agriculture's Food Patterns Equivalents data (n = 15,674). Physical activity was measured by Global Physical Activity questionnaire; dietary quality was analyzed using the Healthy Eating Index 2015; diabetes prevalence was determined by reported diagnosis and glycohemoglobin or fasting glucose. Data were analyzed using multiple logistic regression adjusted for demographic variables and weight status. Results revealed that although no statistically significant or non-substantial relationships were observed between dietary quality or physical activity and diabetes prevalence, respondents who did not meet physical activity recommendations regardless of dietary quality had a higher odds of diabetes prevalence than those who met physical activity recommendations and had a higher dietary quality (p < 0.05). In conclusion, meeting physical activity recommendations is an important protective factor for diabetes especially in combination with a higher quality diet. A healthy lifestyle appears to have the greater impact on diabetes prevention in middle-aged men and women.

The Relationship of Physical Activity and Dietary Quality with Android Fat Composition and Distribution in US Adults.

This study examined the relationship of physical activity (PA) and dietary quality to android fat composition and distribution using a national representative adult sample and determined sex-based differences in these relationships. It is a cross-sectional (n = 10,014) analysis of the 2011−2018 National Health and Nutrition Examination Survey and the US department of Agriculture's Food Patterns Equivalents datasets. Variables utilized for this analysis include PA, 24-h dietary recalls, android percent fat, and android-gynoid (A/G) ratio measured by dual-energy X-ray absorptiometry. Multiple linear regression was performed to examine the relationship between PA and/or dietary quality and android percent fat and A/G ratio adjusted for confounding factors. The study results revealed that PA and/or dietary quality were inversely related to android percent fat and A/G ratio (p < 0.05), but the sex effect was only seen between PA and A/G ratio (p = 0.003). Participants who met PA recommendations and had higher dietary quality had 2.12% lower android fat than those who did not meet PA recommendations and had lower dietary quality (p < 0.001). Both PA and dietary quality are associated with android fat reduction regardless of sex. Given the direct connection between android fat and cardiovascular and metabolic diseases, it is important to increase both PA and dietary quality.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

Endocrine Therapy-Resistant Breast Cancer Cells Are More Sensitive to Ceramide Kinase Inhibition and Elevated Ceramide Levels Than Therapy-Sensitive Breast Cancer Cells.

ET resistance is a critical problem for estrogen receptor-positive (ER+) breast cancer. In this study, we have investigated how alterations in sphingolipids promote cell survival in ET-resistant breast cancer. We have performed LC-MS-based targeted sphingolipidomics of tamoxifen-sensitive and -resistant MCF-7 breast cancer cell lines. Follow-up studies included treatments of cell lines and patient-derived xenograft organoids (PDxO) with small molecule inhibitors; cytometric analyses to measure cell death, proliferation, and apoptosis; siRNA-mediated knockdown; RT-qPCR and Western blot for gene and protein expression; targeted lipid analysis; and lipid addback experiments. We found that tamoxifen-resistant cells have lower levels of ceramides and hexosylceramides compared to their tamoxifen-sensitive counterpart. Upon perturbing the sphingolipid pathway with small molecule inhibitors of key enzymes, we identified that CERK is essential for tamoxifen-resistant breast cancer cell survival, as well as a fulvestrant-resistant PDxO. CERK inhibition induces ceramide-mediated cell death in tamoxifen-resistant cells. Ceramide-1-phosphate (C1P) partially reverses CERK inhibition-induced cell death in tamoxifen-resistant cells, likely through lowering endogenous ceramide levels. Our findings suggest that ET-resistant breast cancer cells maintain lower ceramide levels as an essential pro-survival mechanism. Consequently, ET-resistant breast cancer models have a unique dependence on CERK as its activity can inhibit de novo ceramide production.