Alcohol Regulates Genes that Are Associated with Response to Endocrine Therapy and Attenuates the Actions of Tamoxifen in Breast Cancer Cells

Hereditary, hormonal, and behavioral factors contribute to the development of breast cancer. Alcohol consumption is a modifiable behavior that is linked to increased breast cancer risks and is associated with the development of hormone-dependent breast cancers as well as disease progression and recurrence following endocrine treatment. In this study we examined the molecular mechanisms of action of alcohol by applying molecular, genetic, and genomic approaches in characterizing its effects on estrogen receptor (ER)-positive breast cancer cells. Treatments with alcohol promoted cell proliferation, increased growth factor signaling, and up-regulated the transcription of the ER target gene GREB1 but not the canonical target TFF1/pS2. Microarray analysis following alcohol treatment identified a large number of alcohol-responsive genes, including those which function in apoptotic and cell proliferation pathways. Furthermore, expression profiles of the responsive gene sets in tumors were strongly associated with clinical outcomes in patients who received endocrine therapy. Correspondingly, alcohol treatment attenuated the anti-proliferative effects of the endocrine therapeutic drug tamoxifen in ER-positive breast cancer cells. To determine the contribution and functions of responsive genes, their differential expression in tumors were assessed between outcome groups. The proto-oncogene BRAF was identified as a novel alcohol- and estrogen-induced gene that showed higher expression in patients with poor outcomes. Knock-down of BRAF, moreover, prevented the proliferation of breast cancer cells. These findings not only highlight the mechanistic basis of the effects of alcohol on breast cancer cells and increased risks for disease incidents and recurrence, but may facilitate the discovery and characterization of novel oncogenic pathways and markers in breast cancer research and therapeutics

Chitosan nanoparticle-mediated delivery of miRNA-34a decreases prostate tumor growth in the bone and its expression induces non-canonical autophagy

While several new therapies are FDA-approved for bone-metastatic prostate cancer (PCa), patient survival has only improved marginally. Here, we report that chitosan nanoparticle-mediated delivery of miR-34a, a tumor suppressive microRNA that downregulates multiple gene products involved in PCa progression and metastasis, inhibited prostate tumor growth and preserved bone integrity in a xenograft model representative of established PCa bone metastasis. Expression of miR-34a induced apoptosis in PCa cells, and, in accord with downregulation of targets associated with PCa growth, including MET and Axl and c-Myc, also induced a form of non-canonical autophagy that is independent of Beclin-1, ATG4, ATG5 and ATG7. MiR-34a-induced autophagy is anti-proliferative in prostate cancer cells, as blocking apoptosis still resulted in growth inhibition of tumor cells. Thus, combined effects of autophagy and apoptosis are responsible for miR-34a-mediated prostate tumor growth inhibition, and have translational impact, as this non-canonical form of autophagy is tumor inhibitory. Together, these results provide a new understanding of the biological effects of miR-34a and highlight the clinical potential for miR-34a delivery as a treatment for bone metastatic prostate cancer

Global burden and strength of evidence for 88 risk factors in 204 countries and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Understanding the health consequences associated with exposure to risk factors is necessary to inform public health policy and practice. To systematically quantify the contributions of risk factor exposures to specific health outcomes, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 aims to provide comprehensive estimates of exposure levels, relative health risks, and attributable burden of disease for 88 risk factors in 204 countries and territories and 811 subnational locations, from 1990 to 2021. Methods: The GBD 2021 risk factor analysis used data from 54 561 total distinct sources to produce epidemiological estimates for 88 risk factors and their associated health outcomes for a total of 631 risk–outcome pairs. Pairs were included on the basis of data-driven determination of a risk–outcome association. Age-sex-location-year-specific estimates were generated at global, regional, and national levels. Our approach followed the comparative risk assessment framework predicated on a causal web of hierarchically organised, potentially combinative, modifiable risks. Relative risks (RRs) of a given outcome occurring as a function of risk factor exposure were estimated separately for each risk–outcome pair, and summary exposure values (SEVs), representing risk-weighted exposure prevalence, and theoretical minimum risk exposure levels (TMRELs) were estimated for each risk factor. These estimates were used to calculate the population attributable fraction (PAF; ie, the proportional change in health risk that would occur if exposure to a risk factor were reduced to the TMREL). The product of PAFs and disease burden associated with a given outcome, measured in disability-adjusted life-years (DALYs), yielded measures of attributable burden (ie, the proportion of total disease burden attributable to a particular risk factor or combination of risk factors). Adjustments for mediation were applied to account for relationships involving risk factors that act indirectly on outcomes via intermediate risks. Attributable burden estimates were stratified by Socio-demographic Index (SDI) quintile and presented as counts, age-standardised rates, and rankings. To complement estimates of RR and attributable burden, newly developed burden of proof risk function (BPRF) methods were applied to yield supplementary, conservative interpretations of risk–outcome associations based on the consistency of underlying evidence, accounting for unexplained heterogeneity between input data from different studies. Estimates reported represent the mean value across 500 draws from the estimate's distribution, with 95% uncertainty intervals (UIs) calculated as the 2·5th and 97·5th percentile values across the draws. Findings: Among the specific risk factors analysed for this study, particulate matter air pollution was the leading contributor to the global disease burden in 2021, contributing 8·0% (95% UI 6·7–9·4) of total DALYs, followed by high systolic blood pressure (SBP; 7·8% [6·4–9·2]), smoking (5·7% [4·7–6·8]), low birthweight and short gestation (5·6% [4·8–6·3]), and high fasting plasma glucose (FPG; 5·4% [4·8–6·0]). For younger demographics (ie, those aged 0–4 years and 5–14 years), risks such as low birthweight and short gestation and unsafe water, sanitation, and handwashing (WaSH) were among the leading risk factors, while for older age groups, metabolic risks such as high SBP, high body-mass index (BMI), high FPG, and high LDL cholesterol had a greater impact. From 2000 to 2021, there was an observable shift in global health challenges, marked by a decline in the number of all-age DALYs broadly attributable to behavioural risks (decrease of 20·7% [13·9–27·7]) and environmental and occupational risks (decrease of 22·0% [15·5–28·8]), coupled with a 49·4% (42·3–56·9) increase in DALYs attributable to metabolic risks, all reflecting ageing populations and changing lifestyles on a global scale. Age-standardised global DALY rates attributable to high BMI and high FPG rose considerably (15·7% [9·9–21·7] for high BMI and 7·9% [3·3–12·9] for high FPG) over this period, with exposure to these risks increasing annually at rates of 1·8% (1·6–1·9) for high BMI and 1·3% (1·1–1·5) for high FPG. By contrast, the global risk-attributable burden and exposure to many other risk factors declined, notably for risks such as child growth failure and unsafe water source, with age-standardised attributable DALYs decreasing by 71·5% (64·4–78·8) for child growth failure and 66·3% (60·2–72·0) for unsafe water source. We separated risk factors into three groups according to trajectory over time: those with a decreasing attributable burden, due largely to declining risk exposure (eg, diet high in trans-fat and household air pollution) but also to proportionally smaller child and youth populations (eg, child and maternal malnutrition); those for which the burden increased moderately in spite of declining risk exposure, due largely to population ageing (eg, smoking); and those for which the burden increased considerably due to both increasing risk exposure and population ageing (eg, ambient particulate matter air pollution, high BMI, high FPG, and high SBP). Interpretation: Substantial progress has been made in reducing the global disease burden attributable to a range of risk factors, particularly those related to maternal and child health, WaSH, and household air pollution. Maintaining efforts to minimise the impact of these risk factors, especially in low SDI locations, is necessary to sustain progress. Successes in moderating the smoking-related burden by reducing risk exposure highlight the need to advance policies that reduce exposure to other leading risk factors such as ambient particulate matter air pollution and high SBP. Troubling increases in high FPG, high BMI, and other risk factors related to obesity and metabolic syndrome indicate an urgent need to identify and implement interventions

Novel Mechanisms of Androgen Receptor Signaling in Prostate Cancer

Androgens regulate the physiological development of the prostate and the pathology of prostate cancer. Androgen-receptor (AR)-mediated transcriptional activity is a driver of prostate cancer (PCa) progression. AR-induced transcriptional activity is a dynamic process that is regulated by the binding of ligands that induce distinct conformational changes in AR. These structural alterations lead to the differential recruitment of coregulators (coactivators or corepressors) that control the expression of specific subsets AR-regulated genes. Despite continual improvement in design and enhanced efficacy, PCa cells eventually become resistant to AR-antagonists and anti-androgen treatment. Therefore, in this dissertation, we have proposed and identified two novel mechanisms of harnessing AR-mediated gene transcription in PCa. First, we show that a stretch of proline residues located within the N-terminus of AR is a bona fide coregulator binding surface, the disruption of which reduces the androgen-dependent proliferation and migration of prostate cancer cells. Using T7 phage display, we identified a novel AR-interacting protein, SH3YL1, whose interaction with the receptor is dependent upon this polyproline domain. As with mutations within the AR polyproline domain, knockdown of SH3YL1 attenuated androgen-mediated cell growth and migration. RNA expression analysis revealed that SH3YL1 was required for the induction of a subset of AR-modulated genes. Notable was the observation that ubinuclein1 (UBN1), a key member of a histone H3.3 chaperone complex, was a transcriptional target of the AR/SH3YL1 complex, correlated with aggressive prostate cancer in patients, and was necessary for the maximal androgen-mediated proliferation and migration of prostate cancer cells. Collectively, these data highlight the importance of an amino-terminal activation domain, its associated coregulator, and downstream transcriptional targets in regulating cellular processes of pathological importance in prostate cancer. In the second approach of this dissertation, we studied the downstream AR targets that regulate autophagy. We have determined that 1) androgens regulate overall cell metabolism and cell growth, in part, by increasing autophagy in prostate cancer cells, 2) functional autophagy was clinically detected in metastatic, castration-resistant cancers but not treatment-naïve, localized tumors and 3) autophagy is required for prostate cancer progression in preclinical animal models. Inhibition of autophagy using molecular inhibitors significantly abrogated androgen-induced prostate cancer cell/tumor growth. Autophagy and subsequent cell growth is potentiated by androgen-mediated increases in the expression and activity of several core autophagy genes, including ULK1, ULK2, AT4B, ATG4D, and TFEB. We identify these five genes as direct targets of the androgen receptor (AR) in prostate cancer. Moreover, expression of these five genes is essential for maximal androgen-mediated autophagy and cell proliferation. These findings demonstrate a role for increased autophagy in prostate cancer and highlight the potential of targeting underexplored metabolic pathways for the development of novel therapeutics.Biology and Biochemistry, Department o

Alcohol regulates BRAF, an effector of growth factor signaling, and promotes estrogen-dependent and–independent growth.

<p>(A), Microarray validation demonstrates subtle but highly reproducible effects on gene expression of down-regulated and up-regulated genes. (B) BRAF is up-regulated at the protein level by alcohol and estrogen treatment. (C) BRAF is targetable with siRNA knockdown for functional studies. MTS assays demonstrate the anti-proliferative effect of BRAF knockdown on MCF-7 cells, suggesting that BRAF promotes basal cell proliferation in the absence of estradiol, increases estrogen-dependent growth, and potentiates some of the cell’s response to ethanol.</p

High BRAF expression levels correlated ER+ endocrine treated patients with poor prognosis and response to therapy.

<p>(A) BRAF is expressed at higher levels in patients who experience poor disease outcomes as compared to those who did not experience an adverse event. (B) BRAF expression levels separate patients into different DFS groups with nearly significant p-value (<i>p</i> = 0.074). Statistically different (C) DMFS and (D) DSS groups are observed in ER+ endocrine treated patients based on high expression levels of BRAF.</p

Alcohol increases cell proliferation in an estrogen-dependent manner, promotes the activation of ERK1/2, as well as known ER target genes.

<p>(A), Trypan blue exclusion assays demonstrate that estrogen potentiates cell proliferation increases by alcohol. DMSO treated cells are not statistically different from DMSO and alcohol cotreatment. (B), MTS assays measure statistically significant increases in metabolic rate at 21.7, 43.4 and 65.1 mmol/L ethanol concentrations. Treatment with 86.8 mmol/L EtOH did not result in an increase in cell proliferation. (C) Alcohol promotes the phosphorylation of ERK1/2, a key effector of growth factor signaling and of G1-S progression, regardless of estrogen treatment. Quantification comprises data of experiments in triplicate. (D) The effect of alcohol was tested on ER responsive genes TFF1/pS2 and GREB1 in MCF-7 cells. Only GREB1 responds to alcohol treatment, suggesting a possible overlap between cellular estrogen signaling and alcohol response.</p

Transcriptional regulation of core autophagy and lysosomal genes by the androgen receptor promotes prostate cancer progression

<p>AR (androgen receptor) signaling is crucial for the development and maintenance of the prostate as well as the initiation and progression of prostate cancer. Despite the AR's central role in prostate cancer progression, it is still unclear which AR-mediated processes drive the disease. Here, we identified 4 core autophagy genes: <i>ATG4B, ATG4D, ULK1</i>, and <i>ULK2</i>, in addition to the transcription factor <i>TFEB</i>, a master regulator of lysosomal biogenesis and function, as transcriptional targets of AR in prostate cancer. These findings were significant in light of our recent observation that androgens promoted prostate cancer cell growth in part through the induction of autophagy. Expression of these 5 genes was essential for maximal androgen-mediated autophagy and cell proliferation. In addition, expression of each of these 5 genes alone or in combination was sufficient to increase prostate cancer cell growth independent of AR activity. Further, bioinformatic analysis demonstrated that the expression of these genes correlated with disease progression in 3 separate clinical cohorts. Collectively, these findings demonstrate a functional role for increased autophagy in prostate cancer progression, provide a mechanism for how autophagy is augmented, and highlight the potential of targeting this process for the treatment of advanced prostate cancer.</p