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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2009)

ABSTRACT

Phytochemical Disruption of Hormone Receptor Expression and Intracellular Signaling in Human Reproductive Cancer Cells

By

Crystal Nicole Marconett

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Gary Firestone, Chair

ER-alpha is a critical target of therapeutic strategies to control the proliferation of hormone dependent breast cancers. Preferred clinical options have significant adverse side effects that can lead to treatment resistance due to the persistence of active estrogen receptors. This thesis details the molecular mechanism indole-3-carbinol (I3C) initiates to ablate ER-alpha; expression and subsequent downstream targets of ER-alpha transcriptional activity; IRS1, IGF1R, and hTERT and the ability of an aqueous mixture from the Scutellaria Barbata plant, BZL101, to arrest the proliferation of human reproductive cancer cells, regardless of hormonal status. I3C dependent activation of the aryl hydrocarbon receptor (AhR) initiates Rbx-1 E3 ligase-mediated ubiquitination and proteasomal degradation of ER-alpha protein. I3C disrupts endogenous GATA3 interactions with the ER-alpha promoter, leading to a loss of GATA3 and ER-alpha expression. Ectopic expression of GATA3 has no effect on I3C induced ER-alpha protein degradation but does prevent I3C inhibition of ER-alpha promoter activity, demonstrating the importance of GATA3 in this I3C triggered cascade. Our preclinical results implicate I3C as a novel anti-cancer agent in human cancers that co-express ER-alpha, GATA3 and AhR, a combination found in a large percentage of breast cancers but not in other critical ER-alpha target tissues essential to patient health. Estrogen signaling stimulates growth and proliferation of these cells by activation of key downstream targets, such as Insulin-like Growth Factor Receptor-1 (IGF1R) and Insulin Receptor Substrate-1 (IRS1) of the Insulin-like Growth Factor (IGF1) signaling pathway. We show that I3C downregulates both IGF1R and IRS1 RNA and protein expression. We established that this decrease in expression was contingent upon ER-alpha ablation by I3C. I3C is able to block hTERT expression through a loss of transcriptional activation. I3C mediated loss of hTERT expression was attributed to disrupted endogenous binding to a composite ER-alpha-Sp1 site. The block in hTERT expression disrupted telomerase activity, and ectopic expression of hTERT was able to restore telomerase activity. We have uncovered a critical role I3C employs to block the proproliferative activities of IGF signaling and telomerase activity in hormone sensitive breast cancer cells. Aqueous extracts from BZL101 were able to disrupt estrogen expression in the hormone MCF7 cell line. Specifically, BZL101 induced a G1 cell cycle arrest and ablation of Cyclin D, CDK2, and CDK4 expression in MCF7 cells. MDA-MB-231 and PC3 cells arrested in S phase with corresponding ablations in Cyclin A2 and CDK2. LNCaP cells arrested in G2/M phase upon exposure to BZL101 with a corresponding decrease in Cyclin B1 and CDK1. Our results indicate that BZL101 exerts cell type specific molecular changes that lead to arrest of reproductive cancer proliferation and the general disruption of ER-alpha by phytochemicals is a potent disruptor of hormone sensitive cancer growth and proliferation.

___________________________________

Gary Firestone, PhD

    Cover page: Phytochemical Disruption of Hormone Receptor Expression and Intracellular Signaling in Human Reproductive Cancer Cells
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2014)
    Cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how changes in transcriptional activation during alveolar epithelial cell (AEC) differentiation determine phenotype. We performed transcriptomic profiling using in vitro differentiation of human and rat primary AEC. This model recapitulates in vitro an in vivo process in which AEC transition from alveolar type 2 (AT2) cells to alveolar type 1 (AT1) cells during normal maintenance and regeneration following lung injury. Here we describe in detail the quality control, preprocessing, and normalization of microarray data presented within the associated study [1]. We also include R code for reproducibility of the referenced data and easily accessible processed data tables.
      Cover page: Transcriptomic profiling of primary alveolar epithelial cell differentiation in human and rat
      • Article
      • Peer Reviewed
      The human body consists of hundreds of kinds of cells specified from a single genome overlaid with cell type-specific epigenetic information. Comprehensively profiling the body's distinct epigenetic landscapes will allow researchers to verify cell types used in regenerative medicine and to determine the epigenetic effects of disease, environmental exposures and genetic variation. Key marks/factors that should be investigated include regions of nucleosome-free DNA accessible to regulatory factors, histone marks defining active enhancers and promoters, DNA methylation levels, regulatory RNAs, and factors controlling the three-dimensional conformation of the genome. Here we use the lung to illustrate the importance of investigating an organ's purified cell epigenomes, and outline the challenges and promise of realizing a comprehensive catalog of primary cell epigenomes.
        Cover page: The importance of detailed epigenomic profiling of different cell types within organs
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2024)
        In this study, the effect of cumulative ACEs experienced on human maternal DNA methylation (DNAm) was estimated while accounting for interaction with domains of ACEs in prenatal peripheral blood mononuclear cell samples from the Maternal and Developmental Risks from Environmental Stressors (MADRES) pregnancy cohort. The intergenerational transmission of ACE-associated DNAm was also explored used paired maternal (N = 120) and neonatal cord blood (N = 69) samples. Replication in buccal samples was explored in the Childrens Health Study (CHS) among adult parental (N = 31) and pediatric (N = 114) samples. We used a four-level categorical indicator variable for ACEs exposure: none (0 ACEs), low (1-3 ACEs), moderate (4-6 ACEs), and high (>6 ACEs). Effects of ACEs on maternal DNAm (N = 240) were estimated using linear models. To evaluate evidence for intergenerational transmission, mediation analysis (N = 60 mother-child pairs) was used. Analysis of maternal samples displayed some shared but mostly distinct effects of ACEs on DNAm across low, moderate, and high ACEs categories. CLCN7 and PTPRN2 was associated with maternal DNAm in the low ACE group and this association replicated in the CHS. CLCN7 was also nominally significant in the gene expression correlation analysis among maternal profiles (N = 35), along with 11 other genes. ACE-associated methylation was observed in maternal and neonatal profiles in the COMT promoter region, with some evidence of mediation by maternal COMT methylation. Specific genomic loci exhibited mutually exclusive maternal ACE effects on DNAm in either maternal or neonatal population. There is some evidence for an intergenerational effect of ACEs, supported by shared DNAm signatures in the COMT gene across maternal-neonatal paired samples.
          Cover page: Effect of parental adverse childhood experiences on intergenerational DNA methylation signatures from peripheral blood mononuclear cells and buccal mucosa.Creative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          Research Grants Program Office (RGPO) Funded Publications (2022)
          NKX2.1 is a master regulator of lung morphogenesis and cell specification; however, interactions of NKX2.1 with various transcription factors to regulate cell-specific gene expression and cell fate in the distal lung remain incompletely understood. FOXO1 is a key regulator of stem/progenitor cell maintenance/differentiation in several tissues but its role in the regulation of lung alveolar epithelial progenitor homeostasis has not been evaluated. We identified a novel role for FOXO1 in alveolar epithelial cell (AEC) differentiation that results in the removal of NKX2.1 from surfactant gene promoters and the subsequent loss of surfactant expression in alveolar epithelial type I-like (AT1-like) cells. We found that the FOXO1 forkhead domain potentiates a loss of surfactant gene expression through an interaction with the NKX2.1 homeodomain, disrupting NKX2.1 binding to the SFTPC promoter. In addition, blocking PI-3K/AKT signaling reduces phosphorylated FOXO-1 (p-FOXO1), allowing accumulated nuclear FOXO1 to interact with NKX2.1 in differentiating AEC. Inhibiting AEC differentiation in vitro with keratinocyte growth factor (KGF) maintained an AT2 cell phenotype through increased PI3K/AKT-mediated FOXO1 phosphorylation, resulting in higher levels of surfactant expression. Together these results indicate that FOXO1 plays a central role in AEC differentiation by directly binding NKX2.1 and suggests an essential role for FOXO1 in mediating AEC homeostasis.
            Cover page: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells
            • Article
            • Peer Reviewed
            Research Grants Program Office (RGPO) Funded Publications (2018)

            Aim

            To identify functional lung adenocarcinoma (LUAD) risk SNPs.

            Materials & methods

            Eighteen validated LUAD risk SNPs (p ≤ 5 × 10-8) and 930 SNPs in high linkage disequilibrium (r2 > 0.5) were integrated with epigenomic information from primary human alveolar epithelial cells. Enhancer-associated SNPs likely affecting transcription factor-binding sites were predicted. Three SNPs were functionally investigated using luciferase assays, expression quantitative trait loci and cancer-specific expression.

            Results

            Forty-seven SNPs mapped to putative enhancers; 11 located to open chromatin. Of these, seven altered predicted transcription factor-binding motifs. Rs6942067 showed allele-specific luciferase expression and expression quantitative trait loci analysis indicates that it influences expression of DCBLD1, a gene that encodes an unknown membrane protein and is overexpressed in LUAD.

            Conclusion

            Integration of candidate LUAD risk SNPS with epigenomic marks from normal alveolar epithelium identified numerous candidate functional LUAD risk SNPs including rs6942067, which appears to affect DCBLD1 expression. Data deposition: Data are provided in GEO record GSE84273.
              Cover page: Positional integration of lung adenocarcinoma susceptibility loci with primary human alveolar epithelial cell epigenomes
              • Article
              • Peer Reviewed
              Research Grants Program Office (RGPO) Funded Publications (2022)
              Many acute and chronic diseases affect the distal lung alveoli. Alveolar epithelial cell (AEC) lines are needed to better model these diseases. We used de-identified human remnant transplant lungs to develop a method to establish AEC lines. The lines grow well in 2-dimensional (2D) culture as epithelial monolayers expressing lung progenitor markers. In 3-dimensional (3D) culture with fibroblasts, Matrigel, and specific media conditions, the cells form alveolar-like organoids expressing mature AEC markers including aquaporin 5 (AQP5), G-protein-coupled receptor class C group 5 member A (GPRC5A), and surface marker HTII280. Single-cell RNA sequencing of an AEC line in 2D versus 3D culture revealed increased cellular heterogeneity and induction of cytokine and lipoprotein signaling in 3D organoids. Our approach yields lung progenitor lines that retain the ability to differentiate along the alveolar cell lineage despite long-term expansion and provides a valuable system to model and study the distal lung in vitro.
                Cover page: Development of human alveolar epithelial cell models to study distal lung biology and disease
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2018)
                Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.
                  Creative Commons 'BY-NC-ND' version 4.0 license
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2023)
                  Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents clinically with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of LUAD etiology posits alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains unknown. Here, we examine oncogenic transformation in mouse Gram-domain containing 2 (Gramd2)+ AT1 cells via oncogenic KRASG12D. Activation of KRASG12D in AT1 cells induces multifocal LUAD, primarily of papillary histology. Furthermore, KRT8+ intermediate cell states were observed in both AT2- and AT1-derived LUAD, but SCGB3A2+, another intermediate cell marker, was primarily associated with AT1 cells, suggesting different mechanisms of tumor evolution. Collectively, our study reveals that Gramd2+ AT1 cells can serve as a cell of origin for LUAD and suggests that distinct subtypes of LUAD based on cell of origin be considered in the development of therapeutics.
                    Cover page: Alveolar type I cells can give rise to KRAS-induced lung adenocarcinoma
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2016)
                    Epidemiologically related traits may share genetic risk factors, and pleiotropic analysis could identify individual loci associated with these traits. Because of their shared epidemiological associations, we conducted pleiotropic analysis of genome-wide association studies of lung cancer (12 160 lung cancer case patients and 16 838 control subjects) and cardiovascular disease risk factors (blood lipids from 188 577 subjects, type 2 diabetes from 148 821 subjects, body mass index from 123 865 subjects, and smoking phenotypes from 74 053 subjects). We found that 6p22.1 (rs6904596, ZNF184) was associated with both lung cancer (P = 5.50x10(-6)) and blood triglycerides (P = 1.39x10(-5)). We replicated the association in 6097 lung cancer case patients and 204 657 control subjects (P = 2.40 × 10(-4)) and in 71 113 subjects with triglycerides data (P = .01). rs6904596 reached genome-wide significance in lung cancer meta-analysis (odds ratio = 1.15, 95% confidence interval = 1.10 to 1.21 ,: Pcombined = 5.20x10(-9)). The large sample size provided by the lipid GWAS data and the shared genetic risk factors between the two traits contributed to the uncovering of a hitherto unidentified genetic locus for lung cancer.
                      Cover page: Pleiotropic Analysis of Lung Cancer and Blood Triglycerides
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