An Integrated Liquid Biopsy Analysis for Comprehensive Investigation of Uveal Melanoma

https://openworks.mdanderson.org/sumexp23/1107/thumbnail.jp

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

Sensitization of Resistant Cells with a BET Bromodomain Inhibitor in a Cell Culture Model of Deep Intrinsic Resistance in Breast Cancer

We treated highly metabolically adaptable (SUM149-MA) triple-negative inflammatory breast cancer cells and their control parental SUM149-Luc cell line with JQ1 for long periods to determine its efficacy at inhibiting therapy-resistant cells. After 20 days of treatment with 1–2 µM of JQ1, which killed majority of cells in the parental cell line, a large number of SUM149-MA cells survived, consistent with their pan-resistant nature. Interestingly, though, the JQ1 treatment sensitized resistant cancer cells in both the SUM149-MA and SUM149-Luc cell lines to subsequent treatment with doxorubicin and paclitaxel. To measure JQ1-mediated sensitization of resistant cancer cells, we first eradicated approximately 99% of relatively chemotherapy-sensitive cancer cells in culture dishes by long treatments with doxorubicin or paclitaxel, and then analyzed the remaining resistant cells for survival and growth into colonies. In addition, combination, rather than sequential, treatment with JQ1 and doxorubicin was also effective in overcoming resistance. Notably, Western blotting showed that JQ1-treated cancer cells had significantly lower levels of PD-L1 protein than did untreated cells, indicating that JQ1 treatment may reduce tumor-mediated immune suppression and improve the response to immunotherapy targeting PD-L1. Finally, JQ1 treatment with a low 62.5 nM dose sensitized another resistant cell line, FC-IBC02-MA, to treatment with doxorubicin and paclitaxel

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

Antiproliferative Effects and Mechanisms of Liver X Receptor Ligands in Pancreatic Ductal Adenocarcinoma Cells

<div><p>Pancreatic ductal adenocarcinoma (PDAC) is difficult to detect early and is often resistant to standard chemotherapeutic options, contributing to extremely poor disease outcomes. Members of the nuclear receptor superfamily carry out essential biological functions such as hormone signaling and are successfully targeted in the treatment of endocrine-related malignancies. Liver X receptors (LXRs) are nuclear receptors that regulate cholesterol homeostasis, lipid metabolism, and inflammation, and LXR agonists have been developed to regulate LXR function in these processes. Intriguingly, these compounds also exhibit antiproliferative activity in diverse types of cancer cells. In this study, LXR agonist treatments disrupted proliferation, cell-cycle progression, and colony-formation of PDAC cells. At the molecular level, treatments downregulated expression of proteins involved in cell cycle progression and growth factor signaling. Microarray experiments further revealed changes in expression profiles of multiple gene networks involved in biological processes and pathways essential for cell growth and proliferation following LXR activation. These results establish the antiproliferative effects of LXR agonists and potential mechanisms of action in PDAC cells and provide evidence for their potential application in the prevention and treatment of PDAC.</p></div

Microarray analysis of pancreatic cancer cell lines treated with LXR ligands defines common and cell line-specific effects on gene networks.

<p>A, B, Venn diagrams of up-regulated and down-regulated genes (1.1 fold change cutoff) after treatment with GW 3965 for 72 hours. These cell lines show common and cell-line specific transcriptomic responses to ligand treatment. C, Microarray analysis of up-regulated genes show that all cell lines share up-regulation of lipid metabolic, glucose metabolic, and cell proliferation responses. All cell lines down-regulate pathways that regulate response to viral infection, transmembrane support, as well as viral mRNA transcription. Treatments of BxPC-3 and PANC-1 cells down-regulate the expression of genes involved in cell cycle and DNA replication machinery.</p

GW 3965 downregulates oncogenes involved in cancer progression.

<p>A, GW3965 treatment downregulates SKP2 and EGFR protein levels in BxPC-3 and MIA-PaCa-2 cells. Downregulation of EGFR was concomitant with a downregulation of its own phosphorylation in BxPC-3 and MIA-PaCa-2 at 5 uM GW 3965. ERK1/2 and its phosphorylation were not statistically different in any of the cell lines B, C, D Densitometric quantification of SKP2, EGFR, Phospho-EGFR, ERK1/2, and Phospho-ERK1/2 upon treatment with GW3965. Samples were normalized to actin controls. Asterisks indicated statistically significant changes.</p