Combination therapy with WEE1 inhibition and trifluridine/tipiracil against esophageal squamous cell carcinoma.

Despite advanced therapeutics, esophageal squamous cell carcinoma (ESCC) remains one of the deadliest cancers. Here, we propose a novel therapeutic strategy based on synthetic lethality combining trifluridine/tipiracil and MK1775 (WEE1 inhibitor) as a treatment for ESCC. This study demonstrates that trifluridine induces single-strand DNA damage in ESCC cells, as evidenced by phosphorylated replication protein 32. The DNA damage response includes cyclin-dependent kinase 1 (CDK1) (Tyr15) phosphorylation as CDK1 inhibition and a decrease of the proportion of phospho-histone H3 (p-hH3)-positive cells, indicating cell cycle arrest at the G2 phase before mitosis entry. The WEE1 inhibitor remarkedly suppressed CDK1 phosphorylation (Try15) and reactivated CDK1, and also increased the proportion of p-hH3-positive cells, which indicates an increase of the number of cells into mitosis. Trifluridine combined with a WEE1 inhibitor increased trifluridine-mediated DNA damage, namely DNA double-strand breaks, as shown by increased γ-H2AX expression. Moreover, the combination treatment with trifluridine/tipiracil and a WEE1 inhibitor significantly suppressed tumor growth of ESCC-derived xenograft models. Hence, our novel combination treatment with trifluridine/tipiracil and a WEE1 inhibitor is considered a candidate treatment strategy for ESCC.

Intestinal estrogen receptor beta suppresses colon inflammation and tumorigenesis in both sexes.

Estrogen hormones protect against colorectal cancer (CRC) and a preventative role of estrogen receptor beta (ERß) on CRC has been supported using full knockout animals. However, it is unclear through which cells or organ ERß mediates this effect. To investigate the functional role of intestinal ERß during colitis-associated CRC we used intestine-specific ERß knockout mice treated with azoxymethane and dextran sodium sulfate, followed by ex vivo organoid culture to corroborate intrinsic effects. We explored genome-wide impact on TNFα signaling using human CRC cell lines and chromatin immunoprecipitation assay to mechanistically characterize the regulation of ERß. Increased tumor formation in males and tumor size in females was noted upon intestine-specific ERß knockout, accompanied by enhanced local expression of TNFα, deregulation of key NFκB targets, and increased colon ulceration. Unexpectedly, we noted especially strong effects in males. We corroborated that intestinal ERß protects against TNFα-induced damage intrinsically, and characterized an underlying genome-wide signaling mechanism in CRC cell lines whereby ERß binds to cis-regulatory chromatin areas of key NFκB regulators. Our results support a protective role of intestinal ERß against colitis-associated CRC, proposing new therapeutic strategies.

Synthetic Lethality with Trifluridine/Tipiracil and Checkpoint Kinase 1 Inhibitor for Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a disease characterized by a high mutation rate of the TP53 gene, which plays pivotal roles in the DNA damage response (DDR) and is regulated by checkpoint kinase (CHK) 2. CHK1 is another key DDR-related protein, and its selective inhibition is suggested to be particularly sensitive to TP53-mutated cancers, because a loss of both pathways (CHK1 and/or CHK2-p53) is lethal due to the serious impairment of DDR. Such a therapeutic strategy is termed synthetic lethality. Here, we propose a novel therapeutic strategy based on synthetic lethality combining trifluridine/tipiracil and prexasertib (CHK1 inhibitor) as a treatment for ESCC. Trifluridine is a key component of the antitumor drug combination with trifluridine/tipiracil (an inhibitor of trifluridine degradation), also known as TAS-102. In this study, we demonstrate that trifluridine increases CHK1 phosphorylation in ESCC cells combined with a reduction of the S-phase ratio as well as the induction of ssDNA damage. Because CHK1 phosphorylation is considered to be induced as DDR for trifluridine-mediated DNA damage, we examined the effects of CHK1 inhibition on trifluridine treatment. Consequently, CHK1 inhibition by short hairpin RNA or treatment with the CHK1 inhibitor, prexasertib, markedly enhanced trifluridine-mediated DNA damage, represented by an increase of γH2AX expression. Moreover, the combination of trifluridine/tipiracil and CHK1 inhibition significantly suppressed tumor growth of ESCC-derived xenograft tumors. Furthermore, the combination of trifluridine and prexasertib enhanced radiosensitivity both in vitro and in vivo Thus, the combination of trifluridine/tipiracil and a CHK1 inhibitor exhibits effective antitumor effects, suggesting a novel therapeutic strategy for ESCC.

Estrogen receptor beta reduces colon cancer metastasis through a novel miR-205 - PROX1 mechanism.

Colon cancer is a common cause of cancer death in the Western world. Accumulating evidence supports a protective role of estrogen via estrogen receptor beta (ERß) but the mechanism of action is not known. Here, we elucidate a molecular mechanism whereby ERß represses the oncogenic prospero homebox 1 (PROX1) through the upregulation of miR-205. We show that PROX1 is a potential target of miR-205 and that in clinical specimens from The Cancer Genome Atlas data, ERß and miR-205 are decreased in colorectal cancer tissue compared to non-tumorous colon, while PROX1 levels are increased. Through mechanistic studies in multiple colorectal cancer cell lines, we show that ERß upregulates miR-205, and that miR-205 targets and represses PROX1 through direct interaction with its 3'UTR. Through the generation of intestine-specific ERß knockout mice, we establish that this pathway is correspondingly regulated in normal intestinal epithelial cells in vivo. Functionally, we demonstrate that miR-205 decreases cell proliferation and decreases migratory and invasive potential of colon cancer cells, leading to a reduction of micrometastasis in vivo. In conclusion, ERß in both normal and cancerous colon epithelial cells upregulates miRNA-205, which subsequently reduces PROX1 through direct interaction with its 3'UTR. This results in reduced proliferative and metastatic potential of the cells. Our study proposes a novel pathway that may be exploited using ERß-selective agonists and/or miR-205-replacement therapy in order to improve preventive and therapeutic approaches against colon cancer.

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.

Antiproliferative effects and mechanisms of liver X receptor ligands in pancreatic ductal adenocarcinoma cells.

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.

Genetic control of ductal morphology, estrogen-induced ductal growth, and gene expression in female mouse mammary gland.

The uterotropic response of the uterus to 17ß-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous genetic studies from our laboratory using inbred mice that are high (C57BL6/J; B6) or low (C3H/HeJ; C3H) responders to E2 led to the identification of quantitative trait loci (QTL) associated with phenotypic variation in uterine growth and leukocyte infiltration. Like the uterus, phenotypic variation in the responsiveness of the mammary gland to E2 during both normal and pathologic conditions has been reported. In the current experiment, we utilized an E2-specific model of mammary ductal growth combined with a microarray approach to determine the degree to which genotype influences the responsiveness of the mammary gland to E2, including the associated transcriptional programs, in B6 and C3H mice. Our results reveal that E2-induced mammary ductal growth and ductal morphology are genetically controlled. In addition, we observed a paradoxical effect of mammary ductal growth in response to E2 compared with what has been reported for the uterus; B6 is a high responder for the uterus and was a low responder for mammary ductal growth, whereas the reverse was observed for C3H. In contrast, B6 was a high responder for mammary ductal side branching. The B6 phenotype was associated with increased mammary epithelial cell proliferation and apoptosis, and a distinct E2-induced transcriptional program. These findings lay the groundwork for future experiments designed to investigate the genes and mechanisms underlying phenotypic variation in tissue-specific sensitivity to systemic and environmental estrogens during various physiological and disease states.

Coexposure to phytoestrogens and bisphenol a mimics estrogenic effects in an additive manner.

Endocrine-disrupting chemicals (EDC) are abundant in our environment. A number of EDCs, including bisphenol A (BPA) can bind to the estrogen receptors (ER), ERα and ERß, and may contribute to estrogen-linked diseases such as breast cancer. Early exposure is of particular concern; many EDCs cross the placenta and infants have measurable levels of, eg, BPA. In addition, infants are frequently fed soy-based formula (SF) that contains phytoestrogens. Effects of combined exposure to xeno- and phytoestrogens are poorly studied. Here, we extensively compared to what extent BPA, genistein, and an extract of infant SF mimic estrogen-induced gene transcription and cell proliferation. We investigated ligand-specific effects on ER activation in HeLa-ERα and ERß reporter cells; on proliferation, genome-wide gene regulation and non-ER-mediated effects in MCF7 breast cancer cells; and how coexposure influenced these effects. The biological relevance was explored using enrichment analyses of differentially regulated genes and clustering with clinical breast cancer profiles. We demonstrate that coexposure to BPA and genistein, or SF, results in increased functional and transcriptional estrogenic effects. Using statistical modeling, we determine that BPA and phytoestrogens act in an additive manner. The proliferative and transcriptional effects of the tested compounds mimic those of 17ß-estradiol, and are abolished by cotreatment with an ER antagonist. Gene expression profiles induced by each compound clustered with poor prognosis breast cancer, indicating that exposure may adversely affect breast cancer prognosis. This study accentuates that coexposure to BPA and soy-based phytoestrogens results in additive estrogenic effects, and may contribute to estrogen-linked diseases, including breast cancer.

Liver × receptor ligands disrupt breast cancer cell proliferation through an E2F-mediated mechanism.

INTRODUCTION: Liver × receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors and have established functions as regulators of cholesterol, glucose, and fatty acid metabolism and inflammatory responses. Published reports of anti-proliferative effects of synthetic LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment. METHODS: To further determine the effects of LXR ligands and identify their potential mechanisms of action in breast cancer cells, we carried out microarray analysis of gene expression in four breast cancer cell lines following treatments with the synthetic LXR ligand GW3965. Differentially expressed genes were further subjected to gene ontology and pathway analyses, and their expression profiles and associations with disease parameters and outcomes were examined in clinical samples. Response of E2F target genes were validated by real-time PCR, and the posited role of E2F2 in breast cancer cell proliferation was tested by RNA interference experiments. RESULTS: We observed cell line-specific transcriptional responses as well as a set of common responsive genes. In the common responsive gene set, upregulated genes tend to function in the known metabolic effects of LXR ligands and LXRs whereas the downregulated genes mostly include those which function in cell cycle regulation, DNA replication, and other cell proliferation-related processes. Transcription factor binding site analysis of the downregulated genes revealed an enrichment of E2F binding site sequence motifs. Correspondingly, E2F2 transcript levels are downregulated following LXR ligand treatment. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of estrogen receptor positive breast cancer cell proliferation. Ligand treatment also decreased E2F2 binding to cis-regulatory regions of target genes. Hierarchical clustering of breast cancer patients based on the expression profiles of the commonly downregulated LXR ligand-responsive genes showed a strong association of these genes with patient survival. CONCLUSIONS: Taken together, these results indicate that LXR ligands target gene networks, including those regulated by E2F family members, are critical for tumor biology and disease progression and merit further consideration as potential agents in the prevention and treatment of breast cancers.

Estrogen receptor ß expression induces changes in the microRNA pool in human colon cancer cells.

There is epidemiological, animal and in vitro evidence that estrogen receptor ß (ERß) can mediate protective effects against colon cancer, but the mechanism is not completely understood. Previous research has indicated critical pathways whereby ERß acts in an antitumorigenic fashion. In this study, we investigate ERß's impact on the microRNA (miRNA) pool in colon cancer cells using large-scale genomic approaches, bioinformatics and focused functional studies. We detect and confirm 27 miRNAs to be significantly changed following ERß expression in SW480 colon cancer cells. Among these, the oncogenic miR-17-92 cluster and miR-200a/b are strongly downregulated. Using target prediction and anticorrelation to gene expression data followed by focused mechanistic studies, we demonstrate that repression of miR-17 is a secondary event following ERß's downregulatory effect on MYC. We show that re-introduction of miR-17 can reverse the antiproliferative effects of ERß. The repression of miR-17 also influences cell death upon DNA damage and mediates regulation of NCOA3 (SRC-3) and CLU in colon cancer cells. We further determine that the downregulation of miR-200a/b mediates increased ZEB1 while decreasing E-cadherin levels in ERß-expressing colon cancer cells. Changes in these genes correspond to significant alterations in morphology and migration. Our work contributes novel data of ERß and miRNA in the colon. Elucidating the mechanism of ERß and biomarkers of its activity has significant potential to impact colon cancer prevention and treatment.