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Background: Estrogen typically promotes the progression of hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1. While estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed as an unexpected outcome of aromatase inhibitor (AI)-resistance, the molecular mechanisms have not yet been fully defined. Methods: Characterization of estrogen regulation in the two ER+ breast cancer patient-derived xenograft (PDX) models with opposite responses to estrogen offered us an unprecedented opportunity to assess how 17β-estradiol (E2) modulates ER+ cancer. We succeeded in establishing estrogen-stimulating (SC31) and estrogen-suppressing (GS3) PDX models. In vivo tumor promotion or suppression by estrogen was confirmed through experiments by implanting E2 pellets in mice carrying SC31/GS3, and then single cell analysis was performed using SC31/GS3 tumors. To investigate whether GS3 would change its response to E2, we performed intermittent E2 treatment every 28 days (E2 pellet on/off every 28 days) and whole-genome RNA sequencing using GS3 tumors. Results: SC31 and GS3 behaved oppositely regarding estrogen-mediated tumor growth. Immunohistochemistry indicated that the number of ERα+ cells and Ki-67+ cells were increased in SC31 and decreased in GS3 after E2 treatment, but progesterone receptor+ cells appeared in both SC31 and GS3 after E2 treatment at the protein level. The E2-induced suppression of GS3 involves ERα, not ERβ, which was wild-type and not amplified. Single cell RNA sequencing analysis of these PDXs had revealed that E2 upregulated the expression of estrogen-regulated genes (e.g., PGR and AREG) in both SC31 and GS3. However, E2 treatment induced cell cycle promotion in SC31, while E2 induced cell cycle arrest in GS3. These gene-expression changes occurred in both ESR1+ cells and ESR1- cells, demonstrating for the first time the influence of estrogen on ESR1- cells in ER+breast tumors. This result suggests that 100% ER positivity is not essential for endocrine response. E2 also upregulated a tumor suppressor gene, IL24, only in GS3. More IL24+ cells were ESR1+ and in G1 phase than IL24- cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24+ cells after E2 treatment. After three rounds of intermittent E2 treatment on GS3, an E2 independent growth developed. Approximately 60% of genes in the intermittent E2-treated sample had the same trend as E2-treated samples (mainly ER target genes), in which 40% of the genes behaved similarly to the placebo-treated sample (mainly cell cycle progression genes). Furthermore, lower levels of IL24 were linked to estrogen independence. Conclusions: Estrogen/ERα signaling increases the expression of estrogen-regulated genes, but it can modulate HR+ tumor growth in different manners. While E2 should activate ERα and regulate ESR1+ cells, our findings regarding ESR1- cells were new and suggested crosstalk between ESR1+ cells and ESR1- cells in both estrogen-stimulating and -suppressing ER+ tumors. Furthermore, our studies demonstrate the potential roles of tumor suppressor gene, IL24 in HR+ cancer. Our findings point to the need to identify biomarkers for patients with estrogen-suppressing tumors who can benefit from E2 treatment after AI resistance; measurements of ER and PR expression are insufficient. Expression of IL24 in AI-resistant tumors may be one such indicator for favorable response to E2 therapy. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Masafumi Nakamura, Andrea Bild, Shiuan Chen. Estrogen-mediated mechanisms in estrogen receptor-positive breast cancer at the single cell level [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-04.

Supplementary Table 1: List of primers used for quantitative real time PCR. Supplementary Table. 2: List of primers used for construction of pGL2 and pMIR reporter plasmids. Supplementary Table. 3: List of primers used to generate PCR amplicons for pyrosequencing analysis. Supplementary Table. 4: Sequencing primers used for pyrosequencing. Supplementary Table. 5: Primers used for qPCR analysis in ChIP assay.

A subset of primary breast tumors expresses a 308-gene signature that correlated with gene signatures specifically expressed in immune cells.

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more IDO1 than MCF7 cells. The two cell lines had similar upstream JAK/STAT1 signaling and IDO1 mRNA stability. However, using a series of breast cancer cell lines, IFNγ stimulated IDO1 protein expression and enzymatic activity only in ER−, not ER+, cell lines. Treatment with 5-aza-deoxycytidine reversed the suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation was potentially involved in IDO1 induction. By analyzing several breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER+/luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 promoter methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO1 inhibitor–based immunotherapy. Cancer Immunol Res; 5(4); 330–44. ©2017 AACR.

Figure Legends for Supplementary Figures 1-6

Purpose: Therapeutic strategies against hormonal receptor–positive (HR+)/HER2+ breast cancers with poor response to trastuzumab need to be optimized.Experimental Design: Two HR+/HER2+ patient-derived xenograft (PDX) models named as COH-SC1 and COH-SC31 were established to explore targeted therapies for HER2+ breast cancers. RNA sequencing and RPPA (reverse phase protein array) analyses were conducted to decipher molecular features of the two PDXs and define the therapeutic strategy of interest, validated by in vivo drug efficacy examination and in vitro cell proliferation analysis.Results: Estrogen acted as a growth driver of trastuzumab-resistant COH-SC31 tumors but an accelerator in the trastuzumab-sensitive COH-SC1 model. In vivo trastuzumab efficacy examination further confirmed the consistent responses between PDXs and the corresponding tumors. Integrative omics analysis revealed that mammalian target of rapamycin (mTOR) and ERα signaling predominantly regulate tumor growth of the two HR+/HER2+ PDXs. Combination of the dual mTOR complex inhibitor MLN0128 and anti-HER2 trastuzumab strongly suppressed tumor growth of COH-SC1 PDX accompanied by increasing ER-positive cell population in vivo. Instead, MLN0128 in combination with antiestrogen fulvestrant significantly halted the growth of HR+/HER2+ cancer cells in vitro and trastuzumab-resistant COH-SC31 as well as trastuzumab-sensitive COH-SC1 tumors in vivo.Conclusions: Compared with the standard trastuzumab treatment, this study demonstrates alternative therapeutic strategies against HR+/HER2+ tumors through establishment of two PDXs coupled with integrative omics analyses and in vivo drug efficacy examination. This work presents a prototype of future “co-clinical” trials to tailor personalized medicine in clinical practice. Clin Cancer Res; 24(2); 395–406. ©2017 AACR.

Suppl Information Table S1. Clinical characteristics of COH-SC1 and COH-SC31 PDXs

Figure S1. Characterization of two HR+/HER2+ breast patient-derived xenografts (PDXs), COH-SC1 and COH-SC31. Figure S2. Uterus and tumor weight of mice bearing COH-SC1 and COH-SC31 tumors in respond to estrogen treatment. Figure S3. Observations of body weight in response to drug treatment in vivo. Figure S4: Pathway analysis of up-regulated loci in COH-SC1 and COH-SC31 transcriptomes. Figure S5: Expression patterns of up-regulated proteins in COH-SC1 and COH-SC31 PDXs assayed by RPPA. Figure S6 In vivo efficacy examination of trastuzumab and/or MLN0128 on COH-SC1 PDX.

Table S2. Highly expressed loci in COH-SC31 in relation to COH-SC1

Suppl Figure Legends

Supplementary Table 2 from Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells

Supplementary Figures 1-6, Tables 1-15, Methods from Epigenetic Silencing Mediated through Activated PI3K/AKT Signaling in Breast Cancer

Supplementary Table 1 from Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells

Background: Estradiol (E2) is known to promote breast cancer. However, several small-scale prospective clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)- resistant cases of hormone receptor-positive postmenopausal breast cancer. Considering that the practicality and safety of E2 treatments are still undefined, the objective of this study is to uncover the mechanisms of E2-induced tumor regression, which will lead to a better design of E2 therapies. Methods: While most of our ER+ breast cancer patient-derived xenograft (PDX) models are positively regulated by E2, an estrogen-suppressive model (named GS3) was established from an AI resistant ER+/PR−/HER2− brain metastatic breast cancer. E2 pellets (1mg each) or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC), RNA sequencing, and reverse phase protein array (RPPA) analyses of PDX specimens were conducted to decipher molecular changes after E2 treatments. Importantly, since the cancer tissue has a very heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist (MPP), not by ERβ antagonist (PHTPP). IHC showed that PR expression appeared, Ki-67 and CEACAM5 (CEA) expressions decreased, and the number of apoptotic cells significantly increased after E2 treatment. RNA sequencing indicated that estrogen-regulated genes, e.g., PGR, EGR3, PDZK1, and GREB1, were up-regulated, while tumor growth was repressed by E2. Single cell RNAseq analysis demonstrated that cells from E2-treated and Placebo-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes (HVGs). Importantly, after one-week, many cells of E2-treated PDXs were arrested within the G1 phase of the cell cycle (G1, 60%; S, 19%; G2M, 21%), whereas, cells of placebo-treated samples advanced to the S and G2M phases (G1, 47%; S, 27%; G2M, 26%). Gene Set Variation Analysis of cells from E2-treated PDXs revealed enrichment of genes associated with reactive oxygen species (ROS) mechanism. E2 increased cells expressing pro-apoptotic genes, such as TP53, BOK,IL24, and PGLYRP2. Gene expressions including mitogen-activated protein kinase (MAPK) signaling pathway, such as GADD45A, MAP3K5 (ASK1), MAPK9 (JNK2), and especially MAPKAPK2 were also increased in E2 treated PDXs. Conclusions: E2/ERα-mediated suppression of GS3 tumor growth was accompanying the inhibition of the cell cycle progression and increased expression of pro-apoptotic genes. It is known that classical MAPK pathway induces cell proliferation, meanwhile, JNK MAPK pathway induces p53 signaling pathway and apoptosis. We hypothesize that E2 induces ROS and promotes apoptosis of GS3 due to JNK MAPK pathway activation. This could be an unexpected outcome of AI resistance. Using this valuable estrogen-down-regulated ER+ breast cancer PDX and informative integrative omics analyses, critical molecular mechanisms on E2-mediated apoptosis are being revealed in our laboratory. The ongoing research using E2-sensitive PDXs will identify key markers to select AI resistant patients who are expected to respond effectively to estrogen-induced apoptosis treatments as well as to evaluate the efficacy of drug combination with estrogen, including CDK 4/6 inhibitors. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest and apoptosis in aromatase inhibitor-resistant breast cancer: Insights from single cells analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-08-01.

Background: Estrogen is known to promotes hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1. However, several clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)-resistant cases of ER+ breast cancer. Considering potential impact of such clinical observation, we decided to determine the mechanisms of estrogen-induced tumor regression. Methods: A unique estrogen-inhibitory patient-derived xenograft (PDX) tumor, GS3, was established from an AI resistant ER+/HER2– brain metastatic breast cancer. In vivo estrogen suppression was confirmed through experiments by implanting 17β-estradiol (E2) pellets in mice carrying GS3, and then the single-cell analysis was performed using GS3 tumors. In vitro E2 suppression analysis was carried out using organoids from GS3.Results: The E2-induced suppression of GS3 involves ERα, which was wild-type and not amplified. Single cell RNA sequencing analysis of this PDX has revealed that E2 treatment (for 1 week) induces cell cycle arrest in both ESR1+ cells and ESR1– cells, demonstrating the unexpected influence of estrogen on ESR1– cells in ER+ breast cancer. E2 upregulated the expression of estrogen-regulated genes, including a tumor suppressor gene, IL24, and lower levels of IL24 were linked to estrogen independence, after three rounds of intermittent E2 treatment. IL24+ cells included more G1 phase cells of cell cycle compared to IL24– cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24+ cells after E2 treatment. The number of apoptotic cells was significantly increased after long term (for 4 weeks) E2 treatment. Western blotting analysis demonstrated that long term E2 treatment induced expression of apoptosis-associated protein cleaved-PARP and reduction of the pro-survival protein Bcl-xl level.Conclusions: There is the need of markers for patients who can benefit from E2 treatment after AI resistance, and measurements of ER and PR expression are not enough. Analysis of GS3 PDX has revealed that estrogen induces cell cycle arrest and apoptosis. Our study has revealed the cross-talk between ESR1+ and ESR1– cells as well as potential roles of IL24 in estrogen-suppressive tumors.

While ER has multiple biological effects, ER-cyclin D1-CDK4/6-RB is a critical pathway for the action of estrogen on the cell cycle, especially for breast cancers that rely on estrogen for growth. The latest and most efficient CDK4/6 inhibitors target the phosphorylation of retinoblastoma (RB) tumor suppressor gene; thus, altering levels of many cell cycle molecules. Estrogen receptor (ER)+/HER2- breast cancers have shown great progression free survival when CDK4/6 inhibitors are combined with endocrine therapies. Here we report the mechanism of antiestrogen (fulvestrant) combination with CDK4/6 inhibitors is due to synergism in the suppression of ER-mediated cell cycle progression. Furthermore, we performed single cell analysis of cells from an estrogen dependent/hormone receptor-positive patient derived xenograft (PDX) tumor model treated with palbociclib. These single cells expressed various levels of ER and RB which are involved in cell cycle regulation; and the response to palbociclib treatment relies not only on the ER-cyclin D1-CDK4/6-RB pathway but it is also dependent on elevated levels of ER and/or RB. Our preclinical studies show that palbociclib response is dependent on cells with ER, which is directly involved in cell cycle progression in hormone receptor positive (HR+) breast cancer.

Purpose: Therapeutic strategies against hormonal receptor–positive (HR+)/HER2+ breast cancers with poor response to trastuzumab need to be optimized. Experimental Design: Two HR+/HER2+ patient-derived xenograft (PDX) models named as COH-SC1 and COH-SC31 were established to explore targeted therapies for HER2+ breast cancers. RNA sequencing and RPPA (reverse phase protein array) analyses were conducted to decipher molecular features of the two PDXs and define the therapeutic strategy of interest, validated by in vivo drug efficacy examination and in vitro cell proliferation analysis. Results: Estrogen acted as a growth driver of trastuzumab-resistant COH-SC31 tumors but an accelerator in the trastuzumab-sensitive COH-SC1 model. In vivo trastuzumab efficacy examination further confirmed the consistent responses between PDXs and the corresponding tumors. Integrative omics analysis revealed that mammalian target of rapamycin (mTOR) and ERα signaling predominantly regulate tumor growth of the two HR+/HER2+ PDXs. Combination of the dual mTOR complex inhibitor MLN0128 and anti-HER2 trastuzumab strongly suppressed tumor growth of COH-SC1 PDX accompanied by increasing ER-positive cell population in vivo. Instead, MLN0128 in combination with antiestrogen fulvestrant significantly halted the growth of HR+/HER2+ cancer cells in vitro and trastuzumab-resistant COH-SC31 as well as trastuzumab-sensitive COH-SC1 tumors in vivo. Conclusions: Compared with the standard trastuzumab treatment, this study demonstrates alternative therapeutic strategies against HR+/HER2+ tumors through establishment of two PDXs coupled with integrative omics analyses and in vivo drug efficacy examination. This work presents a prototype of future “co-clinical” trials to tailor personalized medicine in clinical practice. Clin Cancer Res; 24(2); 395–406. ©2017 AACR.

Background: Estrogen such as estradiol (E2) is known to promote ER+ breast cancer. However, several clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)-resistant cases of ER+ postmenopausal breast cancer. The objective of this study is to uncover the mechanisms of E2-induced tumor regression, leading to an unconventional treatment of AI resistance. Methods: An E2-suppressive patient-derived xenograft model (named GS3) was established from an AI resistant ER+/PR-/HER2- brain metastatic breast cancer. Placebo or E2 pellets were implanted in mice carrying GS3 for evaluating the effects of E2. Immunohistochemistry (IHC) and RNA sequencing of GS3 were conducted to decipher molecular changes after E2 treatments. Since the cancer tissue has a heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist, not by ERβ antagonist. IHC showed that ER, Ki-67 and CEA expressions decreased and PR expression appeared after E2 treatment. Gene set enrichment analysis (GSEA) using RNAseq results showed that the E2 response gene sets were significantly up-regulated after E2 treatment. However, the cell cycle gene sets and the TNFA/NFKB gene set were down-regulated. GS3 gained an E2 independence after three cycles of intermittent E2 treatment (E2 pellet on/off every 4 weeks; Int-E2). Interestingly, the cell cycle and TNFA/NFKB gene sets were up-regulated after Int-E2 treatment. Single-cell RNAseq analysis revealed that cells from one-week E2-treated and Placebo-treated GS3 were placed in different clusters based on principle component analysis of Highly Variable Genes. Although E2 response genes were up-regulated, the percent of ESR1+ cells decreased after E2 treatment (41.3% vs. 31.5%). The number of cells arrested at the G1 phase increased (+12.5%) after E2 treatment. GSEA using genes expressed in only ESR1+ cells showed that the TNFA/NFKB gene set was significantly down-regulated after E2 treatment. Meanwhile, GSEA using genes expressed in only ESR1– cells showed that cell cycle gene sets were significantly down-regulated. Single-cell trajectory analysis disclosed three major branches; 1) common E2 and placebo, 2) E2, and 3) placebo. In the E2 only branch, the cell cycle arrested at the G1 phase, the E2 response gene sets were up-regulated, and the NFKB gene set was down-regulated in ESR1+ cells. Significantly, E2 response gene sets were also up-regulated and cell cycle genes were down-regulated even in ESR1– cells. In the placebo branch, E2 response gene sets were not up-regulated and cell cycle genes were not down regulated. A group of MKI67+ cells (at G2M phase), including some ESR1+ cells, were present in both E2-treated and placebo-treated tumors. Conclusions: E2-induced suppression is an unexpected outcome of AI resistance. In these cases, elimination of estrogen by AI results in maintaining tumor growth. Analysis of GS3 PDX has revealed that estrogen can induce cell cycle arrest and the expression of estrogen-regulated genes. Our results also suggest the cross-talk between ESR1+ and ESR1- cells as well as potential roles of the TNFA/NFKB pathway. Our findings point out the need of markers for such patients who can benefit from E2 treatment after AI resistance, and measurements of ER and PR expression are not sufficient. An intermittent treatment strategy does not sustain the effect of estrogen-mediated suppression. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest as an unexpected outcome of aromatase inhibitor-resistance: Insights from single-cell trajectory analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-02.

Recruitment of transcription machinery to target promoters for aberrant gene expression has been well studied, but underlying control directed by distant-acting enhancers remains unclear in cancer development. Our previous study demonstrated that distant estrogen response elements (DEREs) located on chromosome 20q13 are frequently amplified and translocated to other chromosomes in ERα-positive breast cancer cells. In this study, we used three-dimensional interphase fluorescence in situ hybridization to decipher spatiotemporal gathering of multiple DEREs in the nucleus. Upon estrogen stimulation, scattered 20q13 DEREs were mobilized to form regulatory depots for synchronized gene expression of target loci. A chromosome conformation capture assay coupled with chromatin immunoprecipitation further uncovered that ERα-bound regulatory depots are tethered to heterochromatin protein 1 (HP1) for coordinated chromatin movement and histone modifications of target loci, resulting in transcription repression. Neutralizing HP1 function dysregulated the formation of DERE-involved regulatory depots and transcription inactivation of candidate tumor-suppressor genes. Deletion of amplified DEREs using the CRISPR/Cas9 genomic-editing system profoundly altered transcriptional profiles of proliferation-associated signaling networks, resulting in reduction of cancer cell growth. These findings reveal a formerly uncharacterized feature wherein multiple copies of the amplicon congregate as transcriptional units in the nucleus for synchronous regulation of function-related loci in tumorigenesis. Disruption of their assembly can be a new strategy for treating breast cancers and other malignancies.

PURPOSE: Therapeutic strategies against hormonal receptor-positive (HR+)/HER2+ breast cancers with poor response to trastuzumab need to be optimized. EXPERIMENTAL DESIGN: Two HR+/HER2+ patient-derived xenograft (PDX) models named as COH-SC1 and COH-SC31 were established to explore targeted therapies for HER2+ breast cancers. RNA sequencing and RPPA (reverse phase protein array) analyses were conducted to decipher molecular features of the two PDXs and define the therapeutic strategy of interest, validated by in vivo drug efficacy examination and in vitro cell proliferation analysis. RESULTS: Estrogen acted as a growth-driver of trastuzumab-resistant COH-SC31 tumors, but an accelerator in trastuzumab-sensitive COH-SC1 model. In vivo trastuzumab efficacy examination further confirmed the consistent responses between PDXs and the corresponding tumors. Integrative omics analysis revealed that mammalian target of rapamycin (mTOR) and ERα signaling predominantly regulate tumor growth of the two HR+/HER2+ PDXs. Combination of dual mTOR complex inhibitor MLN0128 and anti-HER2 trastuzumab strongly suppressed tumor growth of COH-SC1 PDX accompanied with increasing ER-positive cell population in vivo. Instead, MLN0128 in combination with anti-estrogen fulvestrant significantly halted the growth of HR+/HER2+ cancer cells in vitro and trastuzumab-resistant COH-SC31 as well as trastuzumab-sensitive COH-SC1 tumors in vivo. CONCLUSION: Compared to the standard trastuzumab treatment, this study demonstrates alternative therapeutic strategies against HR+/HER2+ tumors through establishment of two PDXs coupling with integrative omics analyses and in vivo drug efficacy examination. This work presents a prototype of future “co-clinical” trials to tailor personalized medicine in clinical practice.

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more IDO1 than MCF7 cells. The two cell lines had similar upstream JAK/STAT1 signaling and IDO1 mRNA stability. However, using a series of breast cancer cell lines, IFNγ stimulated IDO1 protein expression and enzymatic activity only in ER−, not ER+, cell lines. Treatment with 5-aza-deoxycytidine reversed the suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation was potentially involved in IDO1 induction. By analyzing several breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER+/luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 promoter methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO1 inhibitor–based immunotherapy. Cancer Immunol Res; 5(4); 330–44. ©2017 AACR.

SummaryA causal role of gene amplification in tumorigenesis is well known, whereas amplification of DNA regulatory elements as an oncogenic driver remains unclear. In this study, we integrated next-generation sequencing approaches to map distant estrogen response elements (DEREs) that remotely control the transcription of target genes through chromatin proximity. Two densely mapped DERE regions located on chromosomes 17q23 and 20q13 were frequently amplified in estrogen receptor-α-positive luminal breast cancer. These aberrantly amplified DEREs deregulated target gene expression potentially linked to cancer development and tamoxifen resistance. Progressive accumulation of DERE copies was observed in normal breast progenitor cells chronically exposed to estrogenic chemicals. These findings may extend to other DNA regulatory elements, the amplification of which can profoundly alter target transcriptome during tumorigenesis.

Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we show that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future. Cancer Res; 71(5); 1752–62. ©2011 AACR.

Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.

Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect. Expression profiling identified that, relative to control cells, 9.1% of microRNAs (82 of 898 loci) were altered in epithelial progeny derived from mammospheres exposed to a synthetic estrogen, diethylstilbestrol. Repressive chromatin marks, trimethyl Lys27 of histone H3 (H3K27me3) and dimethyl Lys9 of histone H3 (H3K9me2), were found at a down-regulated locus, miR-9-3, in epithelial cells preexposed to diethylstilbestrol. This was accompanied by recruitment of DNA methyltransferase 1 that caused an aberrant increase in DNA methylation of its promoter CpG island in mammosphere-derived epithelial cells on diethylstilbestrol preexposure. Functional analyses suggest that miR-9-3 plays a role in the p53-related apoptotic pathway. Epigenetic silencing of this gene, therefore, reduces this cellular function and promotes the proliferation of breast cancer cells. Promoter hypermethylation of this microRNA may be a hallmark for early breast cancer development, and restoration of its expression by epigenetic and microRNA-based therapies is another viable option for future treatment of this disease. [Cancer Res 2009;69(14):5936–45]

Several mutual information (MI)-based algorithms have been developed to identify dynamic gene-gene and function-function interactions governed by key modulators (genes, proteins, etc.). Due to intensive computation, however, these methods rely heavily on prior knowledge and are limited in genome-wide analysis. We present the modulated gene/gene set interaction (MAGIC) analysis to systematically identify genome-wide modulation of interaction networks. Based on a novel statistical test employing conjugate Fisher transformations of correlation coefficients, MAGIC features fast computation and adaption to variations of clinical cohorts. In simulated datasets MAGIC achieved greatly improved computation efficiency and overall superior performance than the MI-based method. We applied MAGIC to construct the estrogen receptor (ER) modulated gene and gene set (representing biological function) interaction networks in breast cancer. Several novel interaction hubs and functional interactions were discovered. ER+ dependent interaction between TGFβ and NFκB was further shown to be associated with patient survival. The findings were verified in independent datasets. Using MAGIC, we also assessed the essential roles of ER modulation in another hormonal cancer, ovarian cancer. Overall, MAGIC is a systematic framework for comprehensively identifying and constructing the modulated interaction networks in a whole-genome landscape. MATLAB implementation of MAGIC is available for academic uses at https://github.com/chiuyc/MAGIC.

While arsenic trioxide (As2O3) is an infamous carcinogen, it is also an effective chemotherapeutic agent for acute promyelocytic leukemia and some solid tumors. In human epidermoid carcinoma A431 cells, we found that As2O3 induced cell death in time- and dose-dependent manners. Similarly, dependent regulation of the p21WAF1/CIP1 (p21) promoter, mRNA synthesis, and resultant protein expression was also observed. Additionally, transfection of a small interfering RNA of p21 could block the As2O3-induced cell growth arrest. The As2O3-induced p21 activation was attenuated by inhibitors of EGFR and MEK in a dose-dependent manner. Using a reporter assay, we demonstrated the involvement of the EGFR-Ras-Raf-ERK1/2 pathway in the promoter activation. In contrast, JNK inhibitor enhanced the As2O3-induced p21 activation, also in a dose-dependent fashion. Over-expression of a dominant negative JNK plasmid likewise also enhanced this activation. Furthermore, MEK inhibitor attenuated the anti-tumor effect of As2O3. In contrast, in combination with JNK inhibitor and As2O3 enhanced cellular cytotoxicity. Therefore, we conclude that in A431 cells the ERK1/2 and JNK pathways might differentially contribute to As2O3-induced p21 expression and then due to cellular cytotoxicity.

Background Recent genome-wide analysis has shown that DNA methylation spans long stretches of chromosome regions consisting of clusters of contiguous CpG islands or gene families. Hypermethylation of various gene clusters has been reported in many types of cancer. In this study, we conducted methyl-binding domain capture (MBDCap) sequencing (MBD-seq) analysis on a breast cancer cohort consisting of 77 patients and 10 normal controls, as well as a panel of 38 breast cancer cell lines. Results Bioinformatics analysis determined seven gene clusters with a significant difference in overall survival (OS) and further revealed a distinct feature that the conservation of a large gene cluster (approximately 70 kb) metallothionein-1 (MT1) among 45 species is much lower than the average of all RefSeq genes. Furthermore, we found that DNA methylation is an important epigenetic regulator contributing to gene repression of MT1 gene cluster in both ERα positive (ERα+) and ERα negative (ERα−) breast tumors. In silico analysis revealed much lower gene expression of this cluster in The Cancer Genome Atlas (TCGA) cohort for ERα + tumors. To further investigate the role of estrogen, we conducted 17β-estradiol (E2) and demethylating agent 5-aza-2′-deoxycytidine (DAC) treatment in various breast cancer cell types. Cell proliferation and invasion assays suggested MT1F and MT1M may play an anti-oncogenic role in breast cancer. Conclusions Our data suggests that DNA methylation in large contiguous gene clusters can be potential prognostic markers of breast cancer. Further investigation of these clusters revealed that estrogen mediates epigenetic repression of MT1 cluster in ERα + breast cancer cell lines. In all, our studies identify thousands of breast tumor hypermethylated regions for the first time, in particular, discovering seven large contiguous hypermethylated gene clusters. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0045-9) contains supplementary material, which is available to authorized users.

Epidemiological studies reveal that maternal exposure to estrogenic chemicals during pregnancy results in higher risk of breast cancer in offspring. One of possible causal mechanisms is epigenetic reprogramming, but how estrogen/ERα signaling contributes to tumorigenesis through epigenetic machinery remains unclear. In this study, we demonstrate that the epigenetic modulation mediated by estrogen-driven amplification of distant-acting regulatory elements, or enhancers, may be an explanation addressing to this question. Enhancers contain transcription factor binding sites known to remotely regulate transcription through chromatin looping or transvection. Using our in vitro model to mimic maternal estrogen exposure and a "Seq-to-Seek" strategy integrating three next-generation sequencing approaches, we comprehensively mapped distant estrogen response elements (DEREs) that remotely control transcription of target genes through chromatin proximity. Surprisingly, a densely mapped DERE region located on chromosome 20q13 frequently amplifies in ERα-positive breast cancer with poor prognosis. Progressive accumulation of DERE copies was observed in normal breast progenitor and cancer cells chronically exposed to estrogen. Upon estrogen stimulation, these aberrantly amplified DEREs are clustered as a potential transcriptional depot for suppressing target gene expression through altering chromatin interactions, leading to accumulation of repressive histone marks (e.g. trimethylated H3K27 and H3K9), polycomb repressive complex 2, the presence of heterochromatin protein 1 (HP1) and DNA methylation, following prevention of RNA polymerase II and active histone mark- H3K4me3 binding. Furthermore, neutralization of HP1 function can significantly attenuate estrogen-driven DERE clustering and DERE-mediated repressive chromatin interactions, resulting in inhibition of cell proliferation. Deletion of the interested DERE regions using CRISPR/Cas9 genomic editing system further demonstrated that DERE-driven remote transcriptional control play a crucial role in tumor cell growth. Our data support a model in which amplified DEREs preferentially induce repressive epigenetic modulation of target genes. These findings indicate that 20q13 DERE region is a potential transcriptionally repressive domain whose aberrant amplification can result in suppressing expression of tumor suppressor genes, leading to tumorigenesis. In summary, our findings suggest that amplification of DNA regulatory elements can profoundly alter target transcriptome during tumorigenesis and amplified DEREs can be used as potential prognostic markers for endocrine resistance and predicative for progeny at risk of breast cancer. Citation Format: Pei-Yin Hsu, Hang-Kai Hsu, Yi-dong Chen, Victor X Jin, Zelton D Sharp, Tim H-M Huang. Amplified DERE-mediated epigenetic repression in ERα-mediated breast tumorigenesis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-02.

Background An emerging Hi-C protocol has the ability to probe three-dimensional (3D) architecture and capture chromatin interactions in a genome-wide scale. It provides informative results to address how chromatin organization changes contribute to disease/tumor occurrence and progression in response to stimulation of environmental chemicals or hormones. Results In this study, using MCF7 cells as a model system, we found estrogen stimulation significantly impact chromatin interactions, leading to alteration of gene regulation and the associated histone modification states. Many chromosomal interaction regions at different levels of interaction frequency were identified. In particular, the top 10 hot regions with the highest interaction frequency are enriched with breast cancer specific genes. Furthermore, four types of E2-mediated strong differential (gain- or loss-) chromosomal (intra- or inter-) interactions were classified, in which the number of gain-chromosomal interactions is less than the number of loss-chromosomal interactions upon E2 stimulation. Finally, by integrating with eight histone modification marks, DNA methylation, regulatory elements regions, ERα and Pol-II binding activities, associations between epigenetic patterns and high chromosomal interaction frequency were revealed in E2-mediated gene regulation. Conclusions The work provides insight into the effect of chromatin interaction on E2/ERα regulated downstream genes in breast cancer cells.

Background Global profiling of in vivo protein-DNA interactions using ChIP-based technologies has evolved rapidly in recent years. Although many genome-wide studies have identified thousands of ERα binding sites and have revealed the associated transcription factor (TF) partners, such as AP1, FOXA1 and CEBP, little is known about ERα associated hierarchical transcriptional regulatory networks. Results In this study, we applied computational approaches to analyze three public available ChIP-based datasets: ChIP-seq, ChIP-PET and ChIP-chip, and to investigate the hierarchical regulatory network for ERα and ERα partner TFs regulation in estrogen-dependent breast cancer MCF7 cells. 16 common TFs and two common new TF partners (RORA and PITX2) were found among ChIP-seq, ChIP-chip and ChIP-PET datasets. The regulatory networks were constructed by scanning the ChIP-peak region with TF specific position weight matrix (PWM). A permutation test was performed to test the reliability of each connection of the network. We then used DREM software to perform gene ontology function analysis on the common genes. We found that FOS, PITX2, RORA and FOXA1 were involved in the up-regulated genes. We also conducted the ERα and Pol-II ChIP-seq experiments in tamoxifen resistance MCF7 cells (denoted as MCF7-T in this study) and compared the difference between MCF7 and MCF7-T cells. The result showed very little overlap between these two cells in terms of targeted genes (21.2% of common genes) and targeted TFs (25% of common TFs). The significant dissimilarity may indicate totally different transcriptional regulatory mechanisms between these two cancer cells. Conclusions Our study uncovers new estrogen-mediated regulatory networks by mining three ChIP-based data in MCF7 cells and ChIP-seq data in MCF7-T cells. We compared the different ChIP-based technologies as well as different breast cancer cells. Our computational analytical approach may guide biologists to further study the underlying mechanisms in breast cancer cells or other human diseases.

Homodimerization of RON (MST1R), a receptor tyrosine kinase, usually occurs in cells stimulated by a ligand and leads to the downstream activation of signaling pathways. Here we report that bladder cancer cells, in response to physiological stress, use an alternative mechanism for signaling activation. Time-course studies indicated that RON migrated directly from the membrane to the nucleus of bladder cancer cells in response to serum starvation. Biochemical and genetic studies implied that this nuclear internalization was complexed with epidermal growth factor receptor (EGFR) and required the docking of importins. In vivo analysis confirmed that nuclear RON was present in 38.4% (28/73) of primary bladder tumors. Chromatin immunoprecipitation (ChIP) on microarray analysis further revealed that this internalized complex bound to at least 134 target genes known to participate in three stress-responsive networks: p53, stress-activated protein kinase/c-jun N-terminal kinase and phosphatidylinositol 3-kinase/Akt. These findings suggest that RON, in a complex with EGFR, acts as a transcriptional regulator in response to acute disturbances (e.g. serum starvation) imposed on cancer cells. In an attempt to re-establish homeostasis, these cells bypass regular mechanisms required by ligand stimulation and trigger the RON-directed transcriptional response, which confers a survival advantage.

The current concept of epigenetic repression is based on one repressor unit corresponding to one silent gene. This notion, however, cannot adequately explain concurrent silencing of multiple loci observed in large chromosome regions. The long-range epigenetic silencing (LRES) can be a frequent occurrence throughout the human genome. To comprehensively characterize the influence of estrogen signaling on LRES, we analyzed transcriptome, methylome, and estrogen receptor alpha (ESR1)-binding datasets from normal breast epithelia and breast cancer cells. This “omics” approach uncovered 11 large repressive zones (range, 0.35∼5.98 megabases), including a 14-gene cluster located on 16p11.2. In normal cells, estrogen signaling induced transient formation of multiple DNA loops in the 16p11.2 region by bringing 14 distant loci to focal ESR1-docking sites for coordinate repression. However, the plasticity of this free DNA movement was reduced in breast cancer cells. Together with the acquisition of DNA methylation and repressive chromatin modifications at the 16p11.2 loci, an inflexible DNA scaffold may be a novel determinant used by breast cancer cells to reinforce estrogen-mediated repression.

Indoleamine 2, 3-dioxygenase 1, encoded by IDO1, is a key immunosuppressive enzyme that catabolizes essential amino acid tryptophan to kynurenine in the tumor microenvironment. Severe depletion of tryptophan by IDO1 affects proliferation of T cells and tryptophan metabolites cause T-cell anergy and induce apoptosis. In this study, we investigated the epigenetic regulation of IDO1 expression by DNA methylation in breast cancer cells. Bisulfite pyrosequencing analysis of IDO1 promoter methylation performed on a panel of 10 breast cancer cell lines revealed that triple negative breast cancer (TNBC) cell lines (i.e. MDA-MB-231, Hs578t, SUM159) are hypomethylated compared to estrogen receptor positive (ER+) cell lines (i.e. MCF7, BT474, T47D). The same analysis was extended to 30 primary breast tumor and normal control samples and the results demonstrated that TNBC tumors had lower IDO1 promoter methylation compared to ER+ tumors. RT-PCR analysis showed that IDO1 mRNA expression is higher in TNBC cell lines than ER+ cell lines. This inverse correlation between IDO1 promoter methylation and mRNA expression can also be observed in TCGA 450K methylation and RNA-seq data sets in which promoter is hypomethylated and mRNA is up-regulated in basal-like molecular subtypes as compared to other breast cancer subtypes. IDO1 promoter is relatively CpG poor with most CpG sites concentrated near interferon γ (IFNg) responsive GAS and ISRE transcription factor binding sites. To investigate the role of CpG methylation in regulating IDO1 induction by IFNg, we cloned either methylated or unmethylated IDO1 promoter DNA into a luciferase reporter plasmid. Methylated promoter reporter exhibited significantly lower luciferase activity at basal or with IFNg stimulation compared to unmethylated reporter plasmid when transfected into MCF-7 or MDA-MB-231 cell lines. Treatment with a demethylating agent, 5-aza-deoxycytidine, synergistically up-regulated IDO1 mRNA expression with IFNg in MCF7 cells which have hypermethylated IDO1 promoter further supporting the influence of CpG methylation on IDO1 expression. IFNg stimulation or co-culture with activated T-cells significantly induced IDO1 protein expression in MDA-MB-231 cells, but not in MCF7 cells. We found no difference in IDO1 mRNA stability and IFNg induced JAK/STAT signaling pathway between MDA-MB-231 and MCF7 cell lines except promoter methylation. Furthermore, RNA-seq analysis of MDA-MB-231 and MCF7 cell lines co-cultured with activated T-cells revealed an active immune signaling mediated through JAK/STAT pathway shared by both cell lines, but also differential induction of IDO1 transcription between these two cell lines. These findings indicate IDO1 promoter methylation status as an important factor that regulates anti-immune responses by tumor cells towards tumor infiltrating lymphocytes and it could be used as a useful biomarker for IDO inhibitor based immunotherapy. Citation Format: Satish Kumar Reddy Noonepalle, Eun Joon Lee, Maria Ouzounova, Jaejik Kim, Jeong-Hyeon Choi, Austin Shull, Lirong Pei, Ravindra Kolhe, Pei-Yin Hsu, Nagireddy Putluri, Tim Hui-Ming Huang, Arun Sreekumar, Hasan Korkaya, David Munn, Huidong Shi. Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2015-4060

Recruitment of transcriptional machinery to target promoters for aberrant gene expression has been well studied, but underlying control directed by distant-acting enhancers remains unclear in cancer development. Our previous study demonstrated that distant estrogen response elements (DEREs) located on chromosome 20q13 were frequently amplified (50∼60 copies) and translocated to other chromosomes in ERα-positive breast cancer cells. In this study, we used three-dimensional interphase fluorescence in situ hybridization to decipher spatiotemporal gathering of multiple DEREs in the nucleus. Upon estrogen stimulation, the majority of 20q13 DEREs were mobilized to form regulatory depots for synchronized regulation of target loci. A chromosome conformation capture assay coupled with chromatin immunoprecipitation further uncovered that ERα-bound regulatory depots are tethered to heterochromatin protein 1 (HP1) for coordinated chromatin movement and subsequent facilitation of histone modifications of target loci, resulting in transcription repression. Neutralizing HP1 function dissembled these regulatory depots and reversed this transcription inactivation of candidate tumor-suppressor genes. Deletion of amplified DEREs using the CRISPR/Cas9 genomic-editing system profoundly altered transcriptional profiles of JAK/STAT signaling networks, leading to proliferation inhibition of cancer cells with beneficial outcome of breast cancers. These findings reveal a formerly uncharacterized feature of oncogenic amplicons that multiple copies of the amplicon congregate as transcriptional unions in the nucleus for synchronous regulation of function-related loci in tumorigenesis. Disruption of their assembly can be a new strategy for treating breast cancers and other malignancies. Citation Format: Pei-Yin Hsu, Hang-Kai Hsu, Victor X. Jin, Zelton D. Sharp, Tim H.-M. Huang. HP1-mediated spatiotemporal control of estrogen-responsive transcription in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4778. doi:10.1158/1538-7445.AM2015-4778

Since the first amplified gene, MYCN, was identified in neuroblastomas, considerable effort has been directed toward discovery of oncogenes in amplified genomic regions. However, there is no significant correlation between copy number gains and increased expression of corresponding genes within amplicons. We demonstrate that amplification of distant-acting regulatory elements, or enhancers, may be an alternative mechanism contributing to an oncogenic event. Enhancers comprise transcription factor binding sites known to remotely regulate transcription through chromatin looping or transvection. Using a “Seq-to-Seek” strategy integrating three next-generation sequencing approaches, we comprehensively mapped distant estrogen response elements (DEREs) that remotely control transcription of target genes through chromatin proximity. Surprisingly, a densely mapped DERE region located on chromosome 20q13 frequently amplifies in ERα-positive breast cancer with poor prognosis. Progressive accumulation of DERE copies was observed in normal breast progenitor and cancer cells chronically exposed to estrogen or estrogenic chemicals. Pharmacological studies with either an ERα antagonist or an ataxia telangiectasia mutated (ATM) kinase inhibitor further showed that ATM-dependent DNA repair pathway participates in ERα-driven increase of 20q13 DERE copies, suggesting involvement of breakage-fusion-bridge mechanism in DERE amplification. Furthermore, these aberrantly amplified DEREs altered chromatin interactions, leading to transcriptional repression of genes that are associated with tumor-suppressor and apoptosis pathways and potentially linked to cancer development and tamoxifen resistance. Correlation analyses of copy-number variation, occupancy of the repressive histone mark, H3K27me3, and methylation profiling of 20q13 DEREs in ERα-positive cancer cells support a model in which amplified DEREs preferentially induce repressive epigenetic modulation of target genes. In addition, interphase fluorescence in situ hybridization coupled with immunofluorescence analyses revealed that estrogen stimulation leads to DERE clustering in a transcriptional hub nearby in a heterochromatic region. These findings indicate that the 20q13 DERE region is a potential transcriptionally repressive domain whose aberrant amplification can result in suppressing expression of tumor suppressor genes. To determine the precise role of amplified DEREs in epigenetic transcription and their biological significance, our ongoing studies use CRISPR/Cas genomic editing system to delete candidate DEREs at 20q13. In summary, our findings suggest that amplification of DNA regulatory elements can profoundly alter target transcriptome during tumorigenesis and amplified DEREs can be used as potential prognostic markers for endocrine resistance. Citation Format: PEI-YIN HSU, HANG-KAI HSU, Victor X. Jin, Zelton D. Sharp, Tim H.-M. Huang. Amplification of distant estrogen response elements epigenetically deregulates target genes in ERα-mediated breast tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4783. doi:10.1158/1538-7445.AM2014-4783

The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is a critical survival cascade in tumor progression. Our previous study demonstrated that combinational treatment of a potent PI3K inhibitor, LY294002 (LY), and DNA methyltransferase inhibitor, 5’-Aza-deoxycytidine (DAC), compounds effectively reduce tumor formation in the immunodeficiency mouse model, suggesting that PI3K/AKT signaling suppresses tumorigenesis through the epigenetic modulation. While tumor size of the LY-treated xenografts is dramatically decreased compared to the control group, the tumor growth cannot be completely attenuated by either LY or LY/DAC treatment, indicating that there is an alternative route for tumor survival. Here, we performed a microarray analysis to identify LY-mediated expression profiling on an ERα-positive breast cancer cell line, T-47D, the cell line applied in the previous xenograft experiments. A subset of 24 LY-activated genes associated with better clinical outcome are found to be not only direct targets of PI3K/AKT but involved in estrogen (E2)/ERα signaling by ingenuity pathway analysis, suggesting the involvement of ERα signaling in PI3K/AKT-mediated tumor growth. To clarify their relationship, pharmacological experiments using an ERα antagonist, ICI 182, 780 (ICI) and LY coupled with E2 was conducted. We observed that E2 stimulation induces both AKT and ERα activation in a time-dependent manner. Either ICI or LY treatment cannot repress the activation levels of AKT or ERα, respectively. Though, either single or combined inhibitor treatment can effectively restore E2-suppressed gene expression. These findings indicate that cross-talk between PI3K/AKT and ERα signaling cascades may cooperatively regulate cell growth and associated gene expression. In addition, transcription factor motif analysis showed that signal transducer and activator of transcription 3 and 6 (STAT3/6) binding motifs are enriched with highest prediction score on the promoter regions of the examined AKT-directed targets. Inhibitors prohibit E2-mediated assembly of STAT3/6 with ERα and STAT3/6 recruitment onto the promoter regions of target loci using co-immunoprecipitation (co-IP) and chromatin IP-qPCR. Moreover, repressive histone marks, H3K27me3 and H3K9me3, are found to occupy on the same regions as STAT3/6 upon E2 treatment. DAC treatment can effectively restore E2-induced suppression of AKT-directed target genes. Further association studies are ongoing to interrogate whether STAT3/6 is part of repressive transcription complex with H3K27me3 and H3K9me3. In summary, we propose that cross-talk of ERα with PI3K/AKT signaling represents the potential function switch of ERα from genomic binding to no-genomic binding. Combined treatment of PI3K/AKT inhibitors with hormone therapy may shed a light of therapeutic strategy to patients with aggressive or metastatic breast tumors. Citation Format: Hang-Kai Hsu, Pei-Yin Hsu, Yao Wang, Zelton D. Sharp, Tim H.-M. Huang, Victor X. Jin. Cross-talk between PI3K/AKT and ERα signaling leads to STAT3/6-modulated epigenetic transcription in aggressive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2014-3341

One big limitation of computational tools for analyzing ChIP-seq data is that most of them ignore non-unique tags (NUTs) that match the human genome even though NUTs comprise up to 60% of all raw tags in ChIP-seq data. Effectively utilizing these NUTs would increase the sequencing depth and allow a more accurate detection of enriched binding sites, which in turn could lead to more precise and significant biological interpretations. In this study, we have developed a computational tool, LOcating Non-Unique matched Tags (LONUT), to improve the detection of enriched regions from ChIP-seq data. Our LONUT algorithm applies a linear and polynomial regression model to establish an empirical score (ES) formula by considering two influential factors, the distance of NUTs to peaks identified using uniquely matched tags (UMTs) and the enrichment score for those peaks resulting in each NUT being assigned to a unique location on the reference genome. The newly located tags from the set of NUTs are combined with the original UMTs to produce a final set of combined matched tags (CMTs). LONUT was tested on many different datasets representing three different characteristics of biological data types. The detected sites were validated using de novo motif discovery and ChIP-PCR. We demonstrate the specificity and accuracy of LONUT and show that our program not only improves the detection of binding sites for ChIP-seq, but also identifies additional binding sites.