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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.

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.

Rapamycin inhibits mechanistic (or mammalian) target of rapamycin (mTOR) that promotes protein production in cells by facilitating ribosome biogenesis (RiBi) and eIF4E-mediated 5’cap mRNA translation. Chronic treatment with encapsulated rapamycin (eRapa) extended health and life span for wild-type and cancer-prone mice. Yet, the long-term consequences of chronic eRapa treatment are not known at the organ level. Here, we report our observations of chronic eRapa treatment on mTORC1 signaling and RiBi in mouse colon and visceral adipose. As expected, chronic eRapa treatment decreased detection of phosphorylated mTORC1/S6K substrate, ribosomal protein (rpS6) in colon and fat. However, in colon, contrary to expectations, there was an upregulation of 18S rRNA and some ribosomal protein genes (RPGs) suggesting increased RiBi. Among RPGs, eRapa increases rpl22l1 mRNA but not its paralog rpl22 . Furthermore, there was an increase in the cap-binding protein, eIF4E relative to its repressor 4E-BP1 suggesting increased translation. By comparison, in fat, there was a decrease in the level of 18S rRNA (opposite to colon), while overall mRNAs encoding ribosomal protein genes appeared to increase, including rpl22 , but not rpl22l1 (opposite to colon). In fat, there was a decrease in eIF4E relative to actin (opposite to colon) but also an increase in the eIF4E/4E-BP1 ratio likely due to reductions in 4E-BP1 at our lower eRapa dose (similar to colon). Thus, in contrast to predictions of decreased protein production seen in cell-based studies, we provide evidence that colon from chronically treated mice exhibited an adaptive ‘pseudo-anabolic’ state, which is only partially present in fat, which might relate to differing tissue levels of rapamycin, cell-type-specific responses, and/or strain differences. Keywords : mTORC1; rapamycin; translation; ribosome biogenesis (Published: 27 May 2016) To access the supplementary material for this article, please see Supplementary files in the column to the right (under ‘Article Tools’). Citation: Pathobiology of Aging & Age-related Diseases 2016, 6 : 31688 - http://dx.doi.org/10.3402/pba.v6.31688

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.

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