labeling-breast-cancer-patient-derived-xenografts-with-traceable

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

University of Colorado Anschutz Medical Campus

The CHRNA7 gene, which encodes the α7 nicotinic acetylcholine receptor (α7*nAChR), has been implicated as a candidate gene in schizophrenia. Expression of the α7*nAChR mRNA and protein are reduced in multiple regions of post-mortem brain from patients diagnosed with schizophrenia. Transcriptional regulation may therefore be an important mechanism for the regulation of this gene. A 230-bp proximal promoter fragment, necessary for transcription in cultured neuroblastoma cells, was used to study a putative AP-2α binding site. Mutation of the site indicates that AP-2α plays a negative role in regulating CHRNA7 transcription. This was confirmed through knockdown and overexpression of AP-2α. Electrophoretic mobility shift assays (EMSAs) identified positive DNA-protein interaction at this same site, and supershift assays indicate that the complex includes AP-2α. The interaction was confirmed in cells using chromatin immunoprecipitation (ChIP). DNA methylation was discovered as an anomalous mechanism for CHRNA7 regulation in one cell line. These studies suggest a role for AP-2α regulation of CHRNA7 mRNA expression in multiple tissues during development.

Transcriptional repression of the α7 nicotinic acetylcholine receptor subunit gene (CHRNA7) by activating protein-2α (AP-2α).

The α7 nicotinic acetylcholine receptor is known to regulate a wide variety of developmental and secretory functions in neural and non-neural tissues. The mechanisms that regulate its transcription in these varied tissues are not well understood. Epigenetic processes may play a role in the tissue-specific regulation of mRNA expression from the α7 nicotinic receptor subunit gene, CHRNA7. Promoter methylation was correlated with CHRNA7 mRNA expression in various tissue types and the role of DNA methylation in regulating transcription from the gene was tested by using DNA methyltransferase (DNMT1) inhibitors and methyl donors. CHRNA7 mRNA expression was silenced in SH-EP1 cells and bisulfite sequencing PCR revealed the CHRNA7 proximal promoter was hypermethylated. The proximal promoter was hypomethylated in the cell lines HeLa, SH-SY5Y, and SK-N-BE which express varying levels of CHRNA7 mRNA. Expression of CHRNA7 mRNA was present in SH-EP1 cells after treatment with the methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-CdR), and increased in SH-EP1 and HeLa cells using another methylation inhibitor, zebularine (ZEB). Transcription from the CHRNA7 promoter in HeLa cells was increased when the methyl donor methionine (MET) was absent from the media. Using methylation-sensitive restriction enzyme analysis (MSRE), there was a strong inverse correlation between CHRNA7 mRNA levels and promoter DNA methylation across several human tissue types. The results support a role for DNA methylation of the proximal promoter in regulation of CHRNA7 transcription.

Promoter methylation and tissue-specific transcription of the α7 nicotinic receptor gene, CHRNA7.

Bypassing estrogen receptor (ER) signaling during development of endocrine resistance remains the most common cause of disease progression and mortality in breast cancer patients. To date, the majority of molecular research on ER action in breast cancer has occurred in cell line models derived from late stage disease. Here we describe patient-derived ER+ luminal breast tumor models for the study of intratumoral hormone and receptor action. Human breast tumor samples obtained from patients post surgery were immediately transplanted into NOD/SCID or NOD/SCID/ILIIrg(-/-) mice under estrogen supplementation. Five transplantable patient-derived ER+ breast cancer xenografts were established, derived from both primary and metastatic cases. These were assessed for estrogen dependency, steroid receptor expression, cancer stem cell content, and endocrine therapy response. Gene expression patterns were determined in select tumors ±estrogen and ±endocrine therapy. Xenografts morphologically resembled the patient tumors of origin, and expressed similar levels of ER (5-99 %), and progesterone and androgen receptors, over multiple passages. Four of the tumor xenografts were estrogen dependent, and tamoxifen or estrogen withdrawal (EWD) treatment abrogated estrogen-dependent growth and/or tumor morphology. Analysis of the ER transcriptome in select tumors revealed notable differences in ER mechanism of action, and downstream activated signaling networks, in addition to identifying a small set of common estrogen-regulated genes. Treatment of a naïve tumor with tamoxifen or EWD showed similar phenotypic responses, but relatively few similarities in estrogen-dependent transcription, and affected signaling pathways. Several core estrogen centric genes were shared with traditional cell line models. However, novel tumor-specific estrogen-regulated potential target genes, such as cancer/testis antigen 45, were uncovered. These results evoke the importance of mapping both conserved and tumor-unique ER programs in breast cancers. Furthermore, they underscore the importance of primary xenografts for improved understanding of ER+ breast cancer heterogeneity and development of personalized therapies.

Patient-derived luminal breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-dependent gene signatures.

The ovarian hormones progesterone and estrogen play important roles in breast cancer etiology, proliferation, and treatment. Androgens may also contribute to breast cancer risk and progression. In recent years, significant advances have been made in defining the roles of these steroid hormones in stem cell homeostasis in the breast. Stem cells are potential origins of breast cancer and may dictate tumor phenotype. At least a portion of breast cancers are proposed to be driven by cancer stem cells (CSCs), cells that mimic the self-renewing and repopulating properties of normal stem cells, and can confer drug resistance. Progesterone has been identified as the critical hormone regulating normal murine mammary stem cell (MaSC) populations and normal human breast stem cells. Synthetic progestins increase human breast cancer risk; one theory speculates that this occurs through increased stem cells. Progesterone treatment also increases breast CSCs in established breast cancer cell lines. This is mediated in part through progesterone regulation of transcription factors, signal transduction pathways, and microRNAs. There is also emerging evidence that estrogens and androgens can regulate breast CSC numbers. The evolving concept that a breast CSC phenotype is dynamic and can be influenced by cell signaling and external cues emphasizes that steroid hormones could be crucial players in controlling CSC number and function. Here we review recent studies on steroid hormone regulation of breast CSCs, and discuss mechanisms by which this occurs.

Steroid hormones, steroid receptors, and breast cancer stem cells.

Cytokeratin 5 (CK5) is an epithelial cell marker implicated in stem and progenitor cell activity in glandular reproductive tissues and endocrine and chemotherapy resistance in estrogen receptor (ER)(+) breast cancer. The goal of this study was to determine the prevalence of CK5 expression in ovarian cancer and the response of CK5(+) cell populations to cisplatin therapy.

Cytokeratin 5-Positive Cells Represent a Therapy Resistant subpopulation in Epithelial Ovarian Cancer.

Anti-endocrine therapy remains the most effective treatment for ER+ breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer-associated fibroblasts (CAFs), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and anti-endocrine resistance.

Article	Year
Transcriptional repression of the α7 nicotinic acetylcholine receptor subunit gene (CHRNA7) by activating protein-2α (AP-2α). The Journal of biological chemistry\| PubMed ID: 21979958	2011
Promoter methylation and tissue-specific transcription of the α7 nicotinic receptor gene, CHRNA7. Journal of molecular neuroscience : MN\| PubMed ID: 22052086	2012
Patient-derived luminal breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-dependent gene signatures. Breast cancer research and treatment\| PubMed ID: 22821401	2012
Steroid hormones, steroid receptors, and breast cancer stem cells. Journal of mammary gland biology and neoplasia\| PubMed ID: 26265122	2015
Cytokeratin 5-Positive Cells Represent a Therapy Resistant subpopulation in Epithelial Ovarian Cancer. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society\| PubMed ID: 26495758	2015
Fibroblast subtypes regulate responsiveness of luminal breast cancer to estrogen. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 27702820	2016

Jessica Finlay-Schultz

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