Publications by authors named "Carol A Sartorius"

50 Publications

The androgen receptor is a tumor suppressor in estrogen receptor-positive breast cancer.

Nat Med 2021 02 18;27(2):310-320. Epub 2021 Jan 18.

Dame Roma Mitchell Cancer Research Laboratories, Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia.

The role of the androgen receptor (AR) in estrogen receptor (ER)-α-positive breast cancer is controversial, constraining implementation of AR-directed therapies. Using a diverse, clinically relevant panel of cell-line and patient-derived models, we demonstrate that AR activation, not suppression, exerts potent antitumor activity in multiple disease contexts, including resistance to standard-of-care ER and CDK4/6 inhibitors. Notably, AR agonists combined with standard-of-care agents enhanced therapeutic responses. Mechanistically, agonist activation of AR altered the genomic distribution of ER and essential co-activators (p300, SRC-3), resulting in repression of ER-regulated cell cycle genes and upregulation of AR target genes, including known tumor suppressors. A gene signature of AR activity positively predicted disease survival in multiple clinical ER-positive breast cancer cohorts. These findings provide unambiguous evidence that AR has a tumor suppressor role in ER-positive breast cancer and support AR agonism as the optimal AR-directed treatment strategy, revealing a rational therapeutic opportunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-01168-7DOI Listing
February 2021

Enhanced antitumor immunity via endocrine therapy prevents mammary tumor relapse and increases immune checkpoint blockade sensitivity.

Cancer Res 2020 Dec 2. Epub 2020 Dec 2.

Laboratorio de Carcinogénesis Hormonal, Instituto de Biología y Medicina Experimental.

The role of active antitumor immunity in hormone receptor positive (HR+) breast cancer has been historically underlooked. The aim of this study was to determine the contribution of the immune system to antiprogestin-induced tumor growth inhibition using a hormone-dependent breast cancer model. BALB/c-GFP+ bone marrow (BM) cells were transplanted into immunodeficient NSG mice to generate an immunocompetent NSG/BM-GFP+ (NSG-R) mouse model. Treatment with the antiprogestin Mifepristone (MFP) inhibited growth of 59-2-HI tumors with similar kinetics in both animal models. Interestingly, MFP treatment reshaped the tumor microenvironment, enhancing the production of proinflammatory cytokines and chemokines. Tumors in MFP-treated immunocompetent mice showed increased infiltration of F4/80+ macrophages, NK, and CD8 T cells, displaying a central memory phenotype. Mechanistically, MFP induced immunogenic cell death in vivo and in vitro, as depicted by the expression and subcellular localization of the alarmins calreticulin and HMGB-1 and the induction of an immunogenic cell death gene program. Moreover, MFP-treated tumor cells efficiently activated immature dendritic cells, evidenced by enhanced expression of MHC-II and CD86, and induced a memory T cell response, attenuating tumor onset and growth after re-challenge. Finally, MFP treatment increased the sensitivity of HR+ 59-2-HI tumor to PD-L1 blockade, suggesting that antiprogestins may improve immunotherapy response rates. These results contribute to a better understanding of the mechanisms underlying the antitumor effect of hormonal treatment and the rational design of therapeutic combinations based on endocrine and immunomodulatory agents in HR+ breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-20-1441DOI Listing
December 2020

Insulin receptor substrate-1 (IRS-1) mediates progesterone receptor-driven stemness and endocrine resistance in oestrogen receptor+ breast cancer.

Br J Cancer 2021 Jan 4;124(1):217-227. Epub 2020 Nov 4.

Masonic Cancer Center, University of Minnesota, Minneapolis, MN, 55455, USA.

Background: Progesterone receptors (PR) are potent modifiers of endocrine responses. In aberrant signalling cancer contexts, phosphorylation events dramatically alter steroid hormone receptor action.

Methods: The transcriptomes of primary tumours and metastases in mice harbouring ER+ breast cancer patient-derived xenografts (PDXs) were analysed following single-cell RNAseq. In vitro assays were employed to delineate mechanisms of endocrine resistance and stemness.

Results: A 16-gene phospho-Ser294 PR (p-PR) signature predicted poor outcome in ER+ breast cancer. Relative to primary PDX tumours, metastatic lesions expressed abundant p-PR and exhibited an activated PR gene programme with elevated expression of PGR and IRS-1. Breast cancer models of activated PR lost the expression of IGF1R and acquired insulin hypersensitivity with tamoxifen insensitivity. Activated p-PR+ breast cancer cells formed increased tumourspheres with enlarged ALDH+ and CD24-/CD44 populations. E2 induced PR/IRS-1 interaction and exchange of IGF1Rβ for IRS-1 in p-PR-containing transcriptional complexes. Inhibition of IRS-1 or IR and inducible IRS-1 knockdown reduced tumourspheres. Endocrine-resistant models of luminal B breast cancer induced p-PR in 3D cultures and required PR and IRS-1 for tumoursphere formation.

Conclusions: Phospho-PR-B cooperates with IRS-1 to promote outgrowth of endocrine-resistant and stem-like breast cancer cells. Targeting phospho-PR/IRS-1 crosstalk may block the emergence of endocrine resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41416-020-01094-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782753PMC
January 2021

Response to: Progesterone and breast cancer pathogenesis.

J Mol Endocrinol 2021 01;66(1):L1-L2

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/JME-20-0296DOI Listing
January 2021

Exogenous Thyroid Hormone Is Associated with Shortened Survival and Upregulation of High-Risk Gene Expression Profiles in Steroid Receptor-Positive Breast Cancers.

Clin Cancer Res 2021 Jan 23;27(2):585-597. Epub 2020 Oct 23.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Purpose: Thyroid disease is a frequent comorbidity in women with breast cancer, and many require thyroid hormone replacement therapy (THRT). We postulated that THRT has a deleterious clinical effect mechanistically through hormonal interactions, nuclear receptor cross-talk, and upregulation of high-risk breast cancer genes.

Experimental Design: Observational studies of patients with lymph node-negative (LN) breast cancer ( = 820 and = 160) were performed to test interactions between THRT and clinical, histologic, outcome, and treatment variables. Differences between the two cohorts include but are not limited to patient numbers, decades of treatment, duration of follow-up/treatment, tumor sizes, incidence, and type and dose/regimen of antihormonal and/or chemotherapeutic agents. and models, databases, and molecular methods were used to study interactions and define mechanisms underlying THRT effects.

Results: THRT significantly and independently reduced disease-free and breast cancer-specific overall survival of only the steroid receptor (SR)-positive (as compared with SR-negative) node-negative patients in both long-term observational studies. Patients with SR LN breast cancer who received THRT and tamoxifen experienced the shortest survival of all treatment groups. A less potent interaction between THRT and aromatase inhibitors was noted in the second patient cohort. Using and models, TH administration enhanced estrogen and TH-associated gene expression and proliferation, nuclear colocalization of estrogen receptor and thyroid hormone receptor, and activation of genes used clinically to predict tumor aggression in SR breast cancer, including the , , and pathways.

Conclusions: We show clinically significant adverse interactions between THRT, estrogenic, and oncogenic signaling in patients with SR LN breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-20-2647DOI Listing
January 2021

Analysis of circulating breast cancer cell heterogeneity and interactions with peripheral blood mononuclear cells.

Mol Carcinog 2020 10 21;59(10):1129-1139. Epub 2020 Aug 21.

Department of Medicine, Division of Medical Oncology, University of Colorado, Aurora, Colorado.

For solid tumors, extravasation of cancer cells and their survival in circulation represents a critical stage of the metastatic process that lacks complete understanding. Gaining insight into interactions between circulating tumor cells (CTCs) and other peripheral blood mononuclear cells (PBMCs) may provide valuable prognostic information. The purpose of this study was to use single-cell RNA-sequencing (scRNA-seq) of liquid biopsies from breast cancer patients to begin defining intravascular interactions. We captured CTCs from the peripheral blood of breast cancer patients using size-exclusion membranes followed by scRNA-seq of enriched CTCs and carry-over PBMCs. Transcriptome analysis identified two populations of CTCs: one enriched for transcripts indicative of estrogen responsiveness and increased proliferation and another enriched for transcripts characteristic of reduced proliferation and epithelial-mesenchymal transition (EMT). We applied interactome and pathway analysis to determine interactions between CTCs and other captured cells. Our analysis predicted for enhanced immune evasion in the CTC population with EMT characteristics. In addition, PD-1/PD-L1 pathway activation and T cell exhaustion were predicted in T cells isolated from breast cancer patients compared with normal T cells. We conclude that scRNA-seq of breast cancer CTCs generally stratifies them into two types based on their proliferative and epithelial state and differential potential to interact with PBMCs. Better understanding of CTC subtypes and their intravascular interactions may help design treatments directed against CTCs with high metastatic and immune-evasive competence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mc.23242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895311PMC
October 2020

New generation breast cancer cell lines developed from patient-derived xenografts.

Breast Cancer Res 2020 06 23;22(1):68. Epub 2020 Jun 23.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA.

Background: Breast cancer is a highly heterogeneous disease characterized by multiple histologic and molecular subtypes. While a myriad of breast cancer cell lines have been developed over the past 60 years, estrogen receptor alpha (ER)+ disease and some mutations associated with this subtype remain underrepresented. Here we describe six breast cancer cell lines derived from patient-derived xenografts (PDX) and their general characteristics.

Methods: Established breast cancer PDX were processed into cell suspensions and placed into standard 2D cell culture; six emerged into long-term passageable cell lines. Cell lines were assessed for protein expression of common luminal, basal, and mesenchymal markers, growth assessed in response to estrogens and endocrine therapies, and RNA-seq and oncogenomics testing performed to compare relative transcript levels and identify putative oncogenic drivers.

Results: Three cell lines express ER and two are also progesterone receptor (PR) positive; PAM50 subtyping identified one line as luminal A. One of the ER+PR+ lines harbors a D538G mutation in the gene for ER (ESR1), providing a natural model that contains this endocrine-resistant genotype. The third ER+PR cell line has mucinous features, a rare histologic type of breast cancer. The three other lines are ER- and represent two basal-like and a mixed ductal/lobular breast cancer. The cell lines show varied responses to tamoxifen and fulvestrant, and three were demonstrated to regrow tumors in vivo. RNA sequencing confirms all cell lines are human and epithelial. Targeted oncogenomics testing confirmed the noted ESR1 mutation in addition to other mutations (i.e., PIK3CA, BRCA2, CCND1, NF1, TP53, MYC) and amplifications (i.e., FGFR1, FGFR3) frequently found in breast cancers.

Conclusions: These new generation breast cancer cell lines add to the existing repository of breast cancer models, increase the number of ER+ lines, and provide a resource that can be genetically modified for studying several important clinical breast cancer features.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13058-020-01300-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310532PMC
June 2020

90 YEARS OF PROGESTERONE: Progesterone and progesterone receptors in breast cancer: past, present, future.

J Mol Endocrinol 2020 07;65(1):T49-T63

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Progesterone and progesterone receptors (PR) have a storied albeit controversial history in breast cancers. As endocrine therapies for breast cancer progressed through the twentieth century from oophorectomy to antiestrogens, it was recognized in the 1970s that the presence of estrogen receptors (ER) alone could not efficiently predict treatment responses. PR, an estrogen regulated protein, became the first prognostic and predictive marker of response to endocrine therapies. It remains today as the gold standard for predicting the existence of functional, targetable ER in breast malignancies. PRs were subsequently identified as highly structured transcription factors that regulate diverse physiological processes in breast cancer cells. In the early 2000s, the somewhat surprising finding that prolonged use of synthetic progestin-containing menopausal hormone therapies was associated with increased breast cancer incidence raised new questions about the role of PR in 'tumorigenesis'. Most recently, PR have been linked to expansion of cancer stem cells that are postulated to be the principal cells reactivated in occult or dormant disease. Other studies establish PR as dominant modulators of ER activity. Together, these findings mark PR as bona fide targets for progestin or antiprogestin therapies, yet their diverse actions have confounded that use. Here we summarize the early history of PR in breast cancer; debunk the theory that progesterone causes cancer; discuss recent discoveries that PR regulate cell heterogeneity; attempt to unify theories describing PR as either good or bad actors in tumors; and discuss emerging areas of research that may help explain this enigmatic hormone and receptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/JME-20-0104DOI Listing
July 2020

Fibroblast subtypes define a metastatic matrisome in breast cancer.

JCI Insight 2020 02 27;5(4). Epub 2020 Feb 27.

Division of Medical Oncology, Department of Medicine.

Small primary breast cancers can show surprisingly high potential for metastasis. Clinical decision-making for tumor aggressiveness, including molecular profiling, relies primarily on analysis of the cancer cells. Here we show that this analysis is insufficient - that the stromal microenvironment of the primary tumor plays a key role in tumor cell dissemination and implantation at distant sites. We previously described 2 cancer-associated fibroblasts (CAFs) that either express (CD146+) or lack (CD146-) CD146 (official symbol MCAM, alias MUC18). We now find that when mixed with human breast cancer cells, each fibroblast subtype determines the fate of cancer cells: CD146- fibroblasts promoted increased metastasis compared with CD146+ fibroblasts. Potentially novel quantitative and qualitative proteomic analyses showed that CD146+ CAFs produced an environment rich in basement membrane proteins, while CD146- CAFs exhibited increases in fibronectin 1, lysyl oxidase, and tenascin C, all overexpressed in aggressive disease. We also show clinically that CD146- CAFs predicted for likelihood of lymph node involvement even in small primary tumors (<5 cm). Clearly small tumors enriched for CD146- CAFs require aggressive treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.130751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101155PMC
February 2020

Cytokeratin 5 alters β-catenin dynamics in breast cancer cells.

Oncogene 2020 03 27;39(12):2478-2492. Epub 2020 Jan 27.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Estrogen receptor (ER) positive breast cancers often contain subpopulations of cells that express the intermediate filament protein cytokeratin 5 (CK5). CK5+ cells are enriched in cancer stem cell (CSC) properties, can be induced by progestins, and predict poor prognosis in ER+ breast cancer. We established through CK5 knockout and overexpression in ER+ breast cancer cell lines that CK5 is important for tumorsphere formation, prompting us to speculate that CK5 has regulatory activity in CSCs. To interrogate CK5 interacting proteins that may be functionally cooperative, we performed immunoprecipitation-mass spectrometry for CK5 in ER+ breast cancer cells. Focusing on proteins with signaling activity, we identified β-catenin, a key transcription factor of the Wnt signaling pathway and cell adhesion molecule, as a CK5 interactor, which we confirmed by co-immunoprecipitation in several breast cancer models. We interrogated the dual functions of β-catenin in relation to CK5. Knockout or knockdown of CK5 ablated β-catenin transcriptional activity in response to progestins and Wnt stimuli. Conversely, CK5 induced by progestins or overexpression was sufficient to promote the loss of β-catenin at the cell membrane and total E-cadherin loss. A breast cancer patient-derived xenograft showed similar loss of membrane β-catenin and E-cadherin in CK5+ but not intratumoral CK5- cells and single-cell RNA sequencing found the top enriched pathways in the CK5+ cell cluster were cell junction remodeling and signaling. This report highlights that CK5 actively remodels cell morphology and that blockade of CK5-β-catenin interaction may reverse the detrimental properties of CK5+ breast cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-020-1164-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085458PMC
March 2020

Phospho-PR Isoforms and Cancer Stem Cells: What Does the FOXO1 Say?

Endocrinology 2019 05;160(5):1067-1068

Department of Pathology, University of Colorado, Anschutz Medical Campus, Aurora, Colorado.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2019-00158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760320PMC
May 2019

Separation of breast cancer and organ microenvironment transcriptomes in metastases.

Breast Cancer Res 2019 03 6;21(1):36. Epub 2019 Mar 6.

Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA.

Background: The seed and soil hypothesis was proposed over a century ago to describe why cancer cells (seeds) grow in certain organs (soil). Since then, the genetic properties that define the cancer cells have been heavily investigated; however, genomic mediators within the organ microenvironment that mediate successful metastatic growth are less understood. These studies sought to identify cancer- and organ-specific genomic programs that mediate metastasis.

Methods: In these studies, a set of 14 human breast cancer patient-derived xenograft (PDX) metastasis models was developed and then tested for metastatic tropism with two approaches: spontaneous metastases from mammary tumors and intravenous injection of PDX cells. The transcriptomes of the cancer cells when growing as tumors or metastases were separated from the transcriptomes of the microenvironment via species-specific separation of the genomes. Drug treatment of PDX spheroids was performed to determine if genes activated in metastases may identify targetable mediators of viability.

Results: The experimental approaches that generated metastases in PDX models were identified. RNA sequencing of 134 tumors, metastases, and normal non-metastatic organs identified cancer- and organ-specific genomic properties that mediated metastasis. A common genomic response of the liver microenvironment was found to occur in reaction to the invading PDX cells. Genes within the cancer cells were found to be either transiently regulated by the microenvironment or permanently altered due to clonal selection of metastatic sublines. Gene Set Enrichment Analyses identified more than 400 gene signatures that were commonly activated in metastases across basal-like PDXs. A Src signaling signature was found to be extensively upregulated in metastases, and Src inhibitors were found to be cytotoxic to PDX spheroids.

Conclusions: These studies identified that during the growth of breast cancer metastases, there were genomic changes that occurred within both the cancer cells and the organ microenvironment. We hypothesize that pathways upregulated in metastases are mediators of viability and that simultaneously targeting changes within different cancer cell pathways and/or different tissue compartments may be needed for inhibition of disease progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13058-019-1123-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404325PMC
March 2019

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries.

Nucleic Acids Res 2019 02;47(4):e20

RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky1204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393243PMC
February 2019

FGFR1 underlies obesity-associated progression of estrogen receptor-positive breast cancer after estrogen deprivation.

JCI Insight 2018 07 26;3(14). Epub 2018 Jul 26.

Division of Endocrinology, Metabolism, & Diabetes, Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA.

Obesity increases breast cancer mortality by promoting resistance to therapy. Here, we identified regulatory pathways in estrogen receptor-positive (ER-positive) tumors that were shared between patients with obesity and those with resistance to neoadjuvant aromatase inhibition. Among these was fibroblast growth factor receptor 1 (FGFR1), a known mediator of endocrine therapy resistance. In a preclinical model with patient-derived ER-positive tumors, diet-induced obesity promoted a similar gene expression signature and sustained the growth of FGFR1-overexpressing tumors after estrogen deprivation. Tumor FGFR1 phosphorylation was elevated with obesity and predicted a shorter disease-free and disease-specific survival for patients treated with tamoxifen. In both human and mouse mammary adipose tissue, FGF1 ligand expression was associated with metabolic dysfunction, weight gain, and adipocyte hypertrophy, implicating the impaired response to a positive energy balance in growth factor production within the tumor niche. In conjunction with these studies, we describe a potentially novel graft-competent model that can be used with patient-derived tissue to elucidate factors specific to extrinsic (host) and intrinsic (tumor) tissue that are critical for obesity-associated tumor promotion. Taken together, we demonstrate that obesity and excess energy establish a tumor environment with features of endocrine therapy resistance and identify a role for ligand-dependent FGFR1 signaling in obesity-associated breast cancer progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.120594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124402PMC
July 2018

Breast Cancer Suppression by Progesterone Receptors Is Mediated by Their Modulation of Estrogen Receptors and RNA Polymerase III.

Cancer Res 2017 09 20;77(18):4934-4946. Epub 2017 Jul 20.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Greater than 50% of estrogen receptor (ER)-positive breast cancers coexpress the progesterone receptor (PR), which can directly and globally modify ER action to attenuate tumor growth. However, whether this attenuation is mediated only through PR-ER interaction remains unknown. To address this question, we assessed tumor growth in ER/PR-positive patient-derived xenograft models of breast cancer, where both natural and synthetic progestins were found to antagonize the mitogenic effects of estrogens. Probing the genome-wide mechanisms by which this occurs, we documented that chronic progestin treatment blunted ER-mediated gene expression up to 2-fold at the level of mRNA transcripts. Unexpectedly, <25% of all ER DNA binding events were affected by the same treatment. The PR cistrome displayed a bimodal distribution. In one group, >50% of PR binding sites were co-occupied by ER, with a propensity for both receptors to coordinately gain or lose binding in the presence of progesterone. In the second group, PR but not ER was associated with a large fraction of RNA polymerase III-transcribed tRNA genes, independent of hormone treatment. Notably, we discovered that PR physically associated with the Pol III holoenzyme. Select pre-tRNAs and mature tRNAs with PR and POLR3A colocalized at their promoters were relatively decreased in estrogen + progestin-treated tumors. Our results illuminate how PR may indirectly impede ER action by reducing the bioavailability of translational molecules needed for tumor growth. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-16-3541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600857PMC
September 2017

Efficacy and Molecular Mechanisms of Differentiated Response to the Aurora and Angiogenic Kinase Inhibitor ENMD-2076 in Preclinical Models of p53-Mutated Triple-Negative Breast Cancer.

Front Oncol 2017 15;7:94. Epub 2017 May 15.

Department of Medicine, Division of Medical Oncology, University of Colorado Cancer Center, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Purpose: Triple-negative breast cancer (TNBC) is a subtype associated with poor prognosis and for which there are limited therapeutic options. The purpose of this study was to evaluate the efficacy of ENMD-2076 in p53-mutated TNBC patient-derived xenograft (PDX) models and describe patterns of terminal cell fate in models demonstrating sensitivity, intrinsic resistance, and acquired resistance to ENMD-2076.

Experimental Design: p53-mutated, TNBC PDX models were treated with ENMD-2076 and evaluated for mechanisms of sensitivity or resistance to treatment. Correlative tissue testing was performed on tumor tissue to assess for markers of proliferation, apoptosis, senescence, and pathways of resistance after treatment and at the time of acquired resistance.

Results: Sensitivity to ENMD-2076 200 mg/kg daily was associated with induction of apoptosis while models exhibiting intrinsic or acquired resistance to treatment presented with a senescent phenotype. Response to ENMD-2076 was accompanied by an increase in p53 and p73 levels, even within the background of mutant p53. Treatment with ENMD-2076 resulted in a decrease in pAurA and an increase in pHH3. We observed a TNBC subtype switch from the luminal androgen receptor to the basal-like subtype at acquired resistance.

Conclusion: ENMD-2076 has antitumor activity in preclinical models of p53-mutated TNBC. Increased levels of p53 and p73 correlated with sensitivity whereas senescence was associated with resistance to ENMD-2076. The novel finding of a TNBC subtype switch at time of acquired resistance may provide mechanistic insights into the biologic effects of selective pressure of anticancer treatments on TNBC. ENMD-2076 is currently being evaluated in a Phase 2 clinical trial in patients with metastatic, previously treated TNBC where these biologic correlates can be further explored.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2017.00094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430301PMC
May 2017

Patient-derived xenograft (PDX) models in basic and translational breast cancer research.

Cancer Metastasis Rev 2016 12;35(4):547-573

The Lester and Sue Smith Breast Center, Departments of Molecular and Cellular Biology and Radiology, Baylor College of Medicine, Houston,, TX, 77030, USA.

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational preclinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and "Triple-negative" (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward "credentialing" of PDX models as surrogates to represent individual patients for use in preclinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10555-016-9653-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396460PMC
December 2016

The glucose transporter GLUT1 is required for ErbB2-induced mammary tumorigenesis.

Breast Cancer Res 2016 12 20;18(1):131. Epub 2016 Dec 20.

Department of Pathology, MS 8401, University of Colorado Anschutz Medical Campus, 12801 East 17th Avenue, Box 8104, Aurora, CO, 80045, USA.

Background: Altered tumor cell metabolism is an emerging hallmark of cancer; however, the precise role for glucose in tumor initiation is not known. GLUT1 (SLC2A1) is expressed in breast cancer cells and is likely responsible for avid glucose uptake observed in established tumors. We have shown that GLUT1 was necessary for xenograft tumor formation from primary mammary cells transformed with the polyomavirus middle-T antigen but that it was not necessary for growth after tumors had formed in vivo, suggesting a differential requirement for glucose depending on the stage of tumorigenesis.

Methods: To determine whether GLUT1 is required early during mammary tumorigenesis, we crossed MMTV-NIC mice, which express activated HER2/NEU/ERBB2 and Cre recombinase, to Slc2a1 (GLUT1) mice to generate NIC-GLUT1, NIC-GLUT1, and NIC-GLUT1 mice. In addition, we evaluated effects of glucose restriction or GLUT1 inhibition on transformation in MCF10A-ERBB2 breast epithelial cells in three-dimensional culture. Finally, we utilized global gene expression profiling data of primary human breast tumors to determine the relationship between SLC2A1 and stage of tumorigenesis.

Results: All of the NIC-GLUT1 mice developed tumors in less than 200 days. In contrast, only 1 NIC-GLUT1 mouse and 1 NIC-GLUT1 mouse developed mammary tumors, even after 18 months. Mammary gland development was not disrupted in NIC mice lacking GLUT1; however, epithelial content of mature glands was reduced compared to NIC-GLUT1 mice. In MCF10A-ERBB2 cells, glucose restriction or GLUT1 inhibition blocked transformation induced by activated ERBB2 through reduced cell proliferation. In human breast cancers, SLC2A1 was higher in ductal carcinoma in situ compared to the normal breast, but lower in invasive versus in situ lesions, suggesting the requirement for GLUT1 decreases as tumors progress.

Conclusions: This study demonstrates a strict requirement for GLUT1 in the early stages of mammary tumorigenesis in vitro and in vivo. While metabolic adaptation has emerged as a hallmark of cancer, our data indicate that early tumor cells rely heavily on glucose and highlight the potential for glucose restriction as a breast cancer preventive strategy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13058-016-0795-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5168867PMC
December 2016

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

J Vis Exp 2016 11 30(117). Epub 2016 Nov 30.

Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Campus;

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 transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/54944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226329PMC
November 2016

Steroid Hormone Receptor Positive Breast Cancer Patient-Derived Xenografts.

Horm Cancer 2017 02 28;8(1):4-15. Epub 2016 Oct 28.

Department of Pathology, University of Colorado Anschutz Medical Campus, 12801 E 17th Ave MS8104, Aurora, CO, 80045, USA.

The vast majority of breast cancers are positive for estrogen receptor (ER) and depend on estrogens for growth. These tumors are treated with a variety of ER-targeted endocrine therapies, although eventual resistance remains a major clinical problem. Other steroid hormone receptors such as progesterone receptor (PR) and androgen receptor (AR) are emerging as additional prospective targets in breast cancer. The fundamental mechanism of action of these steroid receptors in gene regulation has been defined mainly by several breast cancer cell lines that were established in the late 1970s. More recently, breast cancer patient-derived xenografts (PDX) have been developed by multiple groups at institutions in several countries. These new models capture the large degree of heterogeneity between patients and within tumors and promise to advance our understanding of steroid hormone receptor positive breast cancer and endocrine resistance. Unfortunately, steroid hormone receptor positive breast cancers are much more difficult than their receptor negative counterparts to establish into sustainable PDX. Herein we discuss the derivation of steroid hormone receptor positive breast cancer PDX, several pitfalls in their genesis, and their utility in preclinical and translational steroid hormone receptor research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12672-016-0275-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291783PMC
February 2017

Fibroblast Subtypes Regulate Responsiveness of Luminal Breast Cancer to Estrogen.

Clin Cancer Res 2017 Apr 4;23(7):1710-1721. Epub 2016 Oct 4.

Department of Medicine, Division of Medical Oncology, University of Colorado Denver, Aurora, Colorado.

Antiendocrine therapy remains the most effective treatment for estrogen receptor-positive (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 (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance. Tissues from patients with ER breast cancer were analyzed for the presence of CD146-positive (CD146) and CD146-negative (CD146) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evaluated in ER tumor cells cocultured with CD146 or CD146 fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrence in tamoxifen-treated patients with ER breast cancer. We demonstrate that ER breast cancers contain two CAF subtypes defined by CD146 expression. CD146 CAFs suppress ER expression in ER breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146 CAFs maintains ER expression in ER breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146 CAFs with tamoxifen-resistant breast cancer cells are sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146 CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER breast cancer and should be considered a target for drug development. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-15-2851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378660PMC
April 2017

Cooperative Dynamics of AR and ER Activity in Breast Cancer.

Mol Cancer Res 2016 11 26;14(11):1054-1067. Epub 2016 Aug 26.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha-positive (ER) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared with dihydrotestosterone (DHT). Estradiol-induced AR-binding sites were enriched for estrogen response elements and had significant overlap with ER-binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER/AR breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER/AR patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Finally, in a comparison of ER/AR primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide preclinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single-agent efficacy may be possible in tumors resistant to traditional endocrine therapy, as clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER.

Implications: This study suggests that AR plays a previously unrecognized role in supporting E2-mediated ER activity in ER/AR breast cancer cells, and that enzalutamide may be an effective therapeutic in ER/AR breast cancers. Mol Cancer Res; 14(11); 1054-67. ©2016 AACR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1541-7786.MCR-16-0167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107172PMC
November 2016

Progestin treatment decreases CD133+ cancer stem cell populations in endometrial cancer.

Gynecol Oncol 2016 Mar 28;140(3):518-26. Epub 2015 Dec 28.

Texas Oncology, Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX 75246, USA.

Objectives: Endometrial cancer is a hormonally responsive malignancy. Response to progestins is associated with estrogen receptor (ER) and progesterone receptor (PR) status. CD133 is a marker of endometrial cancer stem cells. We postulated that CD133+ cells express ER and PR and that progestin therapy differentially regulates CD133+ cells.

Methods: The Ishikawa (ER/PR positive) and KLE (ER/PR negative) cell lines were examined for the presence of CD133 populations. Cell lines were treated with 30.4μM medroxyprogesterone 17-acetate (MPA) for 6days. After treatment, cell counts, apoptosis assays and CD133+ populations were examined. In a clinical project, we identified 12 endometrial cancer patients who were treated with progestin drugs at our institution. Using immunohistochemistry, CD133, ER, PR, and androgen receptor (AR) expression was scored and evaluated for change over time on serial biopsies.

Results: CD133+ populations were identified in Ishikawa and KLE cell lines. MPA treatment resulted in a significant reduction in the percentage of live cells (Ishikawa, P=0.036; KLE, P=0.0002), significant increase in apoptosis (Ishikawa, P=0.01; KLE, P=0.0006) and significant decrease in CD133+ populations (Ishikawa, P<0.0001; KLE, P=0.0001). ER, PR, AR and CD133 were present in 96.4%, 96.4%, 89.3% and 100% of patient samples respectively. Paralleling the in vitro results, CD133 expression decreased in patients who had histologic response to progestin treatment.

Conclusion: CD133+ populations decreased after treatment with MPA in an in vitro model and in patients responding to treatment with progestins. Progestin treatment differentially decreases CD133+ cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ygyno.2015.12.022DOI Listing
March 2016

The Six1 oncoprotein downregulates p53 via concomitant regulation of RPL26 and microRNA-27a-3p.

Nat Commun 2015 Dec 21;6:10077. Epub 2015 Dec 21.

Program in Molecular Biology, University of Colorado, Denver, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, Colorado 80045, USA.

TP53 is mutated in 50% of all cancers, and its function is often compromised in cancers where it is not mutated. Here we demonstrate that the pro-tumorigenic/metastatic Six1 homeoprotein decreases p53 levels through a mechanism that does not involve the negative regulator of p53, MDM2. Instead, Six1 regulates p53 via a dual mechanism involving upregulation of microRNA-27a and downregulation of ribosomal protein L26 (RPL26). Mutation analysis confirms that RPL26 inhibits miR-27a binding and prevents microRNA-mediated downregulation of p53. The clinical relevance of this interaction is underscored by the finding that Six1 expression strongly correlates with decreased RPL26 across numerous tumour types. Importantly, we find that Six1 expression leads to marked resistance to therapies targeting the p53-MDM2 interaction. Thus, we identify a competitive mechanism of p53 regulation, which may have consequences for drugs aimed at reinstating p53 function in tumours.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms10077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703841PMC
December 2015

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

Int J Gynecol Cancer 2015 Nov;25(9):1565-73

*Departments of Obstetrics and Gynecology and †Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO; and ‡Texas Oncology, Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX.

Objective: 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.

Materials And Methods: Cytokeratin 5 expression was evaluated in 2 ovarian tissue microarrays, representing 137 neoplasms, and 6 ovarian cancer cell lines. Cell lines were treated with IC(50) (half-maximal inhibitory concentration) cisplatin, and the prevalence of CK5(+) cells pretreatment and posttreatment was determined. Proliferation of CK5(+) versus CK5(-) cell populations was determined using 5-bromo-2'-deoxyuridine incorporation. Chemotherapy-induced apoptosis in CK5(+) versus CK5(-) cells was measured using immunohistochemical staining for cleaved caspase-3.

Results: Cytokeratin 5 was expressed in 39.3% (42 of 107) of epithelial ovarian cancers with a range of 1% to 80% positive cells. Serous and endometrioid histologic subtypes had the highest percentage of CK5(+) specimens. Cytokeratin 5 expression correlated with ER positivity (38 of 42 CK5(+) tumors were also ER(+)). Cytokeratin 5 was expressed in 5 of 6 overall and 4 of 4 ER(+) epithelial ovarian cancer cell lines ranging from 2.4% to 52.7% positive cells. Cytokeratin 5(+) compared with CK5(-) cells were slower proliferating. The prevalence of CK5(+) cells increased after 48-hour cisplatin treatment in 4 of 5 cell lines tested. Cytokeratin 5(+) ovarian cancer cells compared with CK5(-) ovarian cancer cells were more resistant to cisplatin-induced apoptosis.

Conclusions: Cytokeratin 5 is expressed in a significant proportion of epithelial ovarian cancers and represents a slower proliferating chemoresistant subpopulation that may warrant cotargeting in combination therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/IGC.0000000000000553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635519PMC
November 2015

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

J Mammary Gland Biol Neoplasia 2015 Jun 12;20(1-2):39-50. Epub 2015 Aug 12.

Department of Pathology, University of Colorado Anschutz Medical Campus, 12801 East 17th Avenue; MS 8104, Aurora, CO, 80045, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10911-015-9340-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666507PMC
June 2015

p53 Family Members Regulate Phenotypic Response to Aurora Kinase A Inhibition in Triple-Negative Breast Cancer.

Mol Cancer Ther 2015 May 10;14(5):1117-29. Epub 2015 Mar 10.

Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. Advances in the treatment of TNBC have been hampered by the lack of novel effective targeted therapies. The primary goal of this study was to evaluate the efficacy of targeting Aurora kinase A (AurA), a key regulator of mitosis, in TNBC models. A secondary objective was to determine the role of the p53 family of transcriptional regulators, commonly mutated in TNBC, in determining the phenotypic response to the AurA inhibitor alisertib (MLN8237). Alisertib exhibited potent antiproliferative and proapoptotic activity in a subset of TNBC models. The induction of apoptosis in response to alisertib exposure was dependent on p53 and p73 activity. In the absence of functional p53 or p73, there was a shift in the phenotypic response following alisertib exposure from apoptosis to cellular senescence. In addition, senescence was observed in patient-derived tumor xenografts with acquired resistance to alisertib treatment. AurA inhibitors are a promising class of novel therapeutics in TNBC. The role of p53 and p73 in mediating the phenotypic response to antimitotic agents in TNBC may be harnessed to develop an effective biomarker selection strategy in this difficult to target disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-14-0538-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425647PMC
May 2015

Live multicellular tumor spheroid models for high-content imaging and screening in cancer drug discovery.

Curr Chem Genom Transl Med 2014 7;8(Suppl 1):27-35. Epub 2014 Feb 7.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of Colorado, Anschutz Medical Campus, Aurora CO, USA.

The multi cellular tumor spheroid (MCTS) model has been used for decades with proven superiority over monolayer cell culture models at recapitulating in vivo tumor growth. Yet its use in high-throughput drug discovery has been limited, particularly with image based screening, due to practical and technical hurdles. Here we report a significant advance in utilizing live MCTS models for high-content image based drug discovery. Using a validated GFP reporter (CK5Pro-GFP) of luminal breast cancer stem cells (CSC), we developed an algorithm to quantify changes in CK5Pro-GFP expression levels for individual Z-stack planes (local) or as maximal projections of the summed Z-stacks (global) of MCTS. From these image sets, we can quantify the cross-sectional area of GFP positive cells, the fluorescence intensity of the GFP positive cells, and the percent of spheroid cross-sectional area that expresses CK5Pro-GFP.We demonstrate that acquiring data in this manner can be done in real time and is statistically robust (Z'=0.85) for use in primary high-content screening cancer drug discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/2213988501408010027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3941083PMC
March 2014

Progesterone-inducible cytokeratin 5-positive cells in luminal breast cancer exhibit progenitor properties.

Horm Cancer 2013 Feb 27;4(1):36-49. Epub 2012 Nov 27.

Department of Pathology, University of Colorado Denver, Anschutz Medical Center, Aurora, CO, USA.

Progestins play a deleterious role in the onset of breast cancer, yet their influence on existing breast cancer and tumor progression is not well understood. In luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancer, progestins induce a fraction of cells to express cytokeratin 5 (CK5), a marker of basal epithelial and progenitor cells in the normal breast. CK5(+) cells lose expression of ER and PR and are relatively quiescent, increasing their resistance to endocrine and chemotherapy compared to intratumoral CK5(-)ER(+)PR(+) cells. Characterization of live CK5(+) cells has been hampered by a lack of means for their direct isolation. Here, we describe optical (GFP) and bioluminescent (luciferase) reporter models to quantitate and isolate CK5(+) cells in luminal breast cancer cell lines utilizing the human KRT5 gene promoter and a viral vector approach. Using this system, we confirmed that the induction of GFP(+)/CK5(+) cells is specific to progestins, is dependent on PR, can be blocked by antiprogestins, and does not occur with other steroid hormones. Progestin-induced, fluorescence-activated cell sorting-isolated CK5(+) cells had lower ER and PR mRNA, were slower cycling, and were relatively more invasive and sphere forming than their CK5(-) counterparts in vitro. Repeated progestin treatment and selection of GFP(+) cells enriched for a persistent population of CK5(+) cells, suggesting that this transition can be semi-permanent. These data support that in PR(+) breast cancers, progestins induce a subpopulation of CK5(+)ER(-)PR(-) cells with enhanced progenitor properties and have implications for treatment resistance and recurrence in luminal breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12672-012-0127-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549640PMC
February 2013

Canid progesterone receptors lack activation function 3 domain-dependent activity.

Endocrinology 2012 Dec 5;153(12):6104-13. Epub 2012 Oct 5.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, 3584 CM Utrecht, The Netherlands.

Progesterone regulates multiple behavioral, physiological, and pathological aspects of female reproductive biology through its two progesterone receptors (PRs), PR-B and the truncated PR-A. PR-B is necessary for mammary gland development in mice and, compared with PR-A, is overall a stronger transactivator of target genes due to an additional activation function 3 (AF3) domain. In dogs, known for their high sensitivity to progesterone-induced mammary cancer, the PR-B function was studied. Canine PR (cPR)-B appeared to contain multiple mutations within AF3 core sequence motifs and lacks N-terminal ligand-independent posttranslational modifications. Consequently, cPR-B has a weak transactivation potential on progesterone-responsive mouse mammary tumor virus-luc and progesterone response element 2-luc reporters transiently transfected in hamster, human, or canine cells and also on known target genes FKBP5 and SGK in doxycycline-inducible, stable transfected cPR-B in canine mammary cells. The cPR-B function was restored to the level of human PR-B by the replacement of canine AF3 domain with the human one. The lack of AF3 domain-dependent transcriptional activity was unique for canids (gray wolf, red fox, and raccoon dog) and not present in closely related caniform species (brown bear, gray seal, and domestic ferret). Despite the limited transactivation potential, canids develop normal mammary glands and frequently mammary tumors. Therefore, these results question the role of PR-B in breast cancer development and may explain unique features of canid reproduction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2012-1793DOI Listing
December 2012