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.
.
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.
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.
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.
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.
Triple-negative breast cancer (TNBC) remains an aggressive breast cancer subtype with limited treatment options. ENMD-2076 is a small-molecule inhibitor of Aurora and angiogenic kinases with ...proapoptotic and antiproliferative activity in preclinical models of TNBC.
This dual-institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC previously treated with one to three prior lines of chemotherapy in the advanced setting. Patients were treated with ENMD-2076 250 mg orally once daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary endpoint was 6-month clinical benefit rate (CBR), and secondary endpoints included progression-free survival, pharmacokinetic profile, safety, and biologic correlates in archival and fresh serial tumor biopsies in a subset of patients.
Forty-one patients were enrolled. The 6-month CBR was 16.7% (95% CI, 6-32.8%) and included two partial responses. The 4-month CBR was 27.8% (95% CI, 14-45.2%), and the average duration of benefit was 6.5 cycles. Common adverse events included hypertension, fatigue, diarrhea, and nausea. Treatment with ENMD-2076 resulted in a decrease in cellular proliferation and microvessel density and an increase in p53 and p73 expression, consistent with preclinical observations.
Single-agent ENMD-2076 treatment resulted in partial response or clinical benefit lasting more than 6 months in 16.7% of patients with pretreated, advanced, or metastatic TNBC. These results support the development of predictive biomarkers using archival and fresh tumor tissue, as well as consideration of mechanism-based combination strategies.
ClinicalTrials.gov, NCT01639248 . Registered on July 12, 2012.
Regulation of hematopoietic stem cell proliferation, lineage commitment, and differentiation in adult vertebrates requires extrinsic signals provided by cells in the marrow microenvironment (ME) ...located within the bone marrow. Both secreted and cell-surface bound factors critical to this regulation have been identified, yet control of their expression by cells within the ME has not been addressed. Herein we hypothesize that microRNAs (miRNAs) contribute to their controlled expression. MiRNAs are small noncoding RNAs that bind to target mRNAs and downregulate gene expression by either initiating mRNA degradation or preventing peptide translation. Testing the role of miRNAs in downregulating gene expression has been difficult since conventional techniques used to define miRNA-mRNA interactions are indirect and have high false-positive and negative rates. In this report, a genome-wide biochemical technique (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation or HITS-CLIP) was used to generate unbiased genome-wide maps of miRNA-mRNA interactions in two critical cellular components of the marrow ME: marrow stromal cells and bone marrow endothelial cells. Analysis of these datasets identified miRNAs as direct regulators of JAG1, WNT5A, MMP2, and VEGFA; four factors that are important to ME function. Our results show the feasibility and utility of unbiased genome-wide biochemical techniques in dissecting the role of miRNAs in regulation of complex tissues such as the marrow ME.
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.
There are two major subtypes of human breast cancers: the luminal, estrogen, and progesterone receptor-positive, cytokeratin 18-positive (ER⁺PR⁺CK18⁺) subtype, and the basal ER⁻PR⁻CK18⁻CK5⁺ subtype. ...Tumor-initiating cells (CD44⁺) have been described for human breast cancers; whether these are common to the two subtypes is unknown. We have identified a rare population of cells that are both CD44⁺ and ER⁻PR⁻CK5⁺ in luminal-like ER⁺PR⁺ T47D human breast tumor xenografts. The tumor-isolated CD44⁺ cell fraction was highly enriched for clonogenic (in vitro culture) and tumorigenic (in vivo reimplantation) cells compared with the CD44⁻ cell fraction. Rare ER⁻PR⁻CK5⁺ cells were present within CD44⁺-derived colonies. Tumor-isolated cells placed in minimal media also contained rare ER⁻PR⁻CK5⁺ cells at early time points (<10 cells); however, this population did not expand with increasing colony size. The number of ER⁺PR⁺CK5⁻ cells, conversely, increased linearly with colony growth. Similary, tumors originating in vivo from CD44⁺ cells contained a rare static ER⁻PR⁻CK5⁺ population, an intermediate ER⁻PR⁻CK5⁻ population, and an expanding ER⁺PR⁺CK5⁻ population. Putative ER⁺PR⁺CK5⁺ transitional cells could be seen only in colonies or tumors treated with a progestin. We propose that luminal ER⁺PR⁺ breast tumors contain a minor ER⁻PR⁻CK5⁺ population that has the capacity to generate the majority of ER⁺PR⁺CK18⁺CK5⁻ cells. Luminal breast cancers are treated with endocrine therapies that target ER. The rare ER⁻PR⁻CK5⁺ progenitor cells would escape such treatments and survive to repopulate the tumor.
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.
Abstract
This nonrandomized, open-label, multi-cohort Phase 1b study (NCT02779751) investigated the safety and efficacy of abemaciclib plus pembrolizumab with/without anastrozole in patients with ...hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2−) metastatic breast cancer (MBC) without prior CDK4 and 6 inhibitor exposure. Patients were divided into two cohorts: treatment naïve (cohort 1) and pretreated (cohort 2). Patients received abemaciclib plus pembrolizumab with (cohort 1) or without (cohort 2) anastrozole over 21-day cycles. The primary objective was safety, and secondary objectives included efficacy and pharmacokinetics (PK). Cohort 1/2 enrolled 26/28 patients, respectively. Neutropenia (30.8/28.6%), AST increase (34.6/17.9%), ALT increase (42.3/10.7%), and diarrhea (3.8/10.7%) were the most frequent grade ≥3 adverse events in cohort 1/2, respectively. A total of two deaths occurred, which investigators attributed to treatment-related adverse events (AEs), both in cohort 1. Higher rates of all grade and grade ≥3 interstitial lung disease (ILD)/pneumonitis were observed compared to previously reported with abemaciclib and pembrolizumab monotherapy. The PK profiles were consistent between cohorts and with previous monotherapy studies. In cohorts 1/2, the overall response rate and disease control rate were 23.1/28.6% and 84.6/82.1%, respectively. Median progression-free survival and overall survivals were 8.9 (95% CI: 3.9–11.1) and 26.3 months (95% CI: 20.0–31.0) for cohort 2; cohort 1 data are immature. Abemaciclib plus pembrolizumab demonstrated antitumor activity, but high rates of ILD/pneumonitis and severe transaminase elevations occurred with/without anastrozole compared to the previous reporting. Benefit/risk analysis does not support further evaluation of this combination in the treatment of HR+, HER2− MBC.
Immuno-oncology (IO) agents have demonstrated efficacy across many tumor types and have led to change in standard of care. In breast cancer, atezolizumab and pembrolizumab were recently FDA-approved ...in combination with chemotherapy specifically for patients with PD-L1-positive metastatic triple-negative breast cancer (TNBC). However, the single agent PD-1/PD-L1 inhibitors demonstrate only modest single agent efficacy in breast cancer. The purpose of this study was to investigate the efficacy of novel IO agents in patients with metastatic breast cancer (MBC), beyond TNBC, treated in phase I clinical trials at the University of Colorado.
We performed a retrospective analysis using a database of patients with MBC who received treatment with IO agents in phase I/Ib clinical trials at the University of Colorado Hospital from January 1, 2012 to July 1, 2018. Patient demographics, treatments and clinical outcomes were obtained.
We identified 43 patients treated with an IO agent either as a single agent or in combination. The average age was 53 years; 55.8% had hormone receptor-positive/HER2-negative breast cancer, 39.5% TNBC and 4.7% HER2-positive. Patients received an average of 2 prior lines of chemotherapy (range 0-7) in the metastatic setting. Most patients (72.1%) received IO alone and 27.9% received IO plus chemotherapy. Median progression-free survival (PFS) was 2.3 months and median overall survival (OS) was 12.1 months. Patients remaining on study ≥ 6 months (20.9%) were more likely to be treated with chemotherapy plus IO compared to patients with a PFS < 6 months (77.8% v. 14.7%). No differences in number of metastatic sites, prior lines of chemotherapy, breast cancer subtype, absolute lymphocyte count, or LDH were identified between patients with a PFS ≥ 6 months vs. < 6 months.
Our phase I experience demonstrates benefit from IO therapy that was not limited to patients with TNBC and confirms improved efficacy from IO agents in combination with chemotherapy. A subset of patients with MBC treated in phase I clinical trials with an IO agent derived prolonged clinical benefit. Predictors of response to immunotherapy in breast cancer remain uncharacterized and further research is needed to identify these factors.
PDF
