Genomic profiling studies suggest that triple-negative breast cancer (TNBC) is a heterogeneous disease. In this study, we sought to define TNBC subtypes and identify subtype-specific markers and ...targets.
RNA and DNA profiling analyses were conducted on 198 TNBC tumors estrogen receptor (ER) negativity defined as Allred scale value ≤ 2 with >50% cellularity (discovery set: n = 84; validation set: n = 114) collected at Baylor College of Medicine (Houston, TX). An external dataset of seven publically accessible TNBC studies was used to confirm results. DNA copy number, disease-free survival (DFS), and disease-specific survival (DSS) were analyzed independently using these datasets.
We identified and confirmed four distinct TNBC subtypes: (i) luminal androgen receptor (AR; LAR), (ii) mesenchymal (MES), (iii) basal-like immunosuppressed (BLIS), and (iv) basal-like immune-activated (BLIA). Of these, prognosis is worst for BLIS tumors and best for BLIA tumors for both DFS (log-rank test: P = 0.042 and 0.041, respectively) and DSS (log-rank test: P = 0.039 and 0.029, respectively). DNA copy number analysis produced two major groups (LAR and MES/BLIS/BLIA) and suggested that gene amplification drives gene expression in some cases FGFR2 (BLIS). Putative subtype-specific targets were identified: (i) LAR: androgen receptor and the cell surface mucin MUC1, (ii) MES: growth factor receptors platelet-derived growth factor (PDGF) receptor A; c-Kit, (iii) BLIS: an immunosuppressing molecule (VTCN1), and (iv) BLIA: Stat signal transduction molecules and cytokines.
There are four stable TNBC subtypes characterized by the expression of distinct molecular profiles that have distinct prognoses. These studies identify novel subtype-specific targets that can be targeted in the future for the effective treatment of TNBCs.
Triple-negative breast cancers (TNBC) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs and ...10 proved critical for anchorage-independent growth. In TNBC cells, an LPA-LPAR2-EZH2 NF-κB signaling cascade was essential for expression of interleukin (IL)-6, IL-8, and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for patients with TNBCs.
Using a syngeneic p53-null mouse mammary gland tumor model that closely mimics human breast cancer, we have identified, by limiting dilution transplantation and in vitro mammosphere assay, a ...Lin(-)CD29(H)CD24(H) subpopulation of tumor-initiating cells. Upon subsequent transplantation, this subpopulation generated heterogeneous tumors that displayed properties similar to the primary tumor. Analysis of biomarkers suggests the Lin(-)CD29(H)CD24(H) subpopulation may have arisen from a bipotent mammary progenitor. Differentially expressed genes in the Lin(-)CD29(H)CD24(H) mouse mammary gland tumor-initiating cell population include those involved in DNA damage response and repair, as well as genes involved in epigenetic regulation previously shown to be critical for stem cell self-renewal. These studies provide in vitro and in vivo data that support the cancer stem cell (CSC) hypothesis. Furthermore, this p53-null mouse mammary tumor model may allow us to identify new CSC markers and to test the functional importance of these markers.
Substantial evidence implicates insulin-like growth factor-I (IGF-I) signaling in the development and progression of breast cancer. To more clearly elucidate the role of IGF in human breast cancer, ...we identified and then examined gene expression patterns of IGF-I-treated breast cancer cells.
MCF-7 cells were stimulated with IGF-I for 3 or 24 hours and were profiled for greater than 22,000 RNA transcripts. We defined an IGF-I signature pattern of more than 800 genes that were up- or downregulated at both time points. The gene signature was examined in clinical breast tumors and in experimental models that represented other oncogenic pathways. The signature was correlated with clinical and pathologic variables and with patient outcome.
IGF-I caused temporal changes in gene expression that were strongly associated with cell proliferation, metabolism, and DNA repair. Genes with early and sustained regulation by IGF-I were highly enriched for transcriptional targets of the estrogen receptor (ER), Ras/extracellular signal-related kinase 1/2, and phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathways. In three large, independent data sets of profiled human breast tumors, the IGF-I signature was manifested in the majority of ER-negative breast tumors and in a subset (approximately 25%) of ER-positive breast tumors. Patients who had tumors that manifested the IGF-I signature (including patients who did not receive adjuvant therapy) had a shorter time to a poor outcome event. The IGF gene signature was highly correlated with numerous poor prognostic factors and was one of the strongest indicators of disease outcome.
Transcriptional targets of IGF-I represent pathways of increased aggressiveness and possibly hormone independence in clinical breast cancers.
Human invasive breast cancers (IBC) show enormous histologic and biological diversity. This study comprehensively evaluated diversity in ductal carcinoma in situ (DCIS), the immediate precursors of ...IBCs.
The extent of diversity for conventional histologic grade and standard prognostic biomarkers assessed by immunohistochemistry was evaluated in a series of pure DCIS (n = 200) compared with a contemporaneous series of IBCs (n = 200). A subset of the DCIS (n = 25) was evaluated by DNA microarrays for the presence of luminal, basal, and erbB2 intrinsic subtypes. The extent of diversity within individual cases of DCIS (n = 120) was determined by assessing multiple regions independently for histologic (nuclear) grade and several biomarkers by immunohistochemistry, which approximate microarrays in determining intrinsic subtypes.
DCIS showed a broad distribution of conventional histologic grades and standard biomarkers ranging from well to poorly differentiated, nearly identical to IBCs. Microarrays showed the same intrinsic subtypes in DCIS as in IBCs. However, higher resolution analysis showed that multiple histologic grades, biomarker phenotypes, and intrinsic subtypes often coexist within the same DCIS, and these diverse regions probably compete for dominance. Diversity within cases of DCIS was highly correlated with mutated p53 (P = 0.0007).
These results support the hypothesis that poorly differentiated DCIS gradually evolve from well-differentiated DCIS by randomly acquiring genetic defects resulting in increasingly abnormal cellular features. This diversity is amplified by defects resulting in genetic instability (e.g., p53 mutation), and the alterations are propagated to IBC in a manner independent of progression to invasion.
Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer, and confer clinical resistance to aromatase inhibitors. Expression of the ESR1 Y537S mutation ...induced an epithelial-mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1 mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estradiol (E
) withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of E
; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a nine-gene expression signature, which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1 mutant tumors.
Basal-like breast cancers (BLBCs) are aggressive breast cancers associated with poor survival. Defining the key drivers of BLBC growth will allow identification of molecules for targeted therapy. In ...this study, we performed a primary screen integrating multiple assays that compare transcription factor expression and activity in BLBC and non-BLBC at the RNA, DNA, and protein levels. This integrated screen identified 33 transcription factors that were elevated in BLBC in multiple assays comparing mRNA expression, DNA cis-element sequences, or protein DNA-binding activity. In a secondary screen to identify transcription factors critical for BLBC cell growth, 8 of the 33 candidate transcription factors (TFs) were found to be necessary for growth in at least two of three BLBC cell lines. Of these 8 transcription factors, SOX11 was the only transcription factor required for BLBC growth, but not for growth of non-BLBC cells. Our studies demonstrate that SOX11 is a critical regulator of multiple BLBC phenotypes, including growth, migration, invasion, and expression of signature BLBC genes. High SOX11 expression was also found to be an independent prognostic indicator of poor survival in women with breast cancer. These results identify SOX11 as a potential target for the treatment of BLBC, the most aggressive form of breast cancer.
Approximately 20% of early-stage breast cancers display amplification or overexpression of the ErbB2/HER2 oncogene, conferring poor prognosis and resistance to endocrine therapy. Targeting HER2(+) ...tumors with trastuzumab or the receptor tyrosine kinase (RTK) inhibitor lapatinib significantly improves survival, yet tumor resistance and progression of metastatic disease still develop over time. Although the mechanisms of cytosolic HER2 signaling are well studied, nuclear signaling components and gene regulatory networks that bestow therapeutic resistance and limitless proliferative potential are incompletely understood. Here, we use biochemical and bioinformatic approaches to identify effectors and targets of HER2 transcriptional signaling in human breast cancer. Phosphorylation and activity of the Steroid Receptor Coactivator-3 (SRC-3) is reduced upon HER2 inhibition, and recruitment of SRC-3 to regulatory elements of endogenous genes is impaired. Transcripts regulated by HER2 signaling are highly enriched with E2F1 binding sites and define a gene signature associated with proliferative breast tumor subtypes, cell-cycle progression, and DNA replication. We show that HER2 signaling promotes breast cancer cell proliferation through regulation of E2F1-driven DNA metabolism and replication genes together with phosphorylation and activity of the transcriptional coactivator SRC-3. Furthermore, our analyses identified a cyclin-dependent kinase (CDK) signaling node that, when targeted using the CDK4/6 inhibitor palbociclib, defines overlap and divergence of adjuvant pharmacologic targeting. Importantly, lapatinib and palbociclib strictly block de novo synthesis of DNA, mostly through disruption of E2F1 and its target genes. These results have implications for rational discovery of pharmacologic combinations in preclinical models of adjuvant treatment and therapeutic resistance.
The effectiveness of therapies targeting specific pathways in breast cancer, such as the estrogen receptor or HER2, is limited because many tumors manifest resistance, either de novo or acquired, ...during the course of treatment. To investigate molecular mechanisms of resistance, we used two xenograft models of estrogen receptor-positive (ER+) breast cancer, one with and one without HER2 overexpression (MCF7/HER2-18 and MCF7 wt, respectively). Mice with established tumors were assigned to the following treatment groups: estrogen supplementation (E2), estrogen deprivation (ED), ED plus tamoxifen (Tam), all with or without the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (G). Another group received ED plus the antiestrogen fulvestrant (MCF7 wt only). Tumors with acquired or de novo resistance to these endocrine therapies were profiled for gene expression and compared with tumors in the E2 control group. One class of genes underexpressed in endocrine-resistant tumors (relative to E2-treated tumors) were estrogen inducible in vitro and associated with ER+ human breast cancers (luminal subtype). Another class of genes overexpressed in tumors with acquired resistance in both models represented transcriptional targets of HER2 signaling and was associated with ER-/HER2+ human cancers (ERBB2+ subtype). A third class of genes overexpressed in MCF7/HER2-18 tumors exhibiting de novo resistance to tamoxifen was associated with ER+ human cancers but not with estrogen-regulated genes. Thus, in response to various endocrine therapy regimens, these xenograft breast tumors shut down classic estrogen signaling and activate alternative pathways such as HER2 that contribute to treatment resistance. Over time, the molecular phenotype of breast cancer can change.
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