An enhancer variant associated with breast cancer susceptibility in Black women regulates TNFSF10 expression and antitumor immunity in triple-negative breast cancer.

Women of African ancestry have the highest mortality from triple-negative breast cancer (TNBC) of all racial groups. To understand the genomic basis of breast cancer in the populations, we previously conducted genome-wide association studies and identified single nucleotide polymorphisms (SNPs) associated with breast cancer in Black women. In this study, we investigated the functional significance of the top associated SNP rs13074711. We found the SNP served as an enhancer variant and regulated TNFSF10 (TRAIL) expression in TNBC cells, with a significant association between the SNP genotype and TNFSF10 expression in breast tumors. Mechanistically, rs13074711 modulated the binding activity of c-MYB at the motif and thereby controlled TNFSF10 expression. Interestingly, TNFSF10 expression in many cancers was consistently lower in African Americans compared with European Americans. Furthermore, TNFSF10 expression in TNBC was significantly correlated with the expression of antiviral immune genes and was regulated by type I interferons (IFNs). Accordingly, loss of TNFSF10 resulted in a profound decrease in apoptosis of TNBC cells in response to type I IFNs and poly(I:C), a synthetic analogue of double stranded virus. Lastly, in a syngeneic mouse model of breast cancer, TNFSF10-deficiency in breast tumors decreased tumor-infiltrated CD4+ and CD8+ T cell quantities. Collectively, our results suggested that TNFSF10 plays an important role in the regulation of antiviral immune responses in TNBC, and the expression is in part regulated by a genetic variant associated with breast cancer in Black women. Our results underscore the important contributions of genetic variants to immune defense mechanisms.

TBCRC 018: phase II study of iniparib in combination with irinotecan to treat progressive triple negative breast cancer brain metastases.

Nearly half of patients with advanced triple negative breast cancer (TNBC) develop brain metastases (BM) and most will also have uncontrolled extracranial disease. This study evaluated the safety and efficacy of iniparib, a small molecule anti-cancer agent that alters reactive oxygen species tumor metabolism and penetrates the blood brain barrier, with the topoisomerase I inhibitor irinotecan in patients with TNBC-BM. Eligible patients had TNBC with new or progressive BM and received irinotecan and iniparib every 3 weeks. Time to progression (TTP) was the primary end point; secondary endpoints were response rate (RR), clinical benefit rate (CBR), overall survival (OS), toxicity, and health-related quality of life. Correlative endpoints included molecular subtyping and gene expression studies on pre-treatment archival tissues, and determination of germline BRCA1/2 status. Thirty-seven patients began treatment; 34 were evaluable for efficacy. Five of 24 patients were known to carry a BRCA germline mutation (4 BRCA1, 1 BRCA2). Median TTP was 2.14 months and median OS was 7.8 months. Intracranial RR was 12 %, while intracranial CBR was 27 %. Treatment was well-tolerated; the most common grade 3/4 adverse events were neutropenia and fatigue. Grade 3/4 diarrhea was rare (3 %). Intrinsic subtyping revealed 19 of 21 tumors (79 %) were basal-like, and intracranial response was associated with high expression of proliferation-related genes. This study suggests a modest benefit of irinotecan plus iniparib in progressive TNBC-BM. More importantly, this trial design is feasible and lays the foundation for additional studies for this treatment-refractory disease.

Characterization of cell lines derived from breast cancers and normal mammary tissues for the study of the intrinsic molecular subtypes.

Five molecular subtypes (luminal A, luminal B, HER2-enriched, basal-like, and claudin-low) with clinical implications exist in breast cancer. Here, we evaluated the molecular and phenotypic relationships of (1) a large in vitro panel of human breast cancer cell lines (BCCLs), human mammary fibroblasts (HMFs), and human mammary epithelial cells (HMECs); (2) in vivo breast tumors; (3) normal breast cell subpopulations; (4) human embryonic stem cells (hESCs); and (5) bone marrow-derived mesenchymal stem cells (hMSC). First, by integrating genomic data of 337 breast tumor samples with 93 cell lines we were able to identify all the intrinsic tumor subtypes in the cell lines, except for luminal A. Secondly, we observed that the cell lines recapitulate the differentiation hierarchy detected in the normal mammary gland, with claudin-low BCCLs and HMFs cells showing a stromal phenotype, HMECs showing a mammary stem cell/bipotent progenitor phenotype, basal-like cells showing a luminal progenitor phenotype, and luminal B cell lines showing a mature luminal phenotype. Thirdly, we identified basal-like and highly migratory claudin-low subpopulations of cells within a subset of triple-negative BCCLs (SUM149PT, HCC1143, and HCC38). Interestingly, both subpopulations within SUM149PT were enriched for tumor-initiating cells, but the basal-like subpopulation grew tumors faster than the claudin-low subpopulation. Finally, claudin-low BCCLs resembled the phenotype of hMSCs, whereas hESCs cells showed an epithelial phenotype without basal or luminal differentiation. The results presented here help to improve our understanding of the wide range of breast cancer cell line models through the appropriate pairing of cell lines with relevant in vivo tumor and normal cell counterparts.

PTPN2 copper-sensing rapidly relays copper level fluctuations into EGFR/CREB activation and associated CTR1 transcriptional repression.

Fluxes in human intra- and extracellular copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate (cAMP). We herein applied an unbiased temporal evaluation of the whole-genome transcriptional activities modulated by fluctuations in copper levels to identify the copper sensor proteins responsible for driving these activities. We found that fluctuations in physiologically-relevant copper levels rapidly modulate EGFR/MAPK/ERK signal transduction and activation of the transcription factor cAMP response element-binding protein (CREB). Both intracellular and extracellular assays support Cu 1+ inhibition of the EGFR-phosphatase PTPN2 (and potentially the homologous PTPN1)-via direct ligation to the PTPN2 active site cysteine side chain-as the underlying mechanism of copper-stimulated EGFR signal transduction activation. Depletion of copper represses this signaling pathway. We additionally show i ) copper supplementation drives transcriptional repression of the copper importer CTR1 and ii ) CREB activity is inversely correlated with CTR1 expression. In summary, our study reveals PTPN2 as a physiological copper sensor and defines a regulatory mechanism linking feedback control of copper-stimulated MAPK/ERK/CREB-signaling and CTR1 expression, thereby uncovering a previously unrecognized link between copper levels and cellular signal transduction.

The BRCA1 Pseudogene Negatively Regulates Antitumor Responses through Inhibition of Innate Immune Defense Mechanisms.

Innate immune defense mechanisms play a pivotal role in antitumor responses. Recent evidence suggests that antiviral innate immunity is regulated not only by exogenous non-self-RNA but also by host-derived pseudogene RNAs. A growing body of evidence also indicates a biological role for pseudogenes as gene expression regulators or immune modulators. Here, we report an important role for BRCA1P1, the pseudogene of the BRCA1 tumor-suppressor gene, in regulating innate immune defense mechanisms in breast cancer cells. BRCA1P1 expresses a long-noncoding RNA (lncRNA) in breast cancer cells through divergent transcription. Expression of lncRNA-BRCA1P1 is increased in breast tumors compared with normal breast tissues. Depletion of BRCA1P1 induces an antiviral defense-like program, including the expression of antiviral genes in breast cancer cells. Furthermore, BRCA1P1-deficient cancer cells mimic virus-infected cells by stimulating cytokines and inducing cell apoptosis. Accordingly, depletion of BRCA1P1 increases host innate immune responses and restricts virus replication. In converse, overexpression of BRCA1P1 reduces cytokine expression in breast cancer cells. Mechanistically, lncRNA-BRCA1P1 is localized in the nucleus, binds to the NF-κB subunit RelA, and negatively regulates antiviral gene expression. Finally, in a xenograft mouse model of breast cancer, depletion of BRCA1P1 stimulates cytokine expression and local immunity, and suppresses tumor growth. Our results suggest an important role for BRCA1P1 in innate immune defense mechanisms and antitumor responses. This mechanism of antiviral immunity regulated by a host-derived pseudogene RNA may guide the development of novel therapies targeting immune responses in breast cancer. SIGNIFICANCE: This study identifies a novel mechanism of innate immunity driven by a host pseudogene RNA that inhibits innate immune defense mechanisms and antitumor responses through regulation of antiviral gene expression.

Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer.

INTRODUCTION: In breast cancer, gene expression analyses have defined five tumor subtypes (luminal A, luminal B, HER2-enriched, basal-like and claudin-low), each of which has unique biologic and prognostic features. Here, we comprehensively characterize the recently identified claudin-low tumor subtype. METHODS: The clinical, pathological and biological features of claudin-low tumors were compared to the other tumor subtypes using an updated human tumor database and multiple independent data sets. These main features of claudin-low tumors were also evaluated in a panel of breast cancer cell lines and genetically engineered mouse models. RESULTS: Claudin-low tumors are characterized by the low to absent expression of luminal differentiation markers, high enrichment for epithelial-to-mesenchymal transition markers, immune response genes and cancer stem cell-like features. Clinically, the majority of claudin-low tumors are poor prognosis estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and epidermal growth factor receptor 2 (HER2)-negative (triple negative) invasive ductal carcinomas with a high frequency of metaplastic and medullary differentiation. They also have a response rate to standard preoperative chemotherapy that is intermediate between that of basal-like and luminal tumors. Interestingly, we show that a group of highly utilized breast cancer cell lines, and several genetically engineered mouse models, express the claudin-low phenotype. Finally, we confirm that a prognostically relevant differentiation hierarchy exists across all breast cancers in which the claudin-low subtype most closely resembles the mammary epithelial stem cell. CONCLUSIONS: These results should help to improve our understanding of the biologic heterogeneity of breast cancer and provide tools for the further evaluation of the unique biology of claudin-low tumors and cell lines.

Inhibition of Copper Transport Induces Apoptosis in Triple-Negative Breast Cancer Cells and Suppresses Tumor Angiogenesis.

Treatment of advanced breast cancer remains challenging. Copper and some of the copper-dependent proteins are emerging therapeutic targets because they are essential for cell proliferation and survival, and have been shown to stimulate angiogenesis and metastasis. Here, we show that DCAC50, a recently developed small-molecule inhibitor of the intracellular copper chaperones, ATOX1 and CCS, reduces cell proliferation and elevates oxidative stress, triggering apoptosis in a panel of triple-negative breast cancer (TNBC) cells. Inhibition of ATOX1 activity with DCAC50 disrupts copper homeostasis, leading to increased copper levels, altered spatial copper redistribution, and accumulation of ATP7B to the cellular perinuclear region. The extent and impact of this disruption to copper homeostasis vary across cell lines and correlate with cellular baseline copper and glutathione levels. Ultimately, treatment with DCAC50 attenuates tumor growth and suppresses angiogenesis in a xenograft mouse model, and prevents endothelial cell network formation in vitro Co-treatment with paclitaxel and DCAC50 enhances cytotoxicity in TNBC and results in favorable dose reduction of both drugs. These data demonstrate that inhibition of intracellular copper transport targets tumor cells and the tumor microenvironment, and is a promising approach to treat breast cancer.

Efficacy and pharmacokinetics of a modified acid-labile docetaxel-PRINT(®) nanoparticle formulation against non-small-cell lung cancer brain metastases.

AIM: Particle Replication in Nonwetting Templates (PRINT(®)) PLGA nanoparticles of docetaxel and acid-labile C2-dimethyl-Si-Docetaxel were evaluated with small molecule docetaxel as treatments for non-small-cell lung cancer brain metastases. MATERIALS & METHODS: Pharmacokinetics, survival, tumor growth and mice weight change were efficacy measures against intracranial A549 tumors in nude mice. Treatments were administered by intravenous injection. RESULTS: Intracranial tumor concentrations of PRINT-docetaxel and PRINT-C2-docetaxel were 13- and sevenfold greater, respectively, than SM-docetaxel. C2-docetaxel conversion to docetaxel was threefold higher in intracranial tumor as compared with nontumor tissues. PRINT-C2-docetaxel increased median survival by 35% with less toxicity as compared with other treatments. CONCLUSION: The decreased toxicity of the PRINT-C2-docetaxel improved treatment efficacy against non-small-cell lung cancer brain metastasis.

Efficacy of Carboplatin Alone and in Combination with ABT888 in Intracranial Murine Models of BRCA-Mutated and BRCA-Wild-Type Triple-Negative Breast Cancer.

Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA-wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation.

SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c controls epithelial-mesenchymal transition by inducing Wnt5a signaling.

We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM-) breast cancer cells to an epithelial EpCAM+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM- breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.

Predicting drug responsiveness in human cancers using genetically engineered mice.

PURPOSE: To use genetically engineered mouse models (GEMM) and orthotopic syngeneic murine transplants (OST) to develop gene expression-based predictors of response to anticancer drugs in human tumors. These mouse models offer advantages including precise genetics and an intact microenvironment/immune system. EXPERIMENTAL DESIGN: We examined the efficacy of 4 chemotherapeutic or targeted anticancer drugs, alone and in combination, using mouse models representing 3 distinct breast cancer subtypes: Basal-like (C3(1)-T-antigen GEMM), Luminal B (MMTV-Neu GEMM), and Claudin-low (T11/TP53-/- OST). We expression-profiled tumors to develop signatures that corresponded to treatment and response, and then tested their predictive potential using human patient data. RESULTS: Although a single agent exhibited exceptional efficacy (i.e., lapatinib in the Neu-driven model), generally single-agent activity was modest, whereas some combination therapies were more active and life prolonging. Through analysis of RNA expression in this large set of chemotherapy-treated murine tumors, we identified a pair of gene expression signatures that predicted pathologic complete response to neoadjuvant anthracycline/taxane therapy in human patients with breast cancer. CONCLUSIONS: These results show that murine-derived gene signatures can predict response even after accounting for common clinical variables and other predictive genomic signatures, suggesting that mice can be used to identify new biomarkers for human patients with cancer.

Pharmacokinetics and efficacy of PEGylated liposomal doxorubicin in an intracranial model of breast cancer.

INTRODUCTION: Breast cancer brain metastases (BCBM) are a challenging consequence of advanced BC. Nanoparticle agents, including liposomes, have shown enhanced delivery to solid tumors and brain. We compared pharmacokinetics (PK) and efficacy of PEGylated liposomal doxorubicin (PLD) with non-liposomal doxorubicin (NonL-doxo) in an intracranial model of BC. METHODS: Athymic mice were inoculated intracerebrally with MDA-MB-231-BR-luciferase-expressing cells. Tumor-bearing mice were administered PLD or NonL-doxo at 6 mg/kg IV × 1 and were euthanized prior to and 0.083, 1, 3, 6, 24, 72 and 96 h post-treatment. Samples were processed to measure sum total doxorubicin via HPLC. PLD and NonL-doxo were administered IV weekly as single agents (6 mg/kg) or in combination (4.5 mg/kg) with the PARP inhibitor, ABT-888, PO 25 mg/kg/day. Efficacy was assessed by survival and bioluminescence. RESULTS: Treatment with PLD resulted in approximately 1,500-fold higher plasma and 20-fold higher intracranial tumor sum total doxorubicin AUC compared with NonL-doxo. PLD was detected at 96 h; NonL-doxo was undetectable after 24 h in plasma and tumor. Median survival of PLD-treated animals was 32 days (d, [CI] 31-38), which was significantly longer than controls (26d [CI 25-28]; p = 0.0012) or NonL-doxo treatment (23.5d [CI 18-28], p = 0.0002). Combination treatment with PLD/ABT-888 yielded improved survival compared to NonL-doxo/ABT-888 (35d [CI 31-38] versus 29.5d [CI 25-34]; p = 0.006). CONCLUSIONS: PLD provides both PK and efficacy advantage over NonL-doxo in the treatment of an in vivo model of BCBM. The results provide preclinical rationale to translate findings into early phase trials of PLD, with or without ABT-888, for patients with BCBM.