FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer.

Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)-chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.

CAPER is vital for energy and redox homeostasis by integrating glucose-induced mitochondrial functions via ERR-α-Gabpa and stress-induced adaptive responses via NF-κB-cMYC.

Ever since we developed mitochondria to generate ATP, eukaryotes required intimate mito-nuclear communication. In addition, since reactive oxygen species are a cost of mitochondrial oxidative phosphorylation, this demands safeguards as protection from these harmful byproducts. Here we identified a critical transcriptional integrator which eukaryotes share to orchestrate both nutrient-induced mitochondrial energy metabolism and stress-induced nuclear responses, thereby maintaining carbon-nitrogen balance, and preserving life span and reproductive capacity. Inhibition of nutrient-induced expression of CAPER arrests nutrient-dependent cell proliferation and ATP generation and induces autophagy-mediated vacuolization. Nutrient signaling to CAPER induces mitochondrial transcription and glucose-dependent mitochondrial respiration via coactivation of nuclear receptor ERR-α-mediated Gabpa transcription. CAPER is also a coactivator for NF-κB that directly regulates c-Myc to coordinate nuclear transcriptome responses to mitochondrial stress. Finally, CAPER is responsible for anaplerotic carbon flux into TCA cycles from glycolysis, amino acids and fatty acids in order to maintain cellular energy metabolism to counter mitochondrial stress. Collectively, our studies reveal CAPER as an evolutionarily conserved 'master' regulatory mechanism by which eukaryotic cells control vital homeostasis for both ATP and antioxidants via CAPER-dependent coordinated control of nuclear and mitochondrial transcriptomic programs and their metabolisms. These CAPER dependent bioenergetic programs are highly conserved, as we demonstrated that they are essential to preserving life span and reproductive capacity in human cells-and even in C. elegans.

ESR1 mutations affect anti-proliferative responses to tamoxifen through enhanced cross-talk with IGF signaling.

The purpose of this study was to address the role of ESR1 hormone-binding mutations in breast cancer. Soft agar anchorage-independent growth assay, Western blot, ERE reporter transactivation assay, proximity ligation assay (PLA), coimmunoprecipitation assay, silencing assay, digital droplet PCR (ddPCR), Kaplan-Meier analysis, and statistical analysis. It is now generally accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic breast cancers; however, we do not yet know how to best treat these patients. We have modeled the three most frequent hormone-binding ESR1 (HBD-ESR1) mutations (Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast cancer cell lines. Effects on growth were examined in response to hormonal and targeted agents, and mutation-specific changes were studied using microarray and Western blot analysis. We determined that the HBD-ESR1 mutations alter anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in activation of the insulin-like growth factor receptor (IGF1R) signaling pathway and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader, fulvestrant, significantly reduced the anchorage-independent growth of ESR1 mutant-expressing cells, while combination treatments with the mTOR inhibitor everolimus, or an inhibitor blocking IGF1R, and the insulin receptor significantly enhanced anti-proliferative responses. Using digital drop (dd) PCR, we identified mutations at high frequencies ranging from 12 % for Y537N, 5 % for Y537S, and 2 % for D538G in archived primary breast tumors from women treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not associated with recurrence-free or overall survival in response in this patient cohort and suggest that knowledge of other cell-intrinsic factors in combination with ESR1 mutation status will be needed determine anti-proliferative responses to Tam.

Analysis of phosphatases in ER-negative breast cancers identifies DUSP4 as a critical regulator of growth and invasion.

Estrogen receptor (ER)-negative cancers have a poor prognosis, and few targeted therapies are available for their treatment. Our previous analyses have identified potential kinase targets critical for the growth of ER-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple-negative" breast cancer (TNBC). Because phosphatases regulate the function of kinase signaling pathways, in this study, we investigated whether phosphatases are also differentially expressed in ER-negative compared to those in ER-positive breast cancers. We compared RNA expression in 98 human breast cancers (56 ER-positive and 42 ER-negative) to identify phosphatases differentially expressed in ER-negative compared to those in ER-positive breast cancers. We then examined the effects of one selected phosphatase, dual specificity phosphatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein microarray analyses of 172 proteins, including phosphoproteins. We identified 48 phosphatase genes are significantly differentially expressed in ER-negative compared to those in ER-positive breast tumors. We discovered that 31 phosphatases were more highly expressed, while 11 were underexpressed specifically in ER-negative breast cancers. The DUSP4 gene is underexpressed in ER-negative breast cancer and is deleted in approximately 50 % of breast cancers. Induced DUSP4 expression suppresses both in vitro and in vivo growths of breast cancer cells. Our studies show that induced DUSP4 expression blocks the cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, JNK1, RB, and NFkB p65 phosphorylation; and inhibits invasiveness of TNBC cells. These results suggest that that DUSP4 is a critical regulator of the growth and invasion of triple-negative breast cancer cells.

Androgen receptor promotes tamoxifen agonist activity by activation of EGFR in ERα-positive breast cancer.

Tamoxifen (Tam) resistance represents a significant clinical problem in estrogen receptor (ER) α-positive breast cancer. We previously showed that decreased expression of Rho guanine nucleotide dissociation inhibitor (Rho GDI) α, a negative regulator of the Rho GTPase pathway, is associated with Tam resistance. We now discover that androgen receptor (AR) is overexpressed in cells with decreased Rho GDIα and seek to determine AR's contribution to resistance. We engineered ERα-positive cell lines with stable knockdown (KD) of Rho GDIα (KD cells). Resistance mechanisms were examined using microarray profiling, protein-interaction studies, growth and reporter gene assays, and Western blot analysis combined with a specific AR antagonist and other signaling inhibitors. Tam-resistant tumors and cell lines with low Rho GDIα levels exhibited upregulated AR expression. Microarray of Rho GDIα KD cells indicated that activation of EGFR and ERα was associated with Tam treatment. When AR levels were elevated, interaction between AR and EGFR was detected. Constitutive and Tam-induced phosphorylation of EGFR and ERK1/2 was blocked by the AR antagonist Enzalutamide, suggesting that AR-mediated EGFR activation was a mechanism of resistance in these cells. Constitutive ERα phosphorylation and transcriptional activity was inhibited by Enzalutamide and the EGFR inhibitor gefitinib, demonstrating that AR-mediated EGFR signaling activated ERα. Tam exhibited agonist activity in AR overexpressing cells, stimulating ERα transcriptional activity and proliferation, which was blocked by Enzalutamide and gefitinib. We describe a novel model of AR-mediated Tam resistance through activation of EGFR signaling leading to ER activation in ERα-positive cells with low expression of Rho GDIα.

Metastasis tumor-associated protein 2 enhances metastatic behavior and is associated with poor outcomes in estrogen receptor-negative breast cancer.

Metastasis remains a major clinical problem in breast cancer. One family of genes previously linked with metastasis is the metastasis tumor-associated (MTA) family, with members MTA1 enhancing and MTA3 inhibiting cancer metastasis. We have previously found that MTA2 enhances anchorage-independent growth in estrogen receptor α (ERα) breast cancers, and, in combination with other genes, performed as a predictive biomarker in ERα-positive breast cancer. We therefore hypothesized that MTA2 enhances breast cancer progression. To test this, cell growth, soft-agar colony formation, migration, and in vivo metastasis were examined in MTA2-overexpressing and Vector control transfected ERα-negative breast cancer cells. Pathways regulating cell-cell interaction, adhesion, and signaling through the Rho pathway were also investigated. Effects of the inhibition of the Rho pathway using a Rho Kinase inhibitor were assessed in soft-agar colony formation and motility assays in MTA2-overexpressing cells. MTA2 expression was associated with poor prognostic markers, and levels of MTA2 were associated with increased risk of early recurrence in retrospective analyses. MTA2 overexpression was associated with enhanced metastasis, and pathways regulating cell-cell interactions in vitro and in vivo. Most critically, MTA2-enhanced motility could be blocked by inhibiting Rho pathway signaling. We present the novel finding that MTA2 defined a subset of ERα-negative patients with a particularly poor outcome.

A gene transcription signature of obesity in breast cancer.

Obesity is thought to contribute to worse disease outcome in breast cancer as a result of increased levels of adipocyte-secreted endocrine factors, insulin, and insulin-like growth factors (IGFs) that accelerate tumor cell proliferation and impair treatment response. We examined the effects of patient obesity on primary breast tumor gene expression, by profiling transcription of a set of 103 tumors for which the patients' body mass index (BMI) was ascertained. Sample profiles were stratified according to patients' obesity phenotype defined as normal (BMI < 25), overweight (BMI 25-29.9), or obese (BMI ≥ 30). Widespread gene expression alterations were evident in breast tumors from obese patients as compared to other tumors, allowing us to define an obesity-associated cancer transcriptional signature of 662 genes. In multiple public expression data sets of breast cancers (representing > 1,500 patients), manifestation of the obesity signature patterns correlated with manifestation of a gene signature for IGF signaling and (to a lesser extent) with lower levels of estrogen receptor. In one patient cohort, manifestation of the obesity signature correlated with shorter time to metastases. A number of small molecules either induced or suppressed the obesity-associated transcriptional program in vitro; estrogens alpha-estradiol, levonorgestrel, and hexestrol induced the program, while several anti-parkinsonian agents targeting neurotransmitter receptor pathways repressed the program. Obesity in breast cancer patients appears to impact the gene expression patterns of the tumor (perhaps as a result of altered body chemistry). These results warrant further investigation of obesity-associated modifiers of breast cancer risk and disease outcome.

Estrogen and insulin-like growth factor-I (IGF-I) independently down-regulate critical repressors of breast cancer growth.

Although estrogen receptor alpha (ERα) and insulin-like growth factor (IGF) signaling are important for normal mammary development and breast cancer, cross-talk between these pathways, particularly at the level of transcription, remains poorly understood. We performed microarray analysis on MCF-7 breast cancer cells treated with estradiol (E2) or IGF-I for 3 or 24 h. IGF-I regulated mRNA of five to tenfold more genes than E2, and many genes were co-regulated by both ligands. Importantly, expression of these co-regulated genes correlated with poor prognosis of human breast cancer. Closer examination revealed enrichment of repressed transcripts. Interestingly, a number of potential tumor suppressors, for example, B-cell linker (BLNK), were down-regulated by IGF-I and E2. Analysis of three down-regulated genes showed that E2-mediated repression occurred independently of IGF-IR, and IGF-I-mediated repression occurred independently of ERα. However, repression by IGF-I or E2 required common kinases, such as PI3K and MEK, suggesting downstream convergence of the two pathways. In conclusion, E2 and IGF-I co-regulate a set of genes that affect breast cancer outcome. There is enrichment of repressed transcripts, and, for some genes, the down-regulation is independent at the receptor level. This may be important clinically, as tumors with active ERα and IGF-IR signaling may require co-targeting of both pathways.

SAFB1 mediates repression of immune regulators and apoptotic genes in breast cancer cells.

The scaffold attachment factors SAFB1 and SAFB2 are paralogs, which are involved in cell cycle regulation, apoptosis, differentiation, and stress response. They have been shown to function as estrogen receptor corepressors, and there is evidence for a role in breast tumorigenesis. To identify their endogenous target genes in MCF-7 breast cancer cells, we utilized a combined approach of chromatin immunoprecipitation (ChIP)-on-chip and gene expression array studies. By performing ChIP-on-chip on microarrays containing 24,000 promoters, we identified 541 SAFB1/SAFB2-binding sites in promoters of known genes, with significant enrichment on chromosomes 1 and 6. Gene expression analysis revealed that the majority of target genes were induced in the absence of SAFB1 or SAFB2 and less were repressed. Interestingly, there was no significant overlap between the genes identified by ChIP-on-chip and gene expression array analysis, suggesting regulation through regions outside the proximal promoters. In contrast to SAFB2, which shared most of its target genes with SAFB1, SAFB1 had many unique target genes, most of them involved in the regulation of the immune system. A subsequent analysis of the estrogen treatment group revealed that 12% of estrogen-regulated genes were dependent on SAFB1, with the majority being estrogen-repressed genes. These were primarily genes involved in apoptosis, such as BBC3, NEDD9, and OPG. Thus, this study confirms the primary role of SAFB1/SAFB2 as corepressors and also uncovers a previously unknown role for SAFB1 in the regulation of immune genes and in estrogen-mediated repression of genes.

A phase II neoadjuvant trial of anastrozole, fulvestrant, and gefitinib in patients with newly diagnosed estrogen receptor positive breast cancer.

Endocrine therapy in patients with breast cancer can be limited by the problem of resistance. Preclinical studies suggest that complete blockade of the estrogen receptor (ER) combined with inhibition of the epidermal growth factor receptor can overcome endocrine resistance. We tested this hypothesis in a phase II neoadjuvant trial of anastrozole and fulvestrant combined with gefitinib in postmenopausal women with newly diagnosed ER-positive breast cancer. After a baseline tumor core biopsy, patients were randomized to receive anastrozole and fulvestrant or anastrozole, fulvestrant, and gefitinib (AFG) for 3 weeks. After a second biopsy at 3 weeks, all patients received AFG for 4 months and surgery was done if the tumor was operable. The primary endpoint was best clinical response by RECIST criteria and secondary endpoints were toxicity and change in biomarkers. The study closed after 15 patients were enrolled because of slow accrual. Median patient age was 67 years and median clinical tumor size was 7 cm. Four patients had metastatic disease present. Three patients withdrew before response was assessed. In the remaining 12 patients, there were two complete clinical responses (17%), three partial responses (25%), five had stable disease (41%), and two (17%) had progressive disease. Most common adverse events were rash in four patients, diarrhea in four, joint symptoms in three, and abnormal liver function tests in three. There were no grade 4 toxicities and all toxicities were reversible. At 3 weeks, cell proliferation as measured by Ki-67 was significantly reduced in the AFG group (P value = 0.01), with a parallel reduction in the expression of the Cyclin D1 (P value = 0.02). RNA microarray data showed a corresponding decrease in the expression of cell cycle genes. These results suggest that AFG was an effective neoadjuvant therapy and consistently reduced proliferation in ER-positive tumors.

A subset of cytotoxic effector memory T cells enhances CAR T cell efficacy in a model of pancreatic ductal adenocarcinoma.

In humans, the natural killer (NK) cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αß T cell receptor-expressing subset with characteristic specificity for tissue-localized viruses. This subset also displays enhanced cytotoxic and memory phenotypes. Here, we characterized this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161-equivalent CD8+ T cell populations (CD8+NK1.1+) revealed substantial up-regulation of granzymes, perforin, killer lectin-like receptors, and innate signaling molecules in comparison to CD8+NK1.1- T cells. Adoptive transfer of CD8+NK1.1+ cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8+NK1.1- cells. Freshly isolated human CD8+CD61+ T cells exhibited heightened allogeneic killing activity in comparison to CD8+CD61- T cells or total peripheral blood mononuclear cells (PBMCs). To determine whether this subset might improve the antitumor efficacy of CAR T cell therapy against solid tumors, we compared bulk PBMCs, CD8+CD161-, and CD8+CD161+ T cells transduced with a human epidermal growth factor receptor-2 (HER2)-specific CAR construct. In vitro, CD8+CD161+ CAR-transduced T cells killed HER2+ targets faster and with greater efficiency. Similarly, these cells mediated enhanced in vivo antitumor efficacy in xenograft models of HER2+ pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.

Hormonal modulation of ESR1 mutant metastasis.

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 (E2) withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of E2; 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.

Decreased TGFbeta signaling and increased COX2 expression in high risk women with increased mammographic breast density.

High mammographic density is associated with a increased risk of breast cancer. We hypothesized that specific pathways exist that are associated with increased mammographic density, and may therefore be used to identify potential targets for chemoprevention. Histologically confirmed normal breast tissue was collected from women undergoing breast surgery who had available demographic data and mammograms for review. Women with low versus high mammographic breast density were compared. Differentially expressed genes using Affymetrix HG U133Plus2 chips were identified in dense versus non-dense tissue. Immunohistochemical analysis (IHC) of estrogen receptor, progesterone receptor, Ki67, and COX2 expression was performed. About 66 women were identified, 28 (42%) had high, and 38 (58%) had low mammographic density. About 73 genes had differential expression between normal breast tissue with high and low mammographic density (P < 0.001, fold change > or = 1.5 with a low false discovery rate (<10%). Network and canonical pathway analysis indicated decreased TGFbeta signaling (TGFBR2, SOS, SMAD3, CD44 and TNFRSF11B) in dense breast tissue relative to non-dense breast. By IHC, only COX2 expression in the stroma was statistically significant on multivariate analysis. TGFbeta ligands are currently the only growth factors known to prevent mammary epithelial cell proliferation. TGFbeta signaling has been reported to be inhibited by COX-2, and these molecules are highly differentially expressed in individuals at high risk of developing breast cancer. These results strongly suggest that COX2 inhibition should be investigated for breast cancer prevention despite possible increase in cardiovascular risk.

Androgen receptor overexpression induces tamoxifen resistance in human breast cancer cells.

Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR and reduced estrogen receptor (ER) alpha mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERalpha-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERalpha that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen's agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

BCL9/STAT3 regulation of transcriptional enhancer networks promote DCIS progression.

The molecular processes by which some human ductal carcinoma in situ (DCIS) lesions advance to the more aggressive form, while others remain indolent, are largely unknown. Experiments utilizing a patient-derived (PDX) DCIS Mouse INtraDuctal (MIND) animal model combined with ChIP-exo and RNA sequencing revealed that the formation of protein complexes between B Cell Lymphoma-9 (BCL9), phosphoserine 727 STAT3 (PS-727-STAT3) and non-STAT3 transcription factors on chromatin enhancers lead to subsequent transcription of key drivers of DCIS malignancy. Downregulation of two such targets, integrin ß3 and its associated metalloproteinase, MMP16, resulted in a significant inhibition of DCIS invasive progression. Finally, in vivo targeting of BCL9, using rosemary extract, resulted in significant inhibition of DCIS malignancy in both cell line and PDX DCIS MIND animal models. As such, our studies provide compelling evidence for future testing of rosemary extract as a chemopreventive agent in breast cancer.

Evaluation of the Predictive Role of Tumor Immune Infiltrate in Patients with HER2-Positive Breast Cancer Treated with Neoadjuvant Anti-HER2 Therapy without Chemotherapy.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are associated with benefit to trastuzumab and chemotherapy in patients with early-stage HER2+ breast cancer. The predictive value of TILs, TIL subsets, and other immune cells in patients receiving chemotherapy-sparing lapatinib plus trastuzumab treatment is unclear.Experimental Design: Hematoxylin and eosin-stained slides (n = 59) were used to score stromal (s-)TILs from pretreatment biopsies of patients enrolled in the neoadjuvant TBCRC006 trial of 12-week lapatinib plus trastuzumab therapy (plus endocrine therapy for ER+ tumors). A 60% threshold was used to define lymphocyte-predominant breast cancer (LPBC). Multiplexed immunofluorescence (m-IF) staining (CD4, CD8, CD20, CD68, and FoxP3) and multispectral imaging were performed to characterize immune infiltrates in single formalin-fixed paraffin-embedded slides (n = 33). RESULTS: The pathologic complete response (pCR) rate was numerically higher in patients with LPBC compared with patients with non-LPBC (50% vs. 19%, P = 0.057). Unsupervised hierarchical clustering of the five immune markers identified two patient clusters with different responses to lapatinib plus trastuzumab treatment (pCR = 7% vs. 50%, for cluster 1 vs. 2 respectively; P = 0.01). In multivariable analysis, cluster 2, characterized by high CD4+, CD8+, CD20+ s-TILs, and high CD20+ intratumoral TILs, was independently associated with a higher pCR rate (P = 0.03). Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by s-CD4, intratumoral (i-) CD4, and i-CD20+ TILs. CONCLUSIONS: LPBC was marginally associated with higher pCR rate than non-LPBC in patients with lapatinib plus trastuzumab treated HER2+ breast cancer. Quantitative assessment of the immune infiltrate by m-IF is feasible and may help correlate individual immune cell subpopulations and immune cell profiles with treatment response.

Ductal carcinoma in situ and the emergence of diversity during breast cancer evolution.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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). CONCLUSIONS: 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.

Targeting the Mevalonate Pathway to Overcome Acquired Anti-HER2 Treatment Resistance in Breast Cancer.

Despite effective strategies, resistance in HER2+ breast cancer remains a challenge. While the mevalonate pathway (MVA) is suggested to promote cell growth and survival, including in HER2+ models, its potential role in resistance to HER2-targeted therapy is unknown. Parental HER2+ breast cancer cells and their lapatinib-resistant and lapatinib + trastuzumab-resistant derivatives were used for this study. MVA activity was found to be increased in lapatinib-resistant and lapatinib + trastuzumab-resistant cells. Specific blockade of this pathway with lipophilic but not hydrophilic statins and with the N-bisphosphonate zoledronic acid led to apoptosis and substantial growth inhibition of R cells. Inhibition was rescued by mevalonate or the intermediate metabolites farnesyl pyrophosphate or geranylgeranyl pyrophosphate, but not cholesterol. Activated Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and mTORC1 signaling, and their downstream target gene product Survivin, were inhibited by MVA blockade, especially in the lapatinib-resistant/lapatinib + trastuzumab-resistant models. Overexpression of constitutively active YAP rescued Survivin and phosphorylated-S6 levels, despite blockade of the MVA. These results suggest that the MVA provides alternative signaling leading to cell survival and resistance by activating YAP/TAZ-mTORC1-Survivin signaling when HER2 is blocked, suggesting novel therapeutic targets. MVA inhibitors including lipophilic statins and N-bisphosphonates may circumvent resistance to anti-HER2 therapy warranting further clinical investigation. IMPLICATIONS: The MVA was found to constitute an escape mechanism of survival and growth in HER2+ breast cancer models resistant to anti-HER2 therapies. MVA inhibitors such as simvastatin and zoledronic acid are potential therapeutic agents to resensitize the tumors that depend on the MVA to progress on anti-HER2 therapies.

Conditional overexpression of Stat3alpha in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern.

Normal neutrophil development requires G-CSF signaling, which includes activation of Stat3. Studies of G-CSF-mediated Stat3 signaling in cell culture and transgenic mice have yielded conflicting data regarding the role of Stat3 in myelopoiesis. The specific functions of Stat3 remain unclear, in part, because two isoforms, Stat3alpha and Stat3beta, are expressed in myeloid cells. To understand the contribution of each Stat3 isoform to myelopoiesis, we conditionally overexpressed Stat3alpha or Stat3beta in the murine myeloid cell line 32Dcl3 (32D) and examined the consequences of overexpression on cell survival and differentiation. 32D cells induced to overexpress Stat3alpha, but not Stat3beta, generated a markedly higher number of neutrophils in response to G-CSF. This effect was a result of decreased apoptosis but not of increased proliferation. Comparison of gene expression profiles of G-CSF-stimulated, Stat3alpha-overexpressing 32D cells with those of cells with normal Stat3alpha expression revealed novel Stat3 gene targets, which may contribute to neutrophil expansion and improved survival, most notably Slc28a2, a purine nucleoside transporter, which is critical for maintenance of intracellular nucleotide levels and prevention of apoptosis, and Gpr65, an acid-sensing, G protein-coupled receptor with pro-oncogenic and antiapoptotic functions.