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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.

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.

A number of epidemiological studies have implicated calcium (Ca(2+)) signaling as a major factor in obesity that contributes to aberrant systems metabolism. Somewhat paradoxically, obesity correlates with decreased circulating Ca(2+) levels, leading to increased release of intracellular Ca(2+) stores from the endoplasmic reticulum. These findings suggest that insulin resistance associated with the obese state is linked to activation of canonical Ca(2+) signaling pathways. Mechanistically, increased intracellular Ca(2+) binds calmodulin (CaM) to activate a set of Ca(2+)/CaM-dependent protein kinases. In this research resource, we explore the metabolic functions and implications of Ca(2+)/CaM-dependent protein kinase kinase 2 (CaMKK2) as a metabolic effector of Ca(2+)/CaM action. We reveal the importance of CaMKK2 for gating insulin release from pancreatic β-cells while concomitantly influencing the sensitivity of insulin-responsive tissues. To provide a better understanding of the metabolic impact of CaMKK2 loss, we performed targeted metabolomic analyses of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice null for CaMKK2. We quantified amino acids and acyl carnitines in 3 insulin-sensitive tissues (liver, skeletal muscle, plasma) isolated from CaMKK2(-/-) mice and their wild-type littermates under conditions of dietary stress (low-fat diet, normal chow, high-fat diet, and fasting), thereby unveiling unique metabolic functions of CaMKK2. Our findings highlight CaMKK2 as a molecular rheostat for insulin action and emphasize the importance of Ca(2+)/CaM/CaMKK2 in regulation of whole-body metabolism. These findings reveal that CaMKK2 may be an attractive therapeutic target for combatting comorbidities associated with perturbed insulin signaling.

Breast cancer research is hampered by difficulties in obtaining and studying primary human breast tissue, and by the lack of in vivo preclinical models that reflect patient tumor biology accurately. To overcome these limitations, we propagated a cohort of human breast tumors grown in the epithelium-free mammary fat pad of severe combined immunodeficient (SCID)/Beige and nonobese diabetic (NOD)/SCID/IL-2γ-receptor null (NSG) mice under a series of transplant conditions. Both models yielded stably transplantable xenografts at comparably high rates (∼21% and ∼19%, respectively). Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet. Overall, 32 stably transplantable xenograft lines were established, representing 25 unique patients. Most tumors yielding xenografts were “triple-negative” [estrogen receptor (ER)−progesterone receptor (PR)−HER2+; n = 19]. However, we established lines from 3 ER−PR−HER2+ tumors, one ER+PR−HER2−, one ER+PR+HER2−, and one “triple-positive” (ER+PR+HER2+) tumor. Serially passaged xenografts show biologic consistency with the tumor of origin, are phenotypically stable across multiple transplant generations at the histologic, transcriptomic, proteomic, and genomic levels, and show comparable treatment responses as those observed clinically. Xenografts representing 12 patients, including 2 ER+ lines, showed metastasis to the mouse lung. These models thus serve as a renewable, quality-controlled tissue resource for preclinical studies investigating treatment response and metastasis. Cancer Res; 73(15); 4885–97. ©2013 AACR.

Introduction Translational breast cancer research is hampered severely by difficulties in obtaining and studying primary human breast tissue, and by the lack of in vivo preclinical models that accurately reflect patient tumor biology. These limitations are due, in part, to the fact that traditional immunocompromised mouse models are not generally permissive for growth. We sought to circumvent some of these limitations by transplanting and growing human mammary tumors in the mammary fat pad of SCID/Beige immunocompromised mice in the absence of exogenous human fibroblasts. Aims and Methods To establish a set of stable human breast cancer xenografts for preclinical studies. Human breast cancer biopsies were received, minced into small fragments and then transplanted directly into “cleared” fat pads of recipient SCID/Beige immunocompromised mice. Transplanted fat pads were checked weekly. After initial tumor was palpated and harvested, tumor fragments were transplanted into new SCID/Beige hosts for subsequent transplant generations. Serial immunohistochemical evaluations were performed to confirm human origin and biomarker status. Analytical flow cytometry for evaluating expression of proposed “cancer stem cell” markers, and gene and protein expression analysis were carried out on all stable lines. Results and Conclusions Xenograft lines were established directly from breast cancer patient samples, without intervening culture in vitro, using the epithelium-free mammary fat pad as the transplantation site. Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet without exogenous human fibroblasts. Thirty six stably transplantable xenograft lines representing 27 patients were established, using pre-treatment, mid-treatment, and/or post-treatment samples. Most patients yielding xenografts were “triple-negative” (ER-PR-HER2−) (n=21), we were able to establish lines from three ER-PR-HER2+ patients, one ER+PR+HER2−, one ER+PR-HER2−and one “triple-positive” (ER+PR+HER2+) patients. Serially passaged xenografts show phenotypic consistency with the tumor of origin at the histopathology level, and remarkable stability across multiple transplant generations at both the genomic, transcriptomic, and proteomic levels. Of 27 lines evaluated fully, thirteen xenografts showed metastasis to the mouse lung. These models thus serve as a renewable, quality-controlled tissue resource, and should prove useful for preclinical evaluation of experimental therapeutics. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-21-01.

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 (BMI25), 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 (representing1,500 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.

Purpose: We previously reported an insulin-like growth factor (IGF) gene expression signature, based on genes induced or repressed by IGF-I, which correlated with poor prognosis in breast cancer. We tested whether the IGF signature was affected by anti–IGF-I receptor (IGF-IR) inhibitors and whether the IGF signature correlated with response to a dual anti–IGF-IR/insulin receptor (InsR) inhibitor, BMS-754807. Experimental Design: An IGF gene expression signature was examined in human breast tumors and cell lines and changes were noted following treatment of cell lines or xenografts with anti–IGF-IR antibodies or tyrosine kinase inhibitors. Sensitivity of cells to BMS-754807 was correlated with levels of the IGF signature. Human primary tumorgrafts were analyzed for the IGF signature and IGF-IR levels and activity, and MC1 tumorgrafts were treated with BMS-754807 and chemotherapy. Results: The IGF gene expression signature was reversed in three different models (cancer cell lines or xenografts) treated with three different anti–IGF-IR therapies. The IGF signature was present in triple-negative breast cancers (TNBC) and TNBC cell lines, which were especially sensitive to BMS-754807, and sensitivity was significantly correlated to the expression of the IGF gene signature. The TNBC primary human tumorgraft MC1 showed high levels of both expression and activity of IGF-IR and IGF gene signature score. Treatment of MC1 with BMS-754807 showed growth inhibition and, in combination with docetaxel, tumor regression occurred until no tumor was palpable. Regression was associated with reduced proliferation, increased apoptosis, and mitotic catastrophe. Conclusions: These studies provide a clear biological rationale to test anti–IGF-IR/InsR therapy in combination with chemotherapy in patients with TNBC. Clin Cancer Res; 17(8); 2314–27. ©2011 AACR.

Estrogen receptor (ER) α is a successful therapeutic target in breast cancer, but patients eventually develop resistance to antiestrogens such as tamoxifen.To identify genes whose expression was associated with the development of tamoxifen resistance and metastasis, we used microarrays to compare gene expression in four primary tumors from tamoxifen-treated patients whose breast cancers did not recur vs five metastatic tumors from patients whose cancers progressed during adjuvant tamoxifen treatment. Because Rho guanine dissociation inhibitor (GDI) α was underexpressed in the tamoxifen-resistant group, we stably transfected ERα-positive MCF-7 breast cancer cells with a plasmid encoding a short hairpin (sh) RNA to silence Rho GDIα expression. We used immunoblots and transcription assays to examine the role of Rho GDIα in ER-related signaling and growth of cells in vitro and as xenografts in treated nude mice (n = 8-9 per group) to examine the effects of Rho GDIα blockade on hormone responsiveness and metastatic behavior. The time to tumor tripling as the time in weeks from randomization to a threefold increase in total tumor volume over baseline was examined in treated mice. The associations of Rho GDIα and MTA2 levels with tamoxifen resistance were examined in microarray data from patients. All statistical tests were two-sided.Rho GDIα was expressed at lower levels in ERα-positive tumors that recurred during tamoxifen treatment than in ERα-positive tamoxifen-sensitive primary tumors. MCF-7 breast cancer cells in which Rho GDIα expression had been silenced were tamoxifen-resistant, had increased Rho GTPase and p21-activated kinase 1 activity, increased phosphorylation of ERα at serine 305, and enhanced tamoxifen-induced ERα transcriptional activity compared with control cells. MCF-7 cells in which Rho GDIα expression was silenced metastasized with high frequency when grown as tumor xenografts. When mice were treated with estrogen or estrogen withdrawal, tripling times for xenografts from cells with Rho GDIα silencing were similar to those from vector-containing control cells; however, tripling times were statistically significantly faster than control when mice were treated with tamoxifen (median tripling time for tumors with Rho GDIα small interfering RNA = 2.34 weeks; for control tumors = not reached, hazard ratio = 4.13, 95% confidence interval = 1.07 to 15.96, P = .040 [adjusted for multiple comparisons, P = .119]). Levels of the metastasis-associated protein MTA2 were also increased upon Rho GDIα silencing, and combined Rho GDIα and MTA2 levels were associated with recurrence in 250 tamoxifen-treated patients.Loss of Rho GDIα enhances metastasis and resistance to tamoxifen via effects on both ERα and MTA2 in models of ERα-positive breast cancer and in tumors of tamoxifen-treated patients.

Background: Estrogen receptor (ESR1) mutations occur at a high frequency in metastatic breast tumors in patients treated with hormonal therapy in the metastatic setting. We do not know if these mutations are involved in metastasis. Experimental design and methods: We generated ESR1 Y537S homozygous mutations using CRISPR Casp-9 technology. Treatment synergy was evaluated using Compusyn. Athymic mice were used in tumor xenograft studies. ChIP-Seq and transcriptome analyses were performed. Results: We generated CRISPR ESR1 Y537S mutation homozygous knock-in clones and lentiviral stable pools in MCF-7 cells. Transcriptome profiling revealed elevated expression of Hallmark pathways, including epithelial mesenchymal transition (EMT) and estrogen-regulated gene expression. Mutant cell growth was resistant to tamoxifen, but responsive to fulvestrant treatment. Synergistic treatment effects were observed with fulvestrant and the mTOR inhibitor everolimus or the CDK4/6 inhibitor palbociclib. CRISPR Y537S mutant knock-in cells grown in the mammary fat-pad of athymic mice spontaneously metastasized to distant organs including the lung, intestine, and kidneys. In the presence of estrogen, there was no difference in the frequency of distant macrometastases between parental wild-type ER and CRISPR Y537S mutant ER mice. However, in the absence of estrogen, mimicking aromatase inhibitor treatment, 80% of CRISPR Y537S mutant ER mice displayed overt distant macrometastases, but none were observed in parental wild-type ER mice (p=0.04). Distant tumors retained ER expression and hormone sensitivity. Comparison of residual tamoxifen-treated metastatic tumors with tumors grown at the primary mammary fat-pad site using immunoblot analysis demonstrated significant reduction in estrogen-regulated gene expression, but no effect on the expression of biomarkers associated with EMT, suggesting a disconnect between EMT and distant metastasis in mutant cells. EMT genes were also identified as direct binding site targets in Y537S mutant cells compared with wide-type ER using ChIPSeq. We discovered that expression of the Y537S mutant was dominant, driving the growth of distant metastatic tumors when co-expressed with wild-type ER cells. A Y537S ER mutant-specific gene expression signature predicted poor disease-free survival of ER-positive patients using the METABRIC database, and lung-specific metastasis-free survival in a Memorial Sloan Kettering dataset. Conclusion: The Y537S ER mutation is a driver of distant metastasis in ER-positive breast cancer cells. A Y537S ER mutant-specific gene expression signature predicted poor disease-free, and distant lung metastasis in ER-positive patients. Mutation status is a potential new predictive factor for hormone therapy of metastatic breast cancer patients on maintenance hormonal therapy. Citation Format: Guowei Gu, Lin Tian, Meng Gao, Yassine Rechoum, Luca Gelsomino, Derek Dustin, Arnoldo Corona-Rodriguez, Amanda R. Beyer, Anna Tsimelzon, Xiang Zhang, Sebastiano Ando', Suzanne Fuqua. The Y537S ESR1 mutation is a dominant driver of distant ER-positive breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 22.

Not all breast cancers respond to tamoxifen, and many develop resistance despite initial benefit. We used an in vivo model of estrogen receptor (ER)–positive breast cancer (MCF-7 xenografts) to investigate mechanisms of this resistance and develop strategies to circumvent it. Epidermal growth factor receptor (EGFR) and HER2, which were barely detected in control estrogen-treated tumors, increased slightly with tamoxifen and were markedly increased when tumors became resistant. Gefitinib, which inhibits EGFR/HER2, improved the antitumor effect of tamoxifen and delayed acquired resistance, but had no effect on estrogen-stimulated growth. Phosphorylated levels of p42/44 and p38 mitogen-activated protein kinases (both downstream of EGFR/HER2) were increased in the tamoxifen-resistant tumors and were suppressed by gefitinib. There was no apparent increase in phosphorylated AKT (also downstream of EGFR/HER2) in resistant tumors, but it was nonetheless suppressed by gefitinib. Phosphorylated insulin-like growth factor-IR (IGF-IR), which can interact with both EGFR and membrane ER, was elevated in the tamoxifen-resistant tumors compared with the sensitive group. However, ER-regulated gene products, including total IGF-IR itself and progesterone receptor, remained suppressed even at the time of acquired resistance. Tamoxifen's antagonism of classic ER genomic function was retained in these resistant tumors and even in tumors that overexpress HER2 (MCF-7 HER2/18) and are de novo tamoxifen-resistant. In conclusion, EGFR/HER2 may mediate tamoxifen resistance in ER-positive breast cancer despite continued suppression of ER genomic function by tamoxifen. IGF-IR expression remains dependent on ER but is activated in the tamoxifen-resistant tumors. This study provides a rationale to combine HER inhibitors with tamoxifen in clinical studies, even in tumors that do not initially overexpress EGFR/HER2. [Cancer Res 2008;68(3):826–33]

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.

Wnt-1 and Neu collaborate to induce mammary tumors in bitransgenic mice carrying both MMTV- Wnt-1 and MMTV- Neu. In this report, gene expression profiles were determined for tumors from these bitransgenic mice, and compared with expression profiles of tumors from mice singly transgenic for MMTV- Wnt-1 or MMTV- Neu. While very different from tumors arising in MMTV- Neu transgenic mice, tumors from these bitransgenic mice were found not to have identifiable differences from tumors from MMTV- Wnt-1 transgenic mice, using clustering and multidimensional scaling analyses (unsupervised and supervised), One-way Analysis of Variance (ANOVA), and two sample t test (the later two of which were combined with false discovery rate computation). These observations suggest that Wnt-1 is dominant over Neu in specifying mammary tumor expression profiles.

Purpose Chemotherapy for operable breast cancer decreases the risk of death. Docetaxel is one of the most active agents in breast cancer, but resistance or incomplete response is frequent. Patients and Methods Core biopsies from 24 patients were obtained before treatment with neoadjuvant docetaxel (four cycles, 100 mg/m2 every 3 weeks), and response was assessed after chemotherapy. After 3 months of neoadjuvant chemotherapy, surgical specimens (n = 13) were obtained, and laser capture microdissection (LCM; n = 8) was performed to enrich for tumor cells. From each core, surgical, and LCM specimen, sufficient total RNA (3 to 6 μg) was extracted for cDNA array analysis using the Affymetrix HgU95-Av2 GeneChip (Affymetrix, Santa Clara, CA). Results From the initial core biopsies, differential patterns of expression of 92 genes correlated with docetaxel response (P = .001). However, the molecular patterns of the residual cancers after 3 months of docetaxel treatment were strikingly similar, independent of initial sensitivity or resistance. This relative genetic homogeneity after treatment was observed in both LCM and non-LCM surgical specimens. The residual tumor after treatment in tumors that were initially sensitive indicates selection of a residual and resistant subpopulation of cells. The gene expression pattern was populated by genes involved in cell cycle arrest at G2M (eg, mitotic cyclins and cdc2) and survival pathways involving the mammalian target of rapamycin. Conclusion A specific and consistent gene expression pattern was found in residual tumors after docetaxel treatment. These profiles provide therapeutic targets that could lead to improved treatment.

A major component of the cardiac stress response is the simultaneous activation of several gene regulatory networks. Interestingly, the transcriptional regulator steroid receptor coactivator-2, SRC-2 is often decreased during cardiac failure in humans. We postulated that SRC-2 suppression plays a mechanistic role in the stress response and that SRC-2 activity is an important regulator of the adult heart gene expression profile. Genome-wide microarray analysis, confirmed with targeted gene expression analyses revealed that genetic ablation of SRC-2 activates the “fetal gene program” in adult mice as manifested by shifts in expression of a) metabolic and b) sarcomeric genes, as well as associated modulating transcription factors. While these gene expression changes were not accompanied by changes in left ventricular weight or cardiac function, imposition of transverse aortic constriction (TAC) predisposed SRC-2 knockout (KO) mice to stress-induced cardiac dysfunction. In addition, SRC-2 KO mice lacked the normal ventricular hypertrophic response as indicated through heart weight, left ventricular wall thickness, and blunted molecular signaling known to activate hypertrophy. Our results indicate that SRC-2 is involved in maintenance of the steady-state adult heart transcriptional profile, with its ablation inducing transcriptional changes that mimic a stressed heart. These results further suggest that SRC-2 deletion interferes with the timing and integration needed to respond efficiently to stress through disruption of metabolic and sarcomeric gene expression and hypertrophic signaling, the three key stress responsive pathways.

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 (EGFR) 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 (AF) 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 twelve 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.

Although estrogen receptor (ER) 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 gene transcription, remains poorly understood. We performed microarray analysis on MCF-7 breast cancer cells treated with estradiol (E2) or IGF-I for 3hr or 24hr. IGF-I regulated mRNA of 5-10-fold 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 patients. Closer examination revealed enrichment of repressed transcripts (compared to induced transcripts).. Interestingly, a number of potential tumor suppressors were down-regulated by IGF-I and estradiol, suchas the B-cell linker BLNK Analysis of three down-regulated genes showed that E2-mediated repression occurred independently of IGF-IR, and IGF-I-mediated repression occurred independently of ER. However, repression by IGF-I or estradiol required common downstream kinases, such as PI3K and MEK, suggesting downstream conversion 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 the down-regulation by E2 and IGF-I is independent at the receptor level. This may be important clinically, as tumors with active ER and IGF-IR signaling may require co-targeting of both pathways.

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.

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 (P0.001, fold changeor = 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.

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. [Cancer Res 2008;68(18):7493–501]

Enlargement of normal terminal duct lobular units (TDLUs) by hyperplastic columnar epithelial cells is one of the most common abnormalities of growth in the adult female human breast. These hyperplastic enlarged lobular units (HELUs) are important clinically as the earliest histologically identifiable potential precursor of breast cancer. The causes of the hyperplasia are unknown but may include estrogen-simulated growth mediated by estrogen receptor-alpha, which is highly elevated in HELUs and may be fundamental to their development. The present study used DNA microarray technology and RNA from microdissected pure epithelial cells to examine changes in gene expression and molecular pathways associated with the development of HELUs from TDLUs. The results suggest that HELUs evolve from TDLUs primarily by reactivation of pathways involved in embryonic development and suppression of terminal differentiation. Changes in ERBB genes were particularly prominent, including a uniform switch in ligands for the ERBB1 receptor (14-fold decrease in epidermal growth factor and 10-fold increase in amphiregulin, respectively) in HELUs compared with TDLUs. Epidermal growth factor regulates terminal differentiation in adult breast and amphiregulin is critical to normal embryonic breast development. Because HELUs are such early potential precursors of breast cancer, targeting some of these alterations may be especially promising strategies for breast cancer prevention.

Purpose: Inflammatory breast cancer (IBC) is associated with very poor prognosis. The aims of this study are (a) to prospectively identify differential gene expression patterns associated with IBC and (b) to confirm these pathways using tissue arrays. Experimental Design: For gene expression analysis, IBC (n = 14) was clinically defined as rapid-onset cancer associated with erythema and skin changes, whereas non-IBC patients (n = 20) had stage III breast cancers, and cDNA analysis was carried out using the Affymetrix (Santa Clara, CA) HG-U133A microarrays. Tissue arrays were constructed from paraffin-embedded material, and the molecular phenotype of 75 IBC was compared with results from >2,000 non-IBC. Results: Gene expression analyses indicated that IBC has higher expression of genes associated with increased metabolic rate, lipid signaling, and cell turnover relative to non-IBC tumors. Consistent with the expression analysis, IBC had statistically higher Ki-67 (93% versus 11%; P < 0.001). BAX expression, reflecting increased apoptosis and cell turnover, was significantly uniformly higher in almost all IBC (98% versus 66%; P < 0.05), whereas the expression of Bcl-2 was not significantly different. IBC tumors were more likely to be steroid hormone receptor negative (estrogen receptor, 49% versus 30%; P = 0.002; progesterone receptor, 68% versus 42%; P = 0.001). The expression of tyrosine kinases was not significantly different. E-cadherin was found to be expressed in 87% of IBC, whereas the expression p53 was not significantly different. Conclusion: This study is one of the largest molecular analyses of IBC. Both IBC and non-IBC are genetically heterogeneous with consistent differences in the molecular phenotype of IBC.

Doxorubicin and cyclophosphamide (Adriamycin/cytoxan, AC) is a standard chemotherapy regimen for breast cancer, but de novo resistance is frequent. We hypothesized that gene expression profiles predictive of AC response may be different from our previously published patterns with docetaxel.Core biopsies from 40 patients were obtained before treatment with AC (6 cycles, 60/600 mg/m2q3 weeks), and clinical responses recorded after treatment. Gene expression patterns were analyzed using Affymetrix U133A chips which comprise approximately 22,200 genes.Clinical complete responses (cCR) were observed in 22, partial responses in 7, stable disease in 11 patients. Differential expression between sensitive cCR and resistant tumors with a low false discovery rate (5%) was obtained. Of these 253 differentially expressed genes, pathways up-regulated in sensitive tumors included cell cycle (BUB3, CDKN1B), survival (BCL2, BAG1, BIRC1, STK39), stress response (CYP2B6, MAPK14), and estrogen-related pathways (ER, IRS1). Resistant tumors expressed gene promoting transcription (GTF3C1, ILF3), differentiation (ST14, CTNNBIP1), signal transduction (EIF1AX, EIF4EBP1), and amino acid metabolism (SRM, PLOD1, PLOD3). With leave-one-out cross validation, 67% of the samples were correctly classified, with a permutation p-value of 0.4. The previously published 92-gene molecular portrait for docetaxel sensitivity could not discriminate AC sensitivity and resistance.This preliminary study supports that molecular profiles for AC response are likely to exist, with unique expression patterns for individual chemotherapy regimens. Larger validation studies are necessary to define and refine patterns for different agents.

We calculated hazard rates for recurrence in patients with primary breast cancer (stage I, II; no adjuvant therapy). Previous publications have indicated a peak in hazard rates for recurrence (or death) at approximately 2–3 years after diagnosis of primary breast cancer. However, there have been conflicting reports concerning the presence of a second peak at 5–7 years after diagnosis. In this study, we estimated hazard functions by the Nelson–Aalen method and fit by cubic–linear and cubic–cubic–linear models to test for the presence of one or two peaks, respectively. We identified two peaks in hazard of recurrence, one at 2 years and another at 5 years. The 5-year peak, though statistically significant, represents very small differences in patient outcome. This additional peak may be an artifact of interval censoring due to a tendency to follow-up patients at specific bench-mark time points.

Summary Background Systemic chemotherapy for operable breast cancer substantially decreases the risk of death. Patients often have de novo resistance or incomplete response to docetaxel, one of the most active agents in this disease. We postulated that gene expression profiles of the primary breast cancer can predict the response to docetaxel. Methods We took core biopsy samples from primary breast tumours in 24 patients before treatment and then assessed tumour response to neoadjuvant docetaxel (four cycles, 100 mg/m 2 daily for 3 weeks) by cDNA analysis of RNA extracted from biopsy samples using HgU95-Av2 GeneChip. Findings From the core biopsy samples, we extracted sufficient total RNA (3-6 μg) for cDNA array analysis using HgU95-Av2 GeneChip. Differential patterns of expression of 92 genes correlated with docetaxel response (p=0·001). Sensitive tumours had higher expression of genes involved in cell cycle, cytoskeleton, adhesion, protein transport, protein modification, transcription, and stress or apoptosis; whereas resistant tumours showed increased expression of some transcriptional and signal transduction genes. In leave-one-out cross-validation analysis, ten of 11 sensitive tumours (90% specificity) and 11 of 13 resistant tumours (85% sensitivity) were correctly classified, with an accuracy of 88%. This 92-gene predictor had positive and negative predictive values of 92% and 83%, respectively. Correlation between RNA expression measured by the arrays and semiquantitative RT-PCR was also ascertained, and our results were validated in an independent set of six patients. Interpretation If validated, these molecular profiles could allow development of a clinical test for docetaxel sensitivity, thus reducing unnecessary treatment for women with breast cancer.

Background: Estrogen receptor (ER) -negative breast cancers are poor prognosis tumors that occur more commonly in young women. Current treatments for ER-negative tumors include cytotoxic chemotherapy, or for those ER-negative breast cancers that overexpress HER2, the anti-HER2 antibody herceptin. Similar therapy for ER-negative, Her2 negative tumors, particularly for (triple-negative breast cancers) is urgently needed. In this project, we investigated whether phosphatases that are differentially expressed in ER-negative as compared to ER-positive breast cancers. We hypothesized that: (1) the specific phosphatases that govern the growth of ER-negative cancers are different from those of ER-positive cancers, (2) Induced expression of some of those phosphatases that are lowly expressed in ER-negative breast cancers will suppress the growth and invasion of ER-negative breast cancers. Methods: Using 102 human breast tumors (57 ER-positive & 45 ER-negative) from the neo-adjuvant studies from the Baylor Breast Center tumor bank, we isolated RNA and performed Affymetrix microarray studies. cDNA was synthesized and Biotin-labeled and hybridized onto an Affymetrix HGU133A GeneChipTM. Statistical analysis was done with dChip software, and phosphatases more highly (>1.5 fold; FDR Results: We identified 20 highly-expressed (>1.5 fold; FDR=0.05), and 29 lowly-expressed ( Conclusions: We identified a set of highly and lowly expressed phosphatases in ER-negative breast cancers as compared to ER-positive cancers. Overexpression of DUSP4 in ER-negative breast cancer cells inhibits growth and invasion of these cells. By identifying the molecules that regulate breast cancer cells growth we are identifying potential new targets for the treatment of these aggressive ER-negative breast cancers. Supported by Komen Promise grant KG081694 (PHB). Citation Format: Abhijit Mazumdar, Petra Den Hollander, Graham Poage, Jamal Hill, Yun Zhang, Anna Tsimelzon, Susan Hilsenbeck, Jenny Chang, Gordon Mills, Powel H. Brown. High expression of DUSP4 in ER-negative breast cancer cells suppresses growth and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 856. doi:10.1158/1538-7445.AM2013-856

Poorly defined molecular heterogeneity and a lack of effective molecular targets hinder advancements in the treatment of ER-/PR-/HER2- breast cancer (TNBC). Meta-analyses of public datasets have revealed new subgroups, but these studies are plagued by samples collected from multiple protocols and poor reproducibility. We aimed to identify stable subsets that could be treated with targeted therapy by employing integrated genomic profiling of a large number of TNBC cases (confirmed by IHC) from a single protocol before applying those subgroups to publicly available TNBC sets. We employed non-negative matrix factorization on two independent gene expression datasets (Discovery n = 84 and Validation n = 118) using the top 1000 variant genes in each set. Cophenetic, dispersion, and silhouette metrics revealed 4 clusters, with significant enrichment of differentially expressed (DE) genes in each subgroup among both sets (all comparisons Fisher Exact p < 2.2E-16). A parsimonious gene centroid signature of 480 total genes was selected based on Goeman's Global Test BH-adjusted p-values and fold change of only the Discovery Set. Subgroups were assigned by Pearson Correlation in the Discovery (Rand Index = 0.97) and Validation Sets (RI = 0.84), as well as an External Set made up of 214 IHC-confirmed cases of TNBCs, and another 5 publicly available studies with clinical outcome data for TNBCs. Ingenuity Pathway Analysis was carried out separately on DE genes from each dataset (BH adjusted p < 0.01). There was overwhelming statistical agreement for all subgroups between the Discovery, Validation, and External Sets. Subgroup 1 was defined by Intermediate Grade LumB tumors and overexpression of ESR1, HER-2, and AR, with activated downstream signaling of ESR1 despite being ER- by IHC. In subgroup 2, CDKN2A and TP53 appeared activated while MYC was inhibited. Dozens of immune cell signaling pathways were downregulated in the third subgroup, a basal-like set of TNBCs, which was driven by inhibition of cytokine gamma-IFN. Disease-free and Overall Survival was the worst for subgroup 3 (log rank p = 0.041 and 0.039). The final subgroup (4) was found to be p53 inactivated, a known feature of TNBC but specific to this second basal-like subgroup. Analysis of 84 and 58 cases with corresponding copy number profiling data from the Discovery and Validation Sets revealed subgroup specific copy number changes in addition to known TNBC patterns. Most notably, loss of chr 6 was unique to subgroup 1, while all other tumors shared common loss of chr 5. We have described four molecular phenotypes of TNBC in two independent sets from the same protocol, and a third set composed of public data. These subgroups have strong agreement of DE genes, pathways, and regulators, and are additionally supported by DNA events and prognostic differences between subgroups. We suggest several promising druggable targets based on overexpressed genes in each subgroup. Citation Format: Matthew D. Burstein, Anna Tsimelzon, Kyle R. Covington, Suzanne AW Fuqua, Jenny C. Chang, Susan G. Hilsenbeck, C Kent Osborne, Gordon B. Mills, Ching C. Lau, Powel H. Brown. Identification of four subgroups of Triple Negative Breast Cancer (TNBC) by genomic profiling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1996. doi:10.1158/1538-7445.AM2013-1996

Background: ER-negative breast cancers are poorly responsive to anti-estrogen therapies. Currently, the only available treatment for ER-negative tumors is either chemotherapy or for a subset of ER-negative breast cancer is herceptin. Thus, there is an urgent need to developed targeted therapy to prevent these life-threatening cancers. In this project, we are investigating whether other signaling molecules, phosphatases, are differentially expressed in ER-negative as compared to ER-positive breast cancers. We hypothesize that: (1) the specific phosphatases that govern the growth of ER-negative cancers are different from those of ER-positive breast cancers, (2) identification of the specific phosphatases that regulate the growth of ER-negative cancer is possible using genomic and proteomic approaches, and (3) inhibition of some of the phosphatases activated in ER-negative cancers, and over-expression of some of the phosphatases under-expressed in ER-negative breast cancers, will suppress the growth of ER-negative breast cancers. Methods: Affymetrix microarray studies: cDNA was synthesized from total RNA isolated from tumor samples followed by Biotin-labeled cRNA and was then hybridized onto an Affymetrix HGU133A GeneChipTM and data analysis as described previously. Analysis was limited by the phosphatase list found using gene ontology annotation. Statistical analysis was done with dChip software. Differentially expressed genes were found using t-test. QRT-PCR: All Taqman assays run on ABI7900HT. siRNA knock-down assay and cell growth: siRNA knockdown was performed using Dharmafect and cell growth was measured by MTS assay. Results: We have identified 22 phosphatases that were upregulated (>1.2 fold with p-value of Conclusion: Several over and under-expressed phosphatases identified in our microarray analysis have been shown to critically regulate the growth of ER-negative breast cancer cells. Our results demonstrate that PPM1A and ACP1 knockdown inhibit ER-negative breast cancer cells growth. By identifying the molecules that regulate breast cancer cell growth we will identify potential new targets for the treatment and prevention of these aggressive breast cancers. Supported by Komen Promise grant KG081694 & Komen SAB grant SAB08-00006 (PB). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1665. doi:10.1158/1538-7445.AM2011-1665

IGF-I signaling is involved in tumor progression and drug resistance and many anti-IGF-IR therapeutic agents are currently in clinical trials. Moreover, growing evidence also suggests that insulin signaling may play an important role in cancer development and progression. However, little is known regarding similarities and difference in IGF-I and insulin signaling in cancer, and there are currently no biomarkers to predict patient response to IGF targeted therapy. To gain a better understanding of these signaling pathways in human breast cancer, we measured the levels of insulin receptor (IR) and IGF-IR, and the activity of insulin and IGF-I, in a large panel of human breast cancer cell lines. Comparative gene expression analysis was performed using publicly available gene expression data and IR and IGF-IR mRNA levels in 17 breast cancer cell lines were measured by Q-RT-PCR. IGF-IR and IR levels were found to be highly variable in breast cancer cell lines. MCF7 and MDA-MB-134 have high levels of IGF-IR expression and MDA-MB-468 and ZR-75-1 have high levels of IR expression, relative to other breast cancer cell lines. Reverse phase protein array (RPPA) analysis using 134 different antibodies was performed on MCF7 cells stimulated with increasing doses of insulin or IGF-I. One Way ANOVA was performed to identify proteins affected by insulin and/or IGF stimulation. Maximal response to insulin and IGF-I stimulation was observed at a 10nM concentration of both ligands. Therefore, we further performed RPPA on 21 breast cancer cell lines treated with 10nM of insulin and IGF-I for 6 time points (5min, 10min, 30 min, 6hrs, 24hrs, and 48hrs). Spearman rank correlation was performed on IR and IGF-IR protein levels to mRNA levels from Q-RT-PCR. There was a significant correlation between IGF-IR protein and mRNA levels (r=0.559, p=0.024), but not between IR protein and mRNA levels. Principle component analysis showed that MCF7, MDA-MB-134, T47D and ZR-75-1 are insulin and IGF-I responsive. Results from ANOVA with contrast in MCF7 cells showed that insulin and IGF-I affected similar set of proteins at 10 minute and 24 hours time points, however, there were several proteins affected only by IGF-I or insulin stimulation. For example, phospho-estrogen receptor (p-ER) was found to be increased by 10 minutes IGF-I stimulation, but not by insulin stimulation. Beta-catenin and c-Myc were significantly suppressed by IGF stimulation while PARP was significantly suppressed by insulin stimulation at the 24 hour time point. Moreover, IGF affected twice the number of proteins compared to insulin at the 48 hour time point. For instance, Bcl2 and SMAD3 were suppressed and Raf and FOXO3a were increased by IGF stimulation after 48 hours of stimulation. Further statistical and system biology analysis of the temporal activation of signaling by IGF-I and insulin in the 21 cell lines is ongoing and will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4133.

BACKGROUND: Insulin and insulin like growth factor-I (IGF-I) are key regulators in growth and metabolism. IGF-I signaling is known to be involved in tumor progression and drug resistance, and growing evidence also suggests that insulin signaling may play an important role in cancer. To gain a better understanding of these signaling pathways in human breast cancer, we measured the levels of IR and IGF-IR, and the activity of insulin and IGF-I, in a large panel of human breast cancer cell lines.METHODS: Comparative gene expression analysis was performed using publicly available gene expression data among a panel of 51 breast cancer cell lines. Q-RT-PCR was used to validate IR and IGF-IR expression differences in 19 breast cell lines. For 8 breast cancer cell lines we immunoblotted for total IR and IGF-IR levels, as well as p-Akt and p-Erk1/2 following insulin and IGF stimulation. Reverse phase protein array (RPPA) analysis was performed on MCF7 cells stimulated with increasing doses of insulin or IGF-I.RESULTS: IR and IGF-IR mRNA levels measured by microarray on breast cancer cell lines showed divergent levels with cells positive or negative for expression of each gene. There was no obvious correlation of levels with breast caner subtype. Q-RT-PCR results were generally consistent with the gene expression data with cell lines categorized into four groups: high IGF-IR and low IR e.g. MCF7 and MDA-MB-134; low IGF-IR and high IR e.g. ZR-75-1 and MDA-MB-468; intermediate levels of IGF-IR and IR e.g. MDA-MB-231 and HCC1954; and low IGF-IR and IR e.g. MDA-MB-415 and HS-578T. IGF-IR protein levels correlated with Q-RT-PCR (Spearman Rank Correlation r=0.93, p=0.0067) whereas IR didn't. We found significant activation of Akt in both insulin and IGF treated MCF7 and MDA-MB-134 cells as well as Akt activation in IGF stimulated MDA-MB-453. Interestingly, significant Erk1/2 activation was observed only in insulin and IGF stimulated MCF7 and insulin stimulated AU565 cells. Cell lines that didn't respond to insulin or IGF stimulation generally had constitutively high p-Akt and/or p-Erk1/2. From our preliminary RPPA analysis in MCF7, we observed an increase in phosphorylation of IGF-IR, Akt, Erk1/2 and p70S6K upon increasing dose of IGF stimulation and a similar effect was also observed following insulin stimulation.DISCUSSION: IGF-IR and IR mRNA and protein levels are divergent in a panel of breast cancer cell lines with cells expressing mRNA for both, one alone, or neither receptor. IGF-IR and IR protein levels indicated similar divergent expression. Interestingly, response to both IGF-IR and insulin stimulation was restricted to cells showing high levels of IGF-IR. To better understand insulin and IGF activities in breast cancer, we are currently performing RPPA on a large panel of 21 breast cancer cell lines. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4168.

Background: Metastasis is the ultimate cause of death for patients succumbing to breast cancer. The metastasis tumor associated (MTA) gene family has been linked with metastasis in breast cancer. Previously, we found that MTA2 overexpression enhanced the metastatic ability of the estrogen receptor-alpha (ERα)-negative cell line MDA-MB-231. This was accompanied by an increase in cell rounding, indicating a change in cytoskeletal organization. Here we explored the molecular pathways contributing to the MTA2-enhanced metastatic phenotype.Materials and Methods: MDA-MB-231 cells were engineered to overexpress MTA2. Changes in protein expression were determined by immunoblot assay. Differential gene expression was determined using Affymetrix microarrays and analyzed using dChip, BRB Array Tools, and R. Pathway analysis was carried out using the Database for Visualization, Annotation, and Integrated Discovery (DAVID). Validation was done by comparing to publicly available datasets. MTA2-overexpressing or control cells were transfected with a Rho GTP dissociation inhibitor alpha (Rho GDIα) or vector control expression constructs. Anchorage-independent growth was measured using soft-agar assays. Analysis of the impact of MTA2 and Rho GDIα combined expression was carried out using publically available datasets.Results: Analysis of enriched ontologies from significantly different genes between MTA2-overexpressing cells and clinical breast cancer samples of the basal subtype previously published by Richardson et al (1) indicated that the focal adhesion pathway was enhanced in MTA2-overexpressing samples. We found that a negative regulator of the focal adhesion pathway, Rho GDIα, was decreased in MTA2-overexpressing cells at the protein level. When a yellow fluorescent protein-tagged Rho GDIα expression vector was re-expressed in MTA2-overexpressing cells, there was a concomitant decrease in the expression of MTA2 and increased expression of endogenous Rho GDIα. When tested in soft agar assays, re-expression of Rho GDIα significantly reduced the colony-forming ability of MTA2-overexpressing cells. We examined the publically available dataset published by Wang et al. (2), and found that ERα-negative patients with high MTA2 expression and low Rho GDIa levels had earlier recurrence compared with all other patients (P=0.02).Conclusion: MTA2 overexpression was associated with an aggressive phenotype in MDA-MB-231 cells. These data indicate that MTA2 overexpression may be associated with increased signaling through the Rho pathway. We found that MTA2 and Rho GDIα form a regulatory loop which reinforces either MTA2 high or MTA2 low expression levels. Furthermore, we found that combined high MTA2 and low Rho GDIα expression had a negative impact on patient outcomes in the ERα-negative population. We are currently exploring the use of Rho pathway inhibitors for their potential to block this phenotype, and to explore the MTA2/Rho GDIα regulatory loop.1) Richardson AL, Wang ZC, De Nicolo A, Lu X et al. X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell 2006 Feb;9(2):121-32.2) Wang Y, Klijn JG, Zhang Y, Sieuwerts AM et al. Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 2005 Feb 19-25;365(9460):671-9. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6143.

Aromatase inhibitors (AIs) are challenging tamoxifen as the treatment of choice for both early and advanced breast cancer in postmenopausal women with estrogen receptor (ER) α-positive diseases. However, resistance frequently occurs. Previously, we identified a lysine to arginine transition at residue 303 (K303R) in ERα in premalignant breast lesions and invasive breast cancers, which confers estrogen hypersensitivity and resistance to the AI Anastrozole (Ana) when transfected in MCF-7 breast cancer cells. To identify genes whose expression was associated with the development of aromatase inhibitor resistance (AIR), we performed microarray analysis, and found a marked increase in the gene expression of different IGF family members in the mutant cells. Immunoblot analysis also revealed elevated constitutive phosphorylation of the IGF-1R/IRS-1/Akt pathway in K303R-expressing cells. Treatment with IGF-1R and Akt inhibitors drastically inhibited proliferation of the K303R-mutant expressing cells, while wild-type ERα-expressing clones showed little reduction of growth by these inhibitors, confirming the increased IGF signaling activation in the mutant cells.Post-translational modifications of ERα, such as phosphorylation, tightly regulate its function, and the K303R mutation resides at a major post-translational modification site, serine (S) residue 305. We found that the mutant was more efficiently phosphorylated than the WT receptor by Akt in vitro, and that the mutant cells exhibited enhanced S305 phosphorylation in vivo. Mutation of S305 to alanine (A) to destroy this phosphorylation site prevented in vitro and in vivo Akt-mediated phosphorylation.The ERα S305 residue is an important site that modifies response to tamoxifen; thus, we questioned whether the S305 site could also influence AI response. To address the role of S305 phosphorylation on AIR, we generated pools of stable transfectants expressing exogenous WT, K303R, or K303R/S305A mutant receptors, and then evaluated for the effects of Ana in soft agar assays. Expression of the K303R/S305A mutant resulted in a significant reduction in the basal non-stimulated growth and sensitivity to Ana compared to K303R ERα-expressing pools, thus mimicking the same response profile of wild-type ERα clones. A selective blocking peptide (S305 peptide, residues 298-308) able to antagonize this phosphorylation reversed AIR. Blockade of S305 phosphorylation also resulted in a specific inhibition of IGF-1R/IRS-1/Akt activation, but only in the mutant cells.Our data suggest that the K303/S305 residues of the ERα mutation may be a novel determinant of aromatase inhibitor response in breast cancer, and blockade of S305 ERα phosphorylation represents a new therapeutic strategy to overcome resistance to hormonal therapies in tumors with the ERα mutation. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5053.

1042 Background: The antiestrogen tamoxifen and aromatase inhibitors are the most frequently prescribed hormonal agents for the treatment of estrogen receptor (ER) α-positive breast cancer. An important question is whether there is a group of hormone resistant, ERα-positive patients who may derive additional benefit from the addition of chemotherapy to endocrine therapy, or who may be candidates for “targeted” biologics. Dicer1 is an RNase III-containing enzyme that processes microRNA precursors into mature microRNA, which have been implicated in breast tumor invasion and metastasis. BCRP1 is a transmembrane transport protein known to efflux a number of chemotherapeutic agents, but also steroid hormones. In the present study, we investigated whether Dicer might affect response to tamoxifen in breast cancer cells, and generated estrogen receptor-positive MCF-7 human breast cancer cells stably overexpressing Dicer1, and they exhibited elevated BCRP1 protein. Methods: We utilized preclinical approaches to study the function of BCRP1 in Dicer-overexpressing breast cancer cells using in vitro growth assays in soft agar, mammosphere formation assays, and in vivo tumor initiation. Results: Microarray analyses of human breast tumors, suggested that Dicer overexpression was associated with tamoxifen resistance. Dicer-overexpressing MCF-7 cells express elevated levels of BCRP1, ALDH, and cErbB2/HER-2 evident by immunoblot analysis. The Dicer1-overexpressing cells formed soft agar colonies in the presence of tamoxifen, however Fumitremorgin C (FTC) or MBLI-97, both BCRP inhibitors, reversed resistance, and sensitized cells to tamoxifen therapy. Preclinical in vivo tumor xenograft experiments confirmed the tamoxifen-resistant phenotype. Mammosphere potential was enhanced in Dicer-overexpressing cells suggesting an enrichment of stem-like breast cancer cells. Conclusions: Our results suggest that Dicer-overexpressing breast cancer cells are a novel preclinical model for an estrogen receptor-positive breast cancer progenitor phenotype and tamoxifen resistance. Based on our data Dicer1 is a potential predictive biomarker in breast cancer, and predicts that clinical BCRP1 inhibition may facilitate tumor sensitization to hormonal therapy. No significant financial relationships to disclose.

Abstract #1107 Background High mammographic density is associated with up to a 6-fold increased risk of breast cancer. Pathways responsible for this increased density are unknown. We hypothesize that specific molecular pathways exist that are associated with increased mammographic density and breast cancer risk, and may therefore be used to identify potential targets for chemoprevention. Methods Histologically confirmed normal breast tissue was collected from women undergoing breast surgery who had available demographic data and mammograms for review. Breast parenchymal density was classified according to the American College of Radiology's Breast Imaging – Reporting and Data System reporting system. Quantitative classification of mammographic parenchyma was performed using thresholding method and percent density. Women with low (less than 50%) versus high (greater than 50%) breast density were compared. Double-stranded cDNA was synthesized from the normal breast tissue using an oligo-dT primer containing a T7 RNA polymerase promoter, followed by in vitro transcription with biotinylated ribonucleotides. The labeled cRNA was hybridized to Affymetrix HG U133Plus2 chips which comprise ∼28,600 genes to determine gene expression patterns. Immunohistochemical analysis (IHC) of estrogen receptor, progesterone receptor, proliferation (Ki67) and COX2 expression was performed. Results Sixty-two women were identified, 26 (42%) had high, and 36 (58%) had low mammographic density. Neither age, menopausal nor hormone receptor status influenced the gene expression pattern. Seventy-three genes had differential expression between normal breast tissue with high and low mammographic density (p1.5) and had a low false discovery rate ( Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1107.

22227 Background: High mammographic density is associated with a 6-fold increased risk of breast cancer. Pathways responsible for this increased density are unknown. We hypothesize that specific mo...

SummaryOxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of β-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.