Estrogen Negative Breast Cancer Research Articles

Abstract PO5-08-06: Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells

Abstract Introduction. Oncogenic factors that are local/in-breast are of great interest as they may be more specifically targetable for breast cancer prevention than systemic factors. We have identified a lipid metabolism gene signature that is enriched in breast tissue at risk for estrogen negative breast cancer (ER- BC). Utilizing the medium chain fatty acid Octanoic acid (OA) to probe lipid metabolism in non-transformed breast epithelial cells, we observed increased flux through several metabolic reactions and altered histone methylation with consequent changes in gene expression (e.g. neural genes). Neuronal signaling and regulatory circuits are observed in cancer cells of multiple origins, not just ones with ontological relationships to neurons. We hypothesize that the first and rate limiting step in the de novo serine pathway, which is catalyzed by Phosphoglycerate Dehydrogenase (PHGDH) is key to these observations. In the forward direction, PHGDH participates in the serine, one-carbon, glycine (SOG) and methionine pathways that produce the methyl donor S-adenosylmethionine (SAM) and in the reverse direction produces the oncometabolite 2-Hydroxyglycerate (2-HG). Methods. Non-transformed MCF-10A cells exposed to OA were utilized for U13C-glucose tracing. SAM and 2-HG concentrations following treatment with OA ± PHGDH inhibitor were measured by liquid chromatography. CUT&RUN for H3K4me3 was performed and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. Single cell RNA-sequencing was carried out using breast microstructures derived from reduction mammoplasty tissue exposed to vehicle or OA. Microstructures were dissociated into single cells and sequenced using the 10x Genomics platform. The digital expression matrix file containing UMIs were analyzed with Seurat. Alkaline comet assay was performed to detect DNA breaks. Results. U13C-glucose tracing in presence of OA revealed that one-carbon-THF was redirected to the methionine cycle increasing flux to methylation. Concentrations of SAM and 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01), these genes are involved in neural pathways, EMT and ER- BC. Motif analysis revealed an overrepresentation of binding sites for transcription factors ATF3/4 (p < 0.05), which are regulators of the serine pathway. Single cell RNA-sequencing revealed OA not only affected the distribution of cell subpopulations but also modulated the expression of many genes within each subcluster. The percentage of luminal progenitor subcluster 3 increased upon OA from less than 1% to about 13%. Within basal subcluster 3, OA drives the expression of ATF3, along with two of the enzymes in the de novo serine pathway: PHGDH and PSAT1. Alkaline comet assay showed DNA breaks in OA- and control 2-HG- treated cells. Conclusions. Metabolism of OA in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM and 2-HG, with implications for oncogenesis: 1. SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express genes consistent with a neural/neural crest-like state. These co-opted neuronal regulatory mechanisms can make critical contributions to the acquired functional capabilities that drive cancer development. 2. 2-HG exposure results in appearance of DNA breaks, which are likely consequent to the inhibition of the alpha-ketoglutarate-dependent dioxygenases KDM 4A/B by 2-HG. Their catalytic activity is required for homologous recombination repair; inhibition results in metabolic “BRCAness”. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Maria Paula Zappia, Elizaveta V. Benevolenskaya, Abul Bashar Mir Md. Khademul Islam, Maxim V. Frolov, Seema Khan, Susan Clare. Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-08-06.

Abstract 444: Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells

Abstract Introduction. A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increases flux through various metabolic reactions including those involved in serine, one-carbon, glycine (SOG) and methionine. We hypothesized that the metabolism of lipids in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing S-adenosylmethionine (SAM) leading to histone methylation increases and changes in gene expression. Methods. MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± the PHGDH inhibitor CBR-5884. ROS-induced redox changes were monitored live in cells transduced with ORP1-roGFP2 vectors. CUT&RUN was performed for H3K4me3. Expression of OA-induced genes was measured by rt-qPCR upon exposure to OA ± CBR-5884 or OA ± the histone methyltransferase (HMT) inhibitor Piribedil. Alkaline comet assay was done to detect DNA breaks. Single-cell RNA-seq (scRNAseq) was performed on tissue-derived breast microstructures exposed to ± OA. Results. Upon OA treatment, the Cancer index increased and 1C-THF was redirected to the methionine cycle increasing flux to methylation. OA significantly increased the production of SAM, GSH and 2-HG after 15 min. Blocking the first enzyme in the serine pathway, PHGDH, prevented these increases. After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p < 0.0001), peaking at 15 min. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01) including neural, EMT and ER- BC related genes. Motif analysis revealed an overrepresentation of binding sites for serine pathway transcriptional activators ATF3/4 (p < 0.05). Blocking PHGDH or HMT prevented OA-induced gene expression changes. Alkaline comet assay showed that OA significantly increased comet tails. scRNAseq revealed OA increased the numbers of cells within all cell clusters expressing ATF3 and PHGDH. Also observed was an increase in the percentage of basal BSL1, luminal progenitor LP3 and hormone sensing HS1 cells. Conclusions. Metabolism of OA results in a metabolic shift toward the SOG and methionine increasing the production of SAM, GSH and 2-HG which have implications for oncogenesis. The increased SAM fosters epigenetic phenotypic plasticity via altered histone methylation. The increased proportion of specific cell types likely reflects the survival of cells able to mitigate oxidative stress. Increased 2-HG may produce metabolic BRCAness by inhibiting 2-oxoglutarate-dependent dioxygenases. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta V. Benevolenskaya, Maxim V. Frolov, Jason Locasale, Marcelo G. Bonini, Navdeep S. Chandel, Seema Khan, Susan Clare. Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 444.

Caffeic acid stimulates breast cancer death through Reactive oxygen species (ROS) formation, Caspase activation and mitochondrial membrane potential depletion

Objectives: The aim of this study is to evaluate the potential effect of caffeic acid (CAF) on the growth of breast cancer cells, in addition to determining the contributing role of caspases, mitochondria, and oxidative status.
 Methods: MCF-7 and MDA-MB-468 breast cancer cells were exposed to varying concentrations of CAF for different periods of time. The potential cytotoxic effect was measured using the MTT assay. The activities of caspase 3 and caspase 8, as well as the cellular level of reactive oxygen species (ROS) and the level of mitochondrial membrane potential (Δψm), were evaluated in different groups of cells.
 Results: The findings showed that CAF decreased the percentage of MCF-7 and MDAMB-468 cells in a manner that depended on the dose and duration of exposure. The death of breast cancer cells induced by CAF was associated with an increase in ROS level in both cell lines. The decrease in mitochondrial membrane potential (Δψm) following CAF treatment suggests that mitochondrial dysfunction may be involved in the death of breast cancer cells induced by CAF. Importantly, the activity of caspase 8 increased after treatment, indicating the potential involvement of the extrinsic apoptosis pathway in the inhibition of breast cancer cell growth by CAF. The dosage of 20µm of CAF following 48 hours of incubation appeared to have the most significant impact on breast cancer cells.
 Conclusion: The study highlights the potential pro-apoptotic effect of CAF in both estrogen-positive and estrogen-negative breast cancer cells. This, in conjunction with other evidence, may lead to new insights for more effective therapeutic approaches in breast cancer

Androgen Receptor Expression in ER and PR Negative Breast Cancer—A Study from a Tertiary Hospital in Northern India

Objectives Breast cancer is the leading cause of cancer-related deaths in women. Estrogen (ER) and progesterone receptor (PR) status and Her2 overexpression are major determinants in therapeutic decision making. Triple-negative breast cancers (TNBCs) have limited treatment options. Androgen receptor (AR) expression opens up therapeutic avenues for these patients. The aim of this article was to study the immunohistochemical expression of ARs in ER and PR Negative breast carcinomas and to correlate AR expression with various clinical, histopathological, and other immunohistochemical parameters. Materials and Methods It is a cross-sectional study including 105 ER and PR Negative cases of breast carcinoma. Clinical parameters, histopathology, and immunohistochemical expression of AR, Her2, and Ki67 were analyzed in all cases. Results AR expression was observed in 63.8% of ER and PR Negative breast cancers. In this group, AR expression was strongly associated with Her2 co-expression (89.2%) as compared to TNBCs (45.8%); p-value = 0.0002. Significant correlation was also observed between AR expression and tumor necrosis (p-value = 0.034) and postmenopausal status (p = 0.007). Conclusion Our study shows that significant proportion of ER and PR Negative breast carcinomas (ER- PR- Her2+ and TNBCs) show AR expression. We strongly recommend routine evaluation of all hormone receptor-negative breast carcinomas for AR status by immunohistochemistry.

Abstract IA017: Targeting the mTOR pathway for the prevention of triple-negative breast cancer

Abstract Phase III cancer prevention clinical trials have shown that breast cancer prevention is feasible using anti-estrogen drugs. However, these drugs have not been widely accepted because of concerns about toxicity. In addition, anti-estrogen drugs do not prevent estrogen-negative breast cancers, including the most aggressive form of breast cancer, triple-negative breast cancer (TNBC), that does not express the estrogen receptor (ER), progesterone receptor, or the human epidermal growth factor receptor 2 (HER2). Our laboratory is focused on identifying growth-regulatory molecules that are essential for the growth of TNBCs. For this study, we identified mTOR as an essential growth-regulatory molecule that is highly expressed in TNBCs. We then investigated whether the mTOR inhibitor everolimus can prevent mammary tumors in transgenic mouse models including 4 models of TNBC and one model of ER-negative/HER2-positive breast cancer. Everolimus treatment significantly delayed mammary tumor formation but with a varying degree in all five mouse models. Everolimus treatment for up to 1 year was well tolerated with no observable toxicity. These results suggest that mTOR inhibitors may be promising drugs for the prevention of ER-negative and triple-negative breast cancers in women at risk of these aggressive breast cancers. Grant support: This research was supported by an NCI-PREVENT contract to P. Brown and A. Mazumdar (HHSN261201500018I/HHSN26100006). Citation Format: Powel H. Brown, Abhijit Mazumdar, William Tahaney, Jamal Hill, Yun Zhang, Sumankalai Ramachandran, Jitesh Kawedia, Jing Qian, Alejandra Contreras, Michelle Savage, Lana Vornik. Targeting the mTOR pathway for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA017.

Oxysterols in catfish skin secretions (Arius bilineatus, Val.) exhibit anti-cancer properties

The edible catfish Arius bilineatus, (Valenciennes) elaborates a proteinaceous gel-like material through its epidermis when threatened or injured. Our on-going studies on this gel have shown it to be a complex mixture of several biologically active molecules. Anti-cancer studies on lipid fractions isolated from the gel-like materials showed them to be active against several cancer cell lines. This prompted us to investigate further the lipid composition of the catfish epidermal gel secretions (EGS). Analysis of the lipid fraction of EGS resulted in identification of 12 oxysterols including cholesterol and 2 deoxygenated steroids i.e., 7α-hydroxy cholesterol, 7β-hydroxycholesterol, 5,6 epoxycholesterol, 3β-hydroxycholest-5-ene-7-one and cholesta-3,5-dien-7-one. Progesterone, cholest-3,5-diene, cholesta-2,4-diene, cholest-3,5,6-triol and 4-cholesten-3-one were found as minor components, and were identified through their MS, 1HNMR and FTIR spectral data and were compared with those of the standards. Cholest-3,6-dione, cholesta-4,6-diene-3-one, cholesta-2,4-diene, and cholesta-5,20(22)-dien-3-ol were found only in trace amounts and were identified by GC/MS/MS spectral data. Since cholesterol is the major component of EGS, the identified oxysterols (OS) are presumably cholesterol oxidation products. Many of the identified OS are known important biological molecules that play vital physiological role in the producer and recipient organisms. We report herein the effects of these sterols on three human cancer cell lines in vitro, i.e., K-562 (CML cell line), MDA MB-231 (estrogen positive breast cancer cell line) and MCF-7 (estrogen negative breast cancer cell line). Interestingly significant (p < 0.05) dose differences were observed between tested OS on cell types used. The presence of these sterols in EGS may help explain some aspects of the physiological activities of fraction B (FB) prepared from EGS, such as enhanced wound and diabetic ulcer healing, anti-inflammatory action and cytotoxic activities reported in our previous studies. The anti-proliferative actions of some of these oxysterols especially the cholesterol 3,5,6-triol (#5) as established on selected cancer cell lines in this study support our previous studies and make them candidates for research for human application.

Hormone receptor binding, selectivity and cytotoxicity of steroid D-homo lactone loaded chitosan nanoparticles for the treatment of breast and prostate cancer cells

Chemically modified steroids have a long history as anti-neoplastic drugs. Incorporation of a lactone moiety in the steroid nucleus, as in previously obtained 3β-acetoxy-17-oxa-17a-homoandrost-5-en-16-one (A) and 3β-hidroxy-17-oxa-17a-homoandrost-5-en-16-one (B), often results in enhanced anticancer properties. In this work, chitosan-based (Ch) nanoparticles were created and loaded with potent anticancer steroidal compounds, A and B. Changes to hormone receptor binding and cytotoxicity were then measured. In agreement with our previous results for A and B, A- and B-loaded Ch displayed cytotoxic properties against cancer cell lines. Both A-Ch and B-Ch showed activity toward estrogen negative breast cancer (MDA-MB-231) and androgen negative prostate cancer cell lines (PC-3). Greater selectivity toward cancer cells versus healthy lung fibroblast (MRC-5) was observed for B-Ch particles. Cell viability and cytotoxicity measurements after a recovery period indicate more robust recovery of healthy cells versus malignant cells. Compounds A and B or their Ch-encapsulated forms were shown to have negligible affinity for the ligand binding domain of estrogen receptor β or the androgen receptor in a fluorescent yeast screen, suggesting a lack of estrogenicity and androgenicity. Steroid-loaded chitosan nanoparticles display strong cytotoxicity towards MDA-MB-231 and PC-3 with a lack of hormone activity, indicating their safety and efficacy.

Comprehensive Analysis of the Expressionand Prognosis for TDO2 in Breast Cancer

A plethora of previous studies have been focused on the role of indoleamine 2,3-dioxygenase 1 (IDO1) in cancer immunity; however, the alternative way of targeting tryptophan 2,3-dioxygenase (TDO2) in cancer immunotherapy has been largely ignored. In particular, the specific role of TDO2 in breast cancer remains unclear. In the present study, we systematically explored and validated the expression and prognostic value of TDO2 in breast cancer using large-scale transcriptome data. We observed overexpression of TDO2 in many types of cancer tissues compared with adjacent normal tissues. TDO2 overexpression was revealed to be positively correlated with malignancy and tumor grade in breast cancer. TDO2 expression was higher in estrogen-negative breast cancer and triple-negative breast cancer, and it was correlated with worse outcome in breast cancer patients. TDO2 expression was correlated with immune infiltrates and tryptophan metabolism-related genes (IDO1 and kynureninase [KYNU]). Therefore, our results indicated that TDO2 plays a pivotal role in regulating the immune microenvironment and tryptophan metabolism in breast cancer, and it predicts poor prognosis in breast cancer, which suggests that TDO2 might be a promising novel immunotherapy target for breast cancer. Additionally, we established the concept that tryptophan-catabolizing enzymes (IDO1, IDO2, TDO2, and KYNU) may function through co-regulating the immunological microenvironment, and thus immunotherapy targeting IDO1 alone might be insufficient.

Competitive ELISA method for novel estrogen-negative breast cancer biomarker quantitation

Estrogen-negative (ER−) breast cancer, is recognized as an aggressive subtype, more difficult to treat, with poor survival and prognosis. They are hormonally unresponsive, with no readily effective and specific target therapy. We have previously identified Nw-hydroxy L-Arginine (NOHA) as a blood-based biomarker to distinguish between ER− and ER+ breast cancer tumors based upon disease burden, progression and molecular phenotype (U.S. Utility Patent 10,073,099). In this study we have demonstrated a competitive ELISA based assay for NOHA measurement using a proprietary monoclonal antibody (mAb) specific for NOHA (U.S. provisional patent 62/754,053). The ELISA assay was evaluated on sensitivity, selectivity, precision, dilution linearity and percent recovery parameters. The assay showed sensitivity at ≥60 pg/ml NOHA antigen with 1 ng/ml NOHA mAb, and maintained NOHA antigen specificity even in the presence of other closely related cationic amino acids (i.e. L-Arginine, D-Arginine, l-Lysine, d-Lysine, L-Ornithine, and L-Citrulline). The reliability of the ELISA protocol was confirmed with the low percent-covariance, for all tested parameters of sensitivity (≤8.2%), selectivity (≤8.6%), precision (≤12.6%), dilution linearity (≤11.2%) and recovery (≤6.7%). Additionally, we can demonstrate NOHA quantification by this ELISA assay to complement the sensitivity achievable with LC-MS (in both assay buffer and with patient plasma samples), thus suggesting it's utility as a simple yet sensitive methodology that might help in ER− breast cancer prognosis, and disease progression monitoring without the need for expensive analytical equipment (such as LC-MS), large lab space, or specialized technical training.

Correction to: Metabolic relevance for N-hydroxy L-arginine reduction in estrogen-negative breast cancer cells.

We found a unit error with our LC-MS lower limit of quantitation (LLOQ) measurement in the Amino Acids Journal.

Sonographic Features of Estrogen-Negative Breast Cancers: A Correlation Study With Human Epidermal Growth Factor Type II Overexpression

The purpose of this study was to retrospectively review sonographic imaging features of estrogen-negative breast cancers and compare tumors with and without human epidermal growth factor type II (HER2) overexpression. Breast sonography findings from a sample of 54 patients with estrogen-negative breast cancer as well as pathological data and HER2 status were reviewed. Sonographic features including size, depth shapes, margin, location, patterns of internal echoes, posterior echoes, orientation, and presence of halo and clinicopathologic data and imaging features were correlated with tumor HER2 status. Based on these 54 patients with estrogen-negative breast cancers, 21 patients were positive for HER2 receptor, and 33 patients were negative for HER2 receptor. Among HER2 positive cancers, irregular shape, microlobulation, indistinct margins, posterior shadowing, a thin halo, heterogeneous internal echoes, and parallel orientation were the most frequent sonographic features. No associations were found between HER-2 status and tumor size, shape, margins, posterior feature, halo, internal echoes, or orientation on sonography.

Metabolic relevance for N-hydroxy L-arginine reduction in estrogen-negative breast cancer cells.

We had shown Nw-hydroxy-L-arginine (NOHA) as a promising blood-based biomarker for estrogen-receptor-negative (ER-) breast cancer (BC) that differentiates ER- BC based on grade and molecular phenotype. In this in vitro study, we assessed the metabolic relevance for ER- BC-specific NOHA modulation and correlated them with NOHA regulatory responses. This study aids future NOHA clinical utility in ER- BC diagnosis and therapy management and would prove useful for potential drug discovery and development process.

Effects of α-zearalenol on the metabolome of two breast cancer cell lines by 1H-NMR approach.

Zearalenone (ZEN) is one of the most widely distributed toxins that contaminates many crops and foods. Its major metabolites are α-Zearalenol (α-zol) and β-Zearalenol. Previous studies showed that ZEN and α-zol have estrogenic properties and are able to induce growth promoting effect in breast tissues. Considering that tumorigenesis is dependent on the reprogramming of cellular metabolism and that the evaluation of the cellular metabolome is useful to understand the metabolic changes that can occur during the cancer development and progression or after treatments, aim of our work is to study, for the first time, the effects of α-zol on the metabolomic profile of an estrogen positive breast cancer cell line, MCF-7, and of an estrogen negative breast cancer cell lines MDA-MB231. Firstly, we tested the effects of α-zol on the cell viability after 24, 48 and 72h of treatments with 10-10, 10-8 and 10-6M concentrations on breast cancer MCF-7 and MDA-MB231 cell lines in comparison to human non-cancerous breast MCF10A cell line. Then, we evaluated cell cycle progression, levels of reactive oxygen species (ROS) and the metabolomic profiling by 1H-NMR approach on MCF-7 and MDA-MB231 before and after 72h treatments. Principal component analysis was used to compare the obtained spectra. α-zol is resulted able to induce: (i) an increase of the cell viability on MCF-7 cells mainly after 72h treatment, (ii) a slight decrease of the cell viability on MDA-MB231 cells, and (iii) an increase of cells in S phase of the cell cycle and of ROS only in MCF-7 cells. Moreover, the evaluation of metabolomics profile evidenced that after treatment with α-zol the levels of some metabolites increased in MCF-7 cells whereas decreased slightly in MDA-MB231 cells. Our results showed that α-zol was able to increase the protein biosynthesis as well as the lipid metabolism in MCF-7 cells, and, hence, to induce an estrogen positive breast cancer progression.

Assessing N w-hydroxy-l-arginine applicability as a novel ethnic specific estrogen-negative breast cancer marker

In our prior study we identified N w-hydroxy-L-arginine (NOHA) as a simple, yet sensitive indicator for estrogen negative (ER-) breast cancer early-prognosis, but not estrogen positive (ER+), and to offer ethnic selectivity for ER- detection. However, the ability of NOHA to assess ER- breast tumor based on disease progression, and tumor severity needs further delineation. Also, the overall NOHA storage stability needs to be validated. To assess the NOHA predictive capability based on disease progression, ER-/ER+ 3D-spheroids (from breast tumor cell lines of human origin) were cultured for 10weeks. We found only ER- 3D-spheroid cultured for 10weeks to show a gradual reduction in NOHA (both in culture medium and 3D-spheroid lysates) that correlated with a progressive increase in cellular NOS2 expression and NOS2 activity (measured as total nitrites). We additionally identified the NOHA-NOS2 correlation to be ethnically selective between ER- African American versus ER- Caucasian groups. Interestingly, such NOHA reduction was observed earlier in ER- culture medium (viz., after week 1) than from ER- 3D-spheroids lysates (viz., at the end of 3weeks). When categorized based on 3D-spheroid grade, we found a ≥68% NOHA reduction in ER- spheroids that were ≤3weeks old, that was categorized as "low-grade" (based on tumor size ≤250µm, and with cellular characteristics identical to healthy cells). A substantial reduction in NOHA of ≥87% occurred with ER- 3D-spheroids grown for 6weeks, which were categorized as "intermediate-grade" (with tumor size of ≥400µm, and with less characteristic similarity to control spheroids). These in vitro findings thus suggest a distinct correlation between NOHA reduction and ER- tumor grade. Such distinctive correlation between NOHA and ER- tumor grade was additionally observed in de-identified clinical samples where a onefold higher reduction in NOHA occurred in grade-2 than with grade-1 de-identified patient plasma (when compared with control), and such correlation offered ethnic selectivity between ER- African American and ER- Caucasian groups. Of additional interest, when NOHA overall storage stability was assessed by incubating patient plasma and culture medium spiked with 75pg/ml NOHA at multiple incubation temperatures and time-points, we found NOHA to maintain its stability for up to 6weeks in culture medium and for 7days in plasma at 4°C and below. These results thus provide the first evidence of NOHA as a stable indicator to monitor ER- disease progression and tumor severity in ethnically distinctive populations.

Prognostic value of histopathology, stromal tumor infiltrating lymphocytes (sTILs) and adjuvant chemotherapy (AdjCT) in early stage triple negative breast cancer (TNBC).

533 Background: Current guidelines define TNBC as complete absence of estrogen (ER) and progesterone receptor (PR), without HER2 amplification. However, the prognostic impact of clinical and histopathological factors, sTILs, and AdjCT in TNBC meeting these strict criteria is unknown. Methods: From a cohort of 9985 women who underwent upfront surgery for M0 breast cancer (BC) at Mayo Clinic Rochester from 1985-2012, 1159 pts with ER negative or low (≤10%) BC were identified for central ER/PR/HER2 staining and HER2 FISH (IHC2+ only) to select those with TNBC by modern definitions. Cox proportional hazards models were used to assess the impact of clinicopathological variables on invasive disease-free (IDFS) and overall survival (OS). Results: Tumors from 605 pts (median age 56.3 yrs) met criteria for TNBC (ER &lt; 1%, PR &lt; 1% and HER2 0, 1 or 2+ and FISH negative). 51% were T1, 65% N0, 88% grade 3, and 75% had Ki67 &gt; 15%. Histologically, 39% were anaplastic, 26% invasive ductal, 16% medullary, 8% metaplastic, 6% apocrine and 5% others. Median sTILs was 20% (0-90%). 55% pts received AdjCT [21% anthracycline (A), 19% A + taxane, and 15% other]. Median follow-up for IDFS and OS were 7.4 and 10.6 yrs, respectively. Multivariate analyses demonstrated that higher N stage (p &lt; 0.01), lower sTILs (p = 0.01) and no AdjCT (p &lt; 0.01) were independently associated with worse IDFS and OS. Histology (medullary subtype) was associated with better IDFS in univariate (HR 0.56, 95% CI, 0.35-0.89) but not in multivariate analyses, once sTILs were accounted for. Among systemically untreated pts (n = 182), higher N (p &lt; 0.01) and lower sTILs (p = 0.04) were associated with worse IDFS. For systemically untreated T1N0 pts (n = 111), the 5-yr IDFS was 70% (95% CI, 61-81) [T1a: 83% (95% CI, 63-100), T1b: 68% (95% CI, 52-88) and T1c: 67% (95% CI, 55-83)], compared to 78% (95% CI, 68-84) for T1N0 pts treated with AdjCT. Conclusions: In TNBC pts, N stage, sTILs and receipt of AdjCT were independently prognostic for IDFS and OS. sTILs remained prognostic for IDFS in systemically untreated TNBC. In N0 TNBC, the risk of recurrence or death was substantial in the absence of chemotherapy, even for those with T1 tumors.

Tamoxifen Directly Inhibits Platelet Activation, Angiogenic Potential and Platelet-Mediated Metastasis

Objective - Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in tumor metastasis and neovascularization. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Platelets carry a plethora of potent angiogenic and metastatic mediators within their alpha-granules and exposure to breast tumor cells induces platelet activation, leading to release of these mediators. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer; its use is associated with a 50% reduction in the risk of invasive and noninvasive breast cancer in women who utilized the drug for at least 5 years. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown to directly impact platelet function. Because platelets are critical for metastatic spread, we posited that tamoxifen or its metabolites may exert anti-tumor effects through direct platelet inhibition. To test this hypothesis, we examined the impact of tamoxifen on platelet activation, angiogenic potential and metastasis both ex vivoand in breast cancer patients utilizing tamoxifen therapy.Approach and Results - We found that ex vivo pretreatment with tamoxifen or its metabolite 4-hydroxytamoxifen (4-OH) lead to a significant inhibition of platelet activation in response to TRAP, ADP or the breast tumor cell lines MDA-MB-231 and MCF-7 in platelets from healthy human donors. Platelets, known to promote tumor angiogenesis, are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. Activated platelets exposed to tamoxifen or 4-OH released significantly lower amounts of the pro-angiogenic regulator VEGF in while anti-angiogenic endostatin release is unaffected, thus shifting the balance of angiogenic regulators that are released. To examine the impact of this alteration in angiogenic protein release, we performed functional angiogenesis assays using releasates generated from tamoxifen or 4-OH treated platelets. These in vitroangiogenesis assays confirmed that tamoxifen pretreatment led to dramatically diminished capillary tube formation and decreased endothelial migration. Tamoxifen also significantly inhibited the ability of platelets to promote metastasis in vitro, causing a dramatic decrease in breast tumor cell invasion and transendothelial migration. Next, we utilized membrane-based cytokine arrays to further interrogate the effect of tamoxifen on the release of stored platelet factors. Using this method, we identified several key proteins known to be associated with metastasis that were lower in releasate from tamoxifen treated platelets including TGF-b, IL-6 and IGF-1 while anti-angiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished VEGF release and lower angiogenic potential.Conclusions - Overall we demonstrate that tamoxifen directly influences the release of specific stored platelet factors leading to decreased tumor cell support. The mechanism is directly linked to tamoxifen's inhibitory role in platelet activation, causing altered of release of key platelet-derived angiogenic and metastatic factors including VEGF, angiopoietin-1, TGF-b, IL-6 and IGF-1 during tumor cell and platelet crosstalk. Furthermore, translational studies confirmed that platelet activation and angiogenic potential are significantly suppressed in breast cancer patients utilizing tamoxifen therapy. Our work stresses the importance of platelets for successful angiogenesis and metastatic spread and, ultimately, supports the idea of utilizing targeted platelet therapies to inhibit the platelet's role in malignancy. DisclosuresNo relevant conflicts of interest to declare.

N w-hydroxy-L-arginine as a novel ethnic specific indicator of estrogen-negative breast cancer.

As a heterogeneous disease, breast cancer can be divided into distinct subtypes. Among the two major subsets of estrogen receptor-negative (ER-) and ER-positive (ER+) tumors, the ER- is a more aggressive subtype, more difficult to treat, has greater ethnic disparity concerns, worse prognosis, and almost twice the risk of mortality. We provide here a fundamental delineation of N w-hydroxy-L-arginine as a sensitive and reliable ethnic specific indicator for ER- breast cancer early-prognosis (United States provisional patent application number 62232816).

A Review of the Recent Developments in Synthetic Anti-Breast Cancer Agents.

The perceptible decrease in the incidence of breast cancer in recent years has not influenced its societal and economic impact and it remains as the most commonly diagnosed malignancies among females. Recent reports of clinical trials in preventive settings suggested chemoprevention as an appealing strategy zeroing heavily on endocrine intervention using selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs). Unfortunately, these drugs are only effective in prevention of endocrine responsive lesions with essentially no effect in reducing the risk of estrogen-negative breast cancer. Further, the existing drugs for breast cancer treatment are invariably associated with several drawbacks such as poor oral bioavailability, non-selectivity and poor pharmacodynamics properties, limiting their clinical utility. Thus, the identification of new molecular targets and the development of agents with better pharmacological profiles will streamline the development of rational, effective and safe anticancer drugs with minimal side-effects. Over the past few years, different research groups have been actively involved in the design and synthesis of novel anti-breast cancer agents. In this review article, the recent developments (2013 onwards) made in the direction of synthesis of new scaffolds with promising anti-breast cancer activity, are briefly described. Hopefully, the data compiled in this article will update scientific community with recent endeavors in this field, and will certainly be encouraging for further research in this direction.

Abstract 2776: Codon 72 and Intron-3 polymorphisms in TP53 are risk factors for breast cancer

Abstract Purpose: A single nucleotide polymorphism (SNP) in exon-4 of the TP53 gene (Arg72Pro) has been associated with an increased risk of developing various human malignancies. Since a 16-base pair polymorphism (insertion/duplication) intron-3 (PIN3) is in linkage disequilibrium with Arg72Pro of TP53, we assessed whether these two polymorphisms serve as breast cancer risk alleles for African Americans (AAs) and non-Hispanic Caucasians (CAs). Methods: Genomic DNA was extracted from formalin-fixed, paraffin-embedded invasive ductal carcinoma tissues [cases, n = 120 (AAs = 55 and CAs = 65)] and from blood of healthy women [controls, n = 152 (AA = 74 and CAs = 78)]. Exon-4 of TP53 was amplified by PCR using exon-specific primers, followed by Sanger sequencing. Identification of SNP was done by using DNASTAR software. TP53 PIN3 polymorphism was detected by amplifying genomic DNA using specific primers followed by gel electrophoresis. The A1 allele (no duplication) resulted in 162 bp fragment and the A2 allele (with 16 bp duplication) resulted in 178 bp fragment. The phenotypes of Arg72Pro (Arg/Arg, Arg/Pro, and Pro/Pro) were correlated with PIN3 status, clinicopathologic features, race/ethnicity, and patient survival using the χ2 test and Kaplan-Meier analyses. A p value of &amp;lt;0.05 was considered statistically significant. Results: In cases, the Arg/Arg/, Pro/Arg, and Pro/Pro phenotypes were 15%, 31%, and 55% in AAs and 57%, 29%, and 14% in CAs (p&amp;lt;0.001), respectively. A similar distribution was observed in controls (16%, 42%, and 42% in AAs and 56%, 37%, and 6% in CAs) (p&amp;lt;0.001). When compared to ER positive, a higher frequency of Arg/Pro or Pro/Pro was noted in estrogen-negative breast cancers (p = 0.03) in both racial groups. However, no significant survival difference in these racial groups was observed when Pro carriers were compared with Arg/Arg cases (log rank, p = 0.565). Frequencies of PIN3 polymorphisms (A1A1, A1A2, and A2A2) were similar in both cases and controls; however, overall A1A1 frequency was higher than the other two genotypes of PIN3. The phenotype Pro/Pro, was strongly associated with A1A1 polymorphism in both racial groups of breast cancer patients (p&amp;lt;0.000078). Conclusion: These preliminary findings suggest that Arg72Pro polymorphism and PIN3 in TP53 are related with higher breast cancer susceptibility for both African American and non-Hispanic Caucasian patients. This work was supported by a pilot grant from UAB Breast SPORE grant of the National Institutes of Health/National Cancer Institute (5P50CA089019). Citation Format: Trafina Jadhav, Jesus Salazar-Gonzalez, Shantel Hebert-Magee, Michael Behring, Balananda-Dhurjati Putcha, Jeehyun Helen Bae, Andra Frost, Isam-Eldin Eltoum, Sejong Bae, Upender Manne. Codon 72 and Intron-3 polymorphisms in TP53 are risk factors for breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2776. doi:10.1158/1538-7445.AM2015-2776

The combined treatment with novel platinum(II) complex and anti-MUC1 increases apoptotic response in MDA-MB-231 breast cancer cells.

New strategy of cancer’s targeting treatment is combining monoclonal antibodies with chemotherapeutic agents. An important goal of targeted therapy appears to be a transmembrane glycoprotein type I—mucin 1 (MUC1), which is overexpressed in tumors of epithelial origin, especially in breast cancer. The goal of the study was to check the effect of monoclonal antibody against MUC1 with novel platinum(II) complex (Pt12) on selected aspects of apoptosis in human MDA-MB-231 breast cancer cells. The number of apoptotic and necrotic cells was measured using annexin V binding assay. The decrease of mitochondrial membrane potential (MMP) and DNA fragmentation was analyzed. Finally, the influence of novel platinum(II) complex (Pt12) used with anti-MUC1 on the concentration of selected markers of apoptosis such as Bax, caspase-8, -9, and caspase-3 was performed using ELISA. The results from combined treatment were compared with those obtained using monotherapy. In our study, we proved that anti-MUC1 used in combination with Pt12 strongly induced apoptosis in MDA-MB-231 breast cancer cell line. The effect was stronger than treatment with Pt12, cisplatin, anti-MUC1, and anti-MUC1 used with cisplatin. We also observed the highest decrease of MMP and the strongest DNA fragmentation after such a combined treatment. The results obtained from ELISA showed increased concentration of Bax, caspases-8, -9, -3 compared to monotherapy. Our study proved that Pt12 together with anti-MUC1 strongly induced apoptosis in estrogen-negative breast cancer cell line (MDA-MB-231). The apoptosis may go through extrinsic pathway associated with caspase-8 as well as intrinsic pathway connected with caspase-9.