Breast Cancer Therapeutics Research Articles

Abstract P6-05-02: Targeting vesicular trafficking machinery for breast cancer therapeutics

Abstract The Rab GTPase family is a key regulator of vesicular import, transport and export. Deregulation of vesicular trafficking promotes undesirable intercellular cross talk via exosomes that contribute to cancer pathophysiology. Preclinical and clinical studies underscore a role for exosomes and their cargo in breast cancer development, progression, metastasis, and resistance to therapy. While many studies have approached the role of exosomes in cancer pathophysiology, the 'rules' governing their production and cargo sorting represents a major unexplored gap in knowledge. Exosomes arise from internal budding of specific endosomal compartments. Rab11 GTPase family members comprised of Rab11a, Rab11b and Rab25 are critical regulators of endocytic cargo trafficking specifically of apical and recycling endosomes. Previously, our lab has shown that Rab25 is amplified and contributes to oncogenesis in both ovarian and Luminal B breast cancers. However, Rab25 is lost and when re-expressed ectopically, it inhibits invasion and migration in Claudin Low tumors suggesting a cell-lineage and context dependent function. Importantly, while in Luminal B cancer, Rab25 and its downstream effector RCP (Rab coupling protein), collaborate in driving oncogenesis, in Her2+ cancers, RCP expression is protective. Further our group recently uncovered the presence of a stroma enriched “reactive protein signature” in ER+/Her- group that is indicative of good outcome for patients. Indeed, many of the “reactive signature” proteins are reported in exosomal cargo by others. Taking together, our ongoing study utilizes a panel of breast cancer cell lines representing ER+ve/Her2+; ER+/Her-, ER-/Her2+, and ER-/Her- to evaluate the role of Rab11 family in modulating vesicular trafficking that alters the exosome biology and how that may contribute to tumor- stroma communications. Our preliminary results show that indeed over expression of Rab25 differentially alters size and number of extracellular vesicles in Luminal B cancers when compared to the Claudin Low subtype. Using a novel class of peptides that interrupt the cellular functions 25, we are investigating if Rab25 expression can be associated with an exosomal cargo profile in each subtype of breast cancer, which could then serve as a biomarker for tumor-stroma interaction. Citation Format: Mitra S, Vykoukal J, Jeong K, Mills G. Targeting vesicular trafficking machinery for breast cancer therapeutics [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-05-02.

Aptamer-conjugated DNA nano-ring as the carrier of drug molecules

Due to its predictable self-assembly and structural stability, structural DNA nanotechnology is considered one of the main interdisciplinary subjects encompassing conventional nanotechnology and biotechnology. Here we have fabricated the mucin aptamer (MUC1)˗conjugated DNA nano˗ring intercalated with doxorubicin (DNRA˗DOX) as potential therapeutics for breast cancer. DNRA˗DOX exhibited significantly higher cytotoxicity to the MCF˗7 breast cancer cells than the controls, including DOX alone and the aptamer deficient DNA nano˗ring (DNR) with doxorubicin. Interactions between DOX and DNRA were studied using spectrophotometric measurements. Dose-dependent cytotoxicity was performed to prove that both DNR and DNRA were non-toxic to the cells. The drug release profile showed a controlled release of DOX at normal physiological pH 7.4, with approximately 61% released, but when exposed to lysosomal of pH 5.5, the corresponding 95% was released within 48 h. Owing to the presence of the aptamer, DNRA˗DOX was effectively taken up by the cancer cells, as confirmed by confocal microscopy, implying that it has potential for use in targeted drug delivery.

A Cell-Permeable Stapled Peptide Inhibitor of the Estrogen Receptor/Coactivator Interaction.

We and others have proposed that coactivator binding inhibitors, which block the interaction of estrogen receptor and steroid receptor coactivators, may represent a potential class of new breast cancer therapeutics. The development of coactivator binding inhibitors has been limited, however, because many of the current molecules which are active in in vitro and biochemical assays are not active in cell-based assays. Our goal in this work was to prepare a coactivator binding inhibitor active in cellular models of breast cancer. To accomplish this, we used molecular dynamics simulations to convert a high-affinity stapled peptide with poor cell permeability into R4K1, a cell-penetrating stapled peptide. R4K1 displays high binding affinity for estrogen receptor α, inhibits the formation of estrogen receptor/coactivator complexes, and distributes throughout the cell with a high percentage of nuclear localization. R4K1 represses native gene transcription mediated by estrogen receptor α and inhibits proliferation of estradiol-stimulated MCF-7 cells. Using RNA-Seq, we demonstrate that almost all of the effects of R4K1 on global gene transcription are estrogen-receptor-associated. This chemical probe provides a significant proof-of-concept for preparing cell-permeable stapled peptide inhibitors of the estrogen receptor/coactivator interaction.

Recombinant nanocomposites by the clinical drugs of Abraxane® and Herceptin® as sequentially dual-targeting therapeutics for breast cancer.

Breast cancer greatly threatens the health of women all over the word despite of several effective drugs. Targeted therapy for breast cancer is limited to human epidermal growth factor receptor 2 (HER2). Herceptin®, monoclonal antibody against HER2, is now widely used in HER2(+) breast cancer. Abraxane®, the current gold standard for paclitaxel (PTX) delivery, has shown superiority in breast cancer based on nanoparticle albumin bound technology. Despite these advances, further novel targeted therapy with more improved anti-tumor efficacy for breast cancer is still urgently needed. Here, we report the recombinant nanocomposites (NPs) composed of the above two clinical drugs of Abraxane® and Herceptin® (Abra/anti-HER2), which at first migrates to the tumor region through the unique targeting mechanism of human serum albumin (HSA) of Abraxane®, and sequentially further precisely recognize the HER2(+) breast cancer cells due to Herceptin®. The Abra/anti-HER2 NPs were fabricated by a “one-step” synthesis using EDC/NHS. In vitro analysis of cell viability, apoptosis and cell cycle revealed that Abra/anti-HER2 NPs showed more anti-tumor efficacy against HER2(+) SK-BR-3 cells than Abraxane® at equivalent PTX concentration. In addition, in HER2(+) breast cancer xenograft model, Abra/anti-HER2 NPs significantly inhibited tumor growth with less side effects. Moreover, the properties of more precise target and delayed release of PTX were proved by NIRF imaging. Thus, our results indicate that Abra/anti-HER2 NPs could represent a next-generation sequentially dual-targeting therapeutic agent for HER2(+) breast cancer.

Galectin-1 Influences Breast Cancer Cell Adhesion to E-selectin Via Ligand Intermediaries.

Invasion of other tissues during bloodborne metastasis in part requires adhesion of cancer cells to vascular endothelium by specific fluid shear-dependent receptor-ligand interactions. This study investigates the hypothesis that the adhesion is mediated by ligands shared between endothelial E-selectin and Galectin-1 (Gal-1), both of which are upregulated during inflammation and cancer. Flow chamber adhesion and dynamic biochemical tissue analysis (DBTA) assays were used to evaluate whether Gal-1 modulates E-selectin adhesive interactions of breast cancer cells and tissues under dynamic flow conditions, while immunocytochemistry, immunohistochemistry, western blotting, and fluorescence anisotropy were used to study molecular interactions under static conditions. Dynamic adhesion assays revealed a shear-dependent binding interaction between Gal-1hFc treated breast cancer cells and tissues and E-selectin-coated beads, causing~300% binding increase of the beads compared to negative controls. Immunocyto- and immunohistochemical analyses showed that Gal-1 and E-selectin fluorescent signals colocalized on cells and tissues at~75% for each assay. Immunoprecipitation and Western blotting of Mac-2BP from breast cancer cell lysates revealed that Gal-1 and E-selectin share Mac-2BP as a ligand, while fluorescence anisotropy and circulating tumor cell model systems exhibited competitive or antagonistic binding between Gal-1 and E-selectin for shared ligands, including Mac-2BP. Furthermore, Mac-2BP functional blockade inhibited the effects of Gal-1 on E-selectin binding. In summary, this investigation reveals a shear-dependent interaction between E-selectin and Gal-1 that may be due to intermediation by a similar or shared ligand(s), including Mac-2BP, which may provide a rational basis for development of novel diagnostics or therapeutics for breast cancer.

Mutant p53 as a therapeutic target for the treatment of triple-negative breast cancer: Preclinical investigation with the anti-p53 drug, PK11007

The identification of a targeted therapy for patients with triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. The p53 gene is mutated in approximately 80% of patients with TNBC, and is a potential therapeutic target for patients with this form of breast cancer. The 2-sulfonylpyrimidine compound, PK11007, preferentially decreases viability in p53-compromised cancer cell lines. We investigated PK11007 as a potential new treatment for TNBC. IC50 values for inhibition of proliferation in a panel of 17 breast cell lines by PK11007 ranged from 2.3 to 42.2 μM. There were significantly lower IC50 values for TNBC than for non-TNBC cell lines (p = 0.03) and for p53-mutated cell lines compared with p53 WT cells (p = 0.003). Response to PK11007 however, was independent of the estrogen receptor (ER) or HER2 status of the cell lines. In addition to inhibiting cell proliferation, PK11007 induced apoptosis in p53 mutant cell lines. Using RNAseq and gene ontology analysis, we found that PK11007 altered the expression of genes enriched in pathways involved in regulated cell death, regulation of apoptosis, signal transduction, protein refolding and locomotion. The observations that PK11007 inhibited cell proliferation, induced apoptosis and altered genes involved in cell death are all consistent with the ability of PK11007 to reactivate mutant p53. Based on our data, we conclude that targeting mutant p53 with PK11007 is a potential approach for treating p53-mutated breast cancer, including the subgroup with TN disease.

RBR-type E3 ubiquitin ligase RNF144A targets PARP1 for ubiquitin-dependent degradation and regulates PARP inhibitor sensitivity in breast cancer cells.

Poly(ADP-ribose) polymerase 1 (PARP1), a critical DNA repair protein, is frequently upregulated in breast tumors with a key role in breast cancer progression. Consequently, PARP inhibitors have emerged as promising therapeutics for breast cancers with DNA repair deficiencies. However, relatively little is known about the regulatory mechanism of PARP1 expression and the determinants of PARP inhibitor sensitivity in breast cancer cells. Here, we report that ring finger protein 144A (RNF144A), a RING-between-RING (RBR)-type E3 ubiquitin ligase with an unexplored functional role in human cancers, interacts with PARP1 through its carboxy-terminal region containing the transmembrane domain, and targets PARP1 for ubiquitination and subsequent proteasomal degradation. Moreover, induced expression of RNF144A decreases PARP1 protein levels and renders breast cancer cells resistant to the clinical-grade PARP inhibitor olaparib. Conversely, knockdown of endogenous RNF144A increases PARP1 protein levels and enhances cellular sensitivity to olaparib. Together, these findings define RNF144A as a novel regulator of PARP1 protein abundance and a potential determinant of PARP inhibitor sensitivity in breast cancer cells, which may eventually guide the optimal use of PARP inhibitors in the clinic.

Alternative Splicing in Breast Cancer and the Potential Development of Therapeutic Tools.

Alternative splicing is a key molecular mechanism now considered as a hallmark of cancer that has been associated with the expression of distinct isoforms during the onset and progression of the disease. The leading cause of cancer-related deaths in women worldwide is breast cancer, and even when the role of alternative splicing in this type of cancer has been established, the function of this mechanism in breast cancer biology is not completely decoded. In order to gain a comprehensive view of the role of alternative splicing in breast cancer biology and development, we summarize here recent findings regarding alternative splicing events that have been well documented for breast cancer evolution, considering its prognostic and therapeutic value. Moreover, we analyze how the response to endocrine and chemical therapies could be affected due to alternative splicing and differential expression of variant isoforms. With all this knowledge, it becomes clear that targeting alternative splicing represents an innovative approach for breast cancer therapeutics and the information derived from current studies could guide clinical decisions with a direct impact in the clinical advances for breast cancer patients nowadays.

From Natural Products to Designer Drugs: Development and Molecular Mechanisms Action of Novel Anti-Microtubule Breast Cancer Therapeutics.

Microtubule-targeted drugs (MTDs) have been on the forefront of breast cancer chemotherapy. Classic MTDs, such as paclitaxel and their semisynthetic derivatives, have achieved considerable success in the clinical management of breast neoplasms. In order to improve the specificity and to reduce undesirable, dose-limiting toxicities of these drugs, a plethora of novel compounds are being synthesized and investigated in laboratories worldwide. Due to their crucial roles during cell division, and to the fact that the suppression of their innate 'dynamic instability' can arrest cell cycle progression, microtubules formed an attractive target for cancer chemotherapy. Kadcyla (ado-trastuzumab emtansine), Halaven (eribulin mesylate), and Ixempra (Ixabepilone) are three relatively-novel, microtubule-targeting antibreast cancer drugs. Kadcyla was developed by conjugating a very potent derivative of the natural product maytansine to trastuzumab, a HER2-targeted monoclonal antibody. Kadcyla is a double-edged weapon, that is, it prevents receptor dimerization to inhibit cell proliferation, and then it enters inside the target tumour cell by receptor-mediated endocytosis and ensures death of the cell. Halaven (eribulin mesylate), created by simplifying the structure of the marine sponge-derived molecule Halichondrin B, works primarily by suppressing the growth rates of microtubules and thereby inducing cell cycle arrest and cell death. Ixabepilone, the semisynthetic analogue of epothilone B, suppresses the shortening rates of dynamic microtubules resulting in cell cycle inhibition and cell death. In order to improve the efficacy and reduce drug-induced side effects, novel therapeutic strategies, including liposome-mediated drug delivery, are being investigated.

Phenformin inhibits growth and epithelial-mesenchymal transition of ErbB2-overexpressing breast cancer cells through targeting the IGF1R pathway.

Reports suggest that metformin, a popular anti-diabetes drug, prevents breast cancer through various systemic effects, including insulin-like growth factor receptor (IGFR) regulation. Although the anti-cancer properties of metformin have been well-studied, reports on a more bioavailable/potent biguanide, phenformin, remain sparse. Phenformin exerts similar functional activity to metformin and has been reported to impede mammary carcinogenesis in rats. Since the effects of phenformin on specific breast cancer subtypes have not been fully explored, we used ErbB2-overexpressing breast cancer cell and animal models to test the anti-cancer potential of phenformin. We report that phenformin (25–75 μM) decreased cell proliferation and impaired cell cycle progression in SKBR3 and 78617 breast cancer cells. Reduced tumor size after phenformin treatment (30 mg/kg/day) was demonstrated in an MMTV-ErbB2 transgenic mouse syngeneic tumor model. Phenformin also blocked epithelial-mesenchymal transition, decreased the invasive phenotype, and suppressed receptor tyrosine kinase signaling, including insulin receptor substrate 1 and IGF1R, in ErbB2-overexpressing breast cancer cells and mouse mammary tumor-derived tissues. Moreover, phenformin suppressed IGF1-stimulated proliferation, receptor tyrosine kinase signaling, and epithelial-mesenchymal transition markers in vitro. Together, our study implicates phenformin-mediated IGF1/IGF1R regulation as a potential anti-cancer mechanism and supports the development of phenformin and other biguanides as breast cancer therapeutics.

Global Analysis of miRNA-mRNA Interaction Network in Breast Cancer with Brain Metastasis.

MicroRNAs (miRNAs) have been linked to a number of cancer types including breast cancer. The rate of brain metastases is 10-30% in patients with advanced breast cancer which is associated with poor prognosis. The potential application of miRNAs in the diagnostics and therapeutics of breast cancer with brain metastasis is an area of intense interest. In an initial effort to systematically address the differential expression of miRNAs and mRNAs in primary breast cancer which may provide clues for early detection of brain metastasis, we analyzed the consequent changes in global patterns of gene expression in Gene Expression Omnibus (GEO) data set obtained by microarray from patients with in situ carcinoma and patients with brain metastasis. The miRNA-pathway regulatory network and miRNA-mRNA regulatory network were investigated in breast cancer specimens from patients with brain metastasis to screen for significantly dysregulated miRNAs followed by prediction of their target genes and pathways by Gene Ontology (GO) analysis. Functional coordination of the changes of gene expression can be modulated by individual miRNAs. Two miRNAs, hsa-miR-17-5p and hsa-miR-16-5p, were identified as having the highest associations with targeted mRNAs [such as B-cell lymphoma 2 (BCL2), small body size/mothers against decapentaplegic 3 (SMAD3) and suppressor of cytokine signaling 1 (SOCS1)] and pathways associated with epithelial-mesenchymal transitions and other processes linked with cancer metastasis (including cell cycle, adherence junctions and extracellular matrix-receptor interaction). mRNAs for two genes [HECT, UBA and WWE domain containing 1 (HUWE1) and BCL2] were found to have the highest associations with miRNAs, which were down-regulated in brain metastasis specimens of breast cancer. The change of 11 selected miRNAs was verified in The Cancer Genome Atlas (TCGA) breast cancer dataset. Up-regulation of hsa-miR-17-5p was detected in triple-negative breast cancer tissues in TCGA. Furthermore, a negative correlation of hsa-miR-17-5p with overall survival and phosphatase and tensin homolog (PTEN) and BCL2 target genes was found in TCGA breast cancer specimens. Our findings provide a functionally coordinated expression pattern of different families of miRNAs that may have potential to provide clinicians with a strategy to treat breast cancer with brain metastasis from a systems-rather than a single-gene perspective.

Methotrexate and beta-carotene loaded-lipid Polymer Hybrid Nanoparticles: A Preclinical Study for Breast Cancer

This work was intended to investigate the targeting potential of fructose-tethered lipid-polymeric hybrid nanoparticles (F-BC-MTX-LPHNPs) co-loaded with beta carotene (BC) and methotrexate (MTX) in breast cancer therapeutics and find out the possible protective role of BC on MTX-induced toxicity. F-BC-MTX-LPHNPs were fabricated using self-assembled nano-precipitation technique. Fructose was conjugated on the surface of the particles. The in vitro cytotoxicity, sub-cellular localization and apoptotic activity of F-BC-MTX-LPHNPs were evaluated against MCF-7 breast cancer cells. The antitumor potential of F-BC-MTX-LPHNPs was further studied. Outcomes suggested that F-BC-MTX-LPHNPs induced the highest apoptosis index (0.89) against MCF-7 cells. Following 30 days of treatment, the residual tumor progression was assessed to be approximately 32%, in animals treated with F-BC-MTX-LPHNPs. F-BC-MTX-LPHNPs are competent to selectively convey the chemotherapeutic agent to the breast cancers. Beta carotene ameliorated MTX-induced hepatic and renal toxicity.

Epigenetic Mechanisms of Tamoxifen Resistance in Luminal Breast Cancer.

Breast cancer is one of the most common cancers and the second leading cause of cancer death in the United States. Estrogen receptor (ER)-positive cancer is the most frequent subtype representing more than 70% of breast cancers. These tumors respond to endocrine therapy targeting the ER pathway including selective ER modulators (SERMs), selective ER downregulators (SERDs) and aromatase inhibitors (AIs). However, resistance to endocrine therapy associated with disease progression remains a significant therapeutic challenge. The precise mechanisms of endocrine resistance remain unclear. This is partly due to the complexity of the signaling pathways that influence the estrogen-mediated regulation in breast cancer. Mechanisms include ER modifications, alteration of coregulatory function and modification of growth factor signaling pathways. In this review, we provide an overview of epigenetic mechanisms of tamoxifen resistance in ER-positive luminal breast cancer. We highlight the effect of epigenetic changes on some of the key mechanisms involved in tamoxifen resistance, such as tumor-cell heterogeneity, ER signaling pathway and cancer stem cells (CSCs). It became increasingly recognized that CSCs are playing an important role in driving metastasis and tamoxifen resistance. Understanding the mechanism of tamoxifen resistance will provide insight into the design of novel strategies to overcome the resistance and make further improvements in breast cancer therapeutics.

Abstract LB-022: PTP1B directed therapeutics for HER2 positive breast cancer

Abstract PTP1B, a validated therapeutic target for diabetes and obesity, has a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We define a new mechanism of allosteric inhibition that targets the C-terminal, non-catalytic segment of PTP1B. We present what is to our knowledge the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small-molecule inhibitor MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. Currently, we are testing MSI-1436 in a Phase 1 clinical trial in HER2-positive breast cancer patients, in collaboration with the Monter Cancer Center at Northwell Health formerly NS-LIJ. Note: This abstract was not presented at the meeting. Citation Format: Nava Krishnan. PTP1B directed therapeutics for HER2 positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-022. doi:10.1158/1538-7445.AM2017-LB-022

Abstract 181: Discovery of novel targeted therapeutics for metastatic breast cancer

Abstract Metastatic disease is the primary cause of breast cancer mortality, due to the lack of effective therapy. The Rho GTPase Rac is integral for the promotion of cancer cell migration/invasion, proliferation, and survival. Since metastatic breast cancers often overexpress or exhibit high Rac activity, inhibition of Rac is a viable strategy against metastatic cancer. Recently, we characterized EHop-016, a small molecule that inhibits Rac activity of metastatic breast cancer cells with an IC50 of 1 µM. EHop-016 is 10-100 times more active than previously available Rac inhibitors, and is the first compound shown to inhibit the activation of Rac by the oncogenic GEF Vav. EHop-016 inhibits the activity of the Rac downstream effector p21 activated kinase (PAK), lamellipodia extension, and cell migration of metastatic breast cancer cells. We also reported that EHop-016 at ≥ 25 mg/kg Body Weight (BW) significantly reduced tumor growth, metastasis, and angiogenesis in a mouse model. However, our recent pharmacokinetic study of EHop-016 in a mouse model demonstrated that the bioavailability of Ehop-016 needs to be improved for further pharmacological development. Therefore our hypothesis is that improvement of the EHop-016 structure will provide probes with increased potency against Rac and, therefore, increased bioavailability. Herein we have tested several Ehop-016 derivatives for their effects on breast cancer cell viability and Rac activation. Using MTT assays we found that the Ehop-016 derivatives, HV-107 and HV-118, significantly inhibit the viability of metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435. The effects of HV-107 and HV-118 on the inhibition of Rac activation were tested by ELISA-based Rac activity assays and pulldown assays. Results show that at 250nM, HV-107 inhibits Rac activation by 55%, whereas HV-118 has a similar effect at 100nM in MDA-MB-231 and MDA-MB-435 cells. Taken together, our findings suggest HV-107 and HV-118 as promising Ehop-016 derivatives with potential as anti-metastatic agents, which should be further characterized. This study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and PRINBRE (NIH/NIGMS P20GM103475-13) Pilot Project to LCP. Citation Format: Cristina Del Valle, Eliud Hernández, Cornelis P. Vlaar, Luis A. Cubano, Suranganie Dharmawardhane, Linette Castillo-Pichardo. Discovery of novel targeted therapeutics for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 181. doi:10.1158/1538-7445.AM2017-181

Abstract 2197: Characterization of immunoliposomes as delivery vehicles for the Rac inhibitor Ehop-016 in HER2 type breast cancer

Abstract Due to the critical need for improved therapeutics against advanced Stage IV breast cancer, we developed and pre-clinically validated the small molecule Ehop-016 as a potential anti-metastatic drug. Ehop-016 is a small molecule inhibitor of the interaction Rac with the guanine nucleotide exchange factor (GEF) Vav2, which consequently decreases invasive actin structures and cellular migration. In a human epidermal growth factor receptor 2 (HER2) type breast cancer model in nude mice, Ehop-016 inhibits mammary tumor growth, angiogenesis, and metastasis. Moreover, Ehop-016 reduces HER2++ cancer cell viability and migration, individually or in combination with anti HER2 or EGFR directed therapeutics. By inhibiting the key signaling intermediate Rac, Ehop-016 has the potential to block signaling downstream of HER2 and EGFRs, as well as signaling via the myriad of receptors that activate Rac, and inhibit cancer progression. Our new results demonstrate that Ehop-016 inhibits both cancer cells and leukocytes in the tumor microenvironment, as well as cytokine levels (IL-6) in mouse plasma. Even though these characteristics are desirable in anticancer agents, systemic therapy with Ehop-016 may adversely affect normal immune system function. Therefore, a strategy was developed for the targeted delivery of Ehop-016 into mammary tumors, using nanoliposomes containing the current breast cancer therapeutic, Trastuzumab, a monoclonal antibody directed at the HER2. We tested various nanoliposome formulations as targeted delivery systems for Ehop-016. First, the encapsulation efficiency of Ehop-016 loaded liposomes was tested using different drug loading methods. To obtain HER2 targeted liposomes, we prepared Trastuzumab-lipid conjugates and incorporated them into the Ehop-016 containing liposomes. The binding, internalization and selectivity of our HER2 targeted delivery system was verified in human metastatic breast cancer cell lines, by using fluorescence microscopy to assess the co-staining of the lipid bilayer and Trastuzumab. In order to evaluate the effectiveness and selectivity of the immunoliposomes in delivering Ehop-016 and inhibiting Rac activity, pull-down assays were performed to detect active Rac-GTP in cells treated with Ehop-016 alone or Trastuzumab-conjugated liposomes with Ehop-016. Results show that the Trastuzumab-conjugated liposomes are more selective at binding to HER2+ breast cancer cells and inhibiting Rac, than treatment with Ehop-016 alone. In summary, our novel delivery system containing Ehop-016 and Trastuzumab has been shown to be effective in breast cancer cells, and needs to be validated in animal models of metastatic breast cancer. The development of this targeted delivery system has implications, not only in the field of breast cancer therapeutics, but may be adapted for the treatment of other diseases, and used to overcome the side effects of current treatments. Citation Format: Michael J. Rivera-Robles, Pablo E. Vivas-Mejia, Linette Castillo-Pichardo, Cornelis Vlaar, Eliud Hernández, Suranganie Dharmawardhane. Characterization of immunoliposomes as delivery vehicles for the Rac inhibitor Ehop-016 in HER2 type breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2197. doi:10.1158/1538-7445.AM2017-2197

Validation and clinical application of MammaPrint ® in patients with breast cancer

Abstract Background In recent years, advances in molecular biology have resulted in innovations in breast cancer diagnostics and therapeutics. The development of genomics has revolutionised our understanding of this disease. MammaPrint® was developed as a diagnostic tool to predict risk of breast cancer recurrence using the expression of 70 genes altering the clinicopathologic paradigm of selection of patients for adjuvant cytotoxic chemotherapy. Materials and methods A study of stages I and II breast cancer patients on whom the MammaPrint® genomic assay was performed. Results The use of the MammaPrint® assay was a decisive factor for the recommendation of adjuvant treatment with chemotherapy and/or hormone therapy in patients with high risk for relapse. In our group, the patients with low-risk have not presented local or systemic recurrences. Discussion The determination of the genetic characteristics and its alterations in breast cancer, is fundamentally important for a better identification of risk, as well as a better selection of cancer therapy. Conclusion MammaPrint® is an effective study to determine risk of recurrence of in early stage breast cancer.

MiR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

The B-lymphoma Moloney murine leukemia virus insertion region-1 protein (BMI1) acts as an oncogene in various cancers, including breast cancer. Recent evidence suggests that BMI1 is rapidly recruited to sites of DNA double strand breaks where it facilitates histone H2A ubiquitination and DNA double strand break repair by homologous recombination. Here we show that miR-15a and miR-16 expressionis decreased during the initial period after DNA damage where it would otherwise down-regulate BMI1, impairing DNA repair. Elevated miR-15a and miR-16 levels down-regulated BMI1 and other polycomb group proteins like RING1A, RING1B, EZH2 and also altered the expression of proteins associated with the BMI1 dependent ubiquitination pathway. Antagonizing the expression of miR-15a and miR-16, enhanced BMI1 protein levels and increased DNA repair. Further, overexpression of miR-15a and miR-16 sensitized breast cancer cells to DNA damage induced by the chemotherapeutic drug doxorubicin. Our results suggest that miR-15a and miR-16 mediate the down-regulation of BMI1, which impedes DNA repair while elevated levels can sensitize breast cancer cells to doxorubicin leading to apoptotic cell death. This data identifies a new target for manipulating DNA damage response that could impact the development of improved therapeutics for breast cancer.

Targeting mutant p53 with PK11007: A new approach for the treatment of patients with triple-negative breast cancer?

e14099 Background:The identification of a targeted therapy for patients with triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. Since the p53 gene is mutated in approximately 80% of TNBC patients, it is a potential therapeutic target for this form of breast cancer. PK11007 is a 2-sulfonypyrimidine that stabilizes and reactivates mutant p53 (Bauer et al, PNAS 2016). The compound recently was reported to preferentially decrease viability in p53-compromised cancer cells. The aim of this investigation was to evaluate PK11007 as a potential new treatment for TNBC. Methods: Cell viability was determined using the MTT assay. Apoptosis was detected using Annexin V Apoptosis Detection Kit. Migration was determined by Transwell migration assay. Knockdowns of p53 protein were carried out using predesigned Flexitube sequences (Qiagen). Results: IC50 values for inhibition of proliferation by PK11007 in the panel of 17 breast cell lines ranged from 2.3 to 42.2 μM. There were significantly lower IC50values for TNBC than for non-TNBC cell lines (p = 0.03) and for p53-mutated cell lines compared with p53 WT cells (p = 0.003). Response to PK11007 however, was independent of ER or HER2 status of the cells. In addition, PK11007 induced apoptosis and inhibited migration in p53 mutant cell lines. Using RNAseq and gene ontogeny analysis, we found that PK11007 altered the expression of genes enriched in pathways involved in regulated cell death, regulation of apoptosis, signal transduction, protein refolding and locomotion. To establish if PK11007 acts by targeting mutant p53, we used siRNA to knockdown p53 in 3 p53-mutated TNBC cell lines. Reduction in p53 protein levels resulted in a significant decrease in the growth inhibitory effects of PK11007, in all 3 cell lines investigated, suggesting that PK11007 mediates growth inhibition via p53. The observations that PK11007 inhibited cell growth, induced apoptosis, blocked cell migration and altered genes involved in cell death, are all consistent with the ability of PK11007 to activate mutant p53. Conclusions: Based on our data, we conclude that targeting mutant p53 with PK11007 is a potential approach for treating p53-mutated TNBC.

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics

Estradiol (E2) and the oestrogen receptor-alpha (ERα) signalling pathway play pivotal roles in the proliferative activity of breast cancer cells. Recent findings show that the brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERα activity-regulator synthetic peptide (ERAP: 165–177 amino acids), derived from α-helical BIG3 sequence, resulted in a significant anti-tumour effect. However, the duration of this effect was very short for viable clinical application. We developed the chemically modified ERAP using stapling methods (stapledERAP) to improve the duration of its antitumour effects. The stapledERAP specifically inhibited the BIG3-PHB2 interaction and exhibited long-lasting suppressive activity. Its intracellular localization without the membrane-permeable polyarginine sequence was possible via the formation of a stable α-helix structure by stapling. Tumour bearing-mice treated daily or weekly with stapledERAP effectively prevented the BIG3-PHB2 interaction, leading to complete regression of E2-dependent tumours in vivo. Most importantly, combination of stapledERAP with tamoxifen, fulvestrant, and everolimus caused synergistic inhibitory effects on growth of breast cancer cells. Our findings suggested that the stapled ERAP may be a promising anti-tumour drug to suppress luminal-type breast cancer growth.