Nanotechnology Platform for the Delivery of Docetaxel and Tyrosine Kinase Inhibitors for HER2-Positive Breast Cancer Therapy.

Identifying novel molecular drug targets continues to be of prime interest in addressing the public health burden of breast cancer in both developed and developing countries alike. In this context, proteomics/pharmacoproteomics approaches offer a new dimension for personalized medicine. We have previously identified differentially expressed proteins with antigenic activity between SKBR3 (ER-, high HER2 expression) and MCF7 (ER+, low HER2 expression) breast cancer cell lines. The aims of the present study were (1) to develop an initial proteome based roadmap of differentially expressed proteins between the two cell lines using two-dimensional electrophoresis (2-DE), and (2) to compare them to those identified by other techniques. SKBR3 and MCF7 cell lysates were subjected to 2-DE and spots of interest were identified by MALDI-TOF/TOF MS. Upregulated proteins (≥2 fold and p<0.05) in MCF7 cells were cellular retinoic acid binding-protein-2, Hsp27, nucleophosmin, electron transfer flavoprotein-α, and profilin-2. In SKBR3 cells, upregulated proteins were RhoGDP dissociation inhibitor-α (RhoGDI-α), voltage-dependent anion channel-2, aldehyde dehydrogeanase-2 (ALDH2), LDH-A, LDH-B, pyrophosphates-1, GAPDH, cathepsin-D preprotein, F–actin capping protein β-subunit, and apolipoprotein A-I binding protein. Differential expression of RhoGDI-α, a molecule with a versatile range of biological activities in different types of breast cancer, was validated using western blotting. In conclusion, these observations using proteomics strategies serve to characterize SKBR3 and MCF7 breast cancer cell lines and offer new insights for personalized medicine on differential expression of putative drug targets between these cancer models. Further studies are warranted to examine the usefulness of SKBR3 cell line as an appropriate model for studying RhoGDI-α activities in HER2+ ER- breast cancer. Finally, we underscore that the findings presented herein also attest to an emerging strand of collaborative proteomics/OMICS studies in developing countries and resource-limited settings towards global personalized medicine, an area of postgenomics data-intensive health research that is in need of greater attention in biomedical literature. Keywords: Breast cancer, developing world OMICS, ER, HER2, personalized medicine and developing countries, pharmacoproteomics, proteomics, RhoGDP dissociation inhibitor-α

Differentially expressed proteins among cancer cell lines fit each cell line as a model for gaining knowledge of heterogeneity in cancer. The aim of our study was to identify differentially expressed proteins between MCF7 (ER+, low HER2 expression) and SKBR3 (ER-, high HER2 expression) cell lines by a proteomic approach. Moreover, a number of proteins in MCF7 cell line were randomly selected and identified. MCF7 and SKBR3 cell lysates were subjected to two dimensional gel electrophoresis and spots of interest were identified by MALDI-TOF/TOF mass spectrometry. Upregulated proteins (≥2 fold and p value &amp;lt;0.05) in MCF7 cells were cellular retinoic acid binding-protein 2, Hsp27, nucleophosmin, electron transfer flavoprotein ≤ subunit, and profilin, and in SKBR3 cells were Rho GDP dissociation inhibitor-α (RhoGDI-α), voltage-dependent anion channel 2, aldehyde dehydrogeanase-2 (ALDH2), LDH- A, LDH-B, pyrophosphates 1, GAPDH, cathepsin D preprotein, and apolipoprotein A-I binding. Most of the identified proteins have been a candidate marker for cancer aggressiveness or drug resistance, but their differential expressions between SKBr3 and MCF7 cells were not known. Apo-lipoprotein binding-protein has not been described in cancer so far. Differential expression of RhoGDI-α was further validated by using western blotting with specific antibody. Further studies are required to clarify the importance of differential expressions of the identified proteins in SKBr3 and MCF7 breast cancer cell line models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4785. doi:1538-7445.AM2012-4785

HER2-positive breast cancers represent approximately 20-25% of all breast cancers and are characterized by an overexpression of the growth factor receptor HER2. Trastuzumab, a monoclonal antibody, is a molecularly targeted therapeutic used in the treatment of this subtype of breast cancer. However, 30% of eligible patients have intrinsic resistance to trastuzumab and approximately 60% of patients who initially responded to this therapeutic, develop resistance within one year. Calcium transporters and modulators are known to be involved in breast cancer and in chemoresistance. However, their role has not been evaluated in HER2-positive trastuzumab resistant breast cancer cells. The aim of this project was to identify possible calcium related proteins associated with trastuzumab resistance. In the first part of this thesis, the expression of Ca2+ transporters and modulators and their role in trastuzumab activity was assessed in the HER2-positive breast cancer cell line SKBR3. Ca2+ signaling profiling was also assessed using fluorescence imaging plate reader (FLIPR) assays. Inhibition of the expression of the Ca2+ channels TPC2, TRPV1 and the Ca2+ channel modulator STIM1 using siRNA decreased SKBR3 cellular proliferation. Silencing of STIM1, the Ca2+ pump SPCA1 and the Ca2+ permeable ion channel TRPM7 increased the anti-proliferative effects of trastuzumab in SKBR3 cells. In the second part of this thesis, trastuzumab resistant and age-matched control cell lines were established from parental SKBR3 cells through seven months of continuous culturing in the presence of trastuzumab. Two trastuzumab treated colonies were selected for their resistance to trastuzumab (RT1 and RT2). Two other colonies were selected from age-matched controls because of their development of de novo resistance to trastuzumab (RV1 and RV2). Two age-matched cell lines that retained their sensitivity to trastuzumab were selected as controls (SV1 and SV2). Levels of mRNA expression of 45 Ca2+ channels, pumps and channel modulators were evaluated using quantitative RT-PCR. An siRNA screen of selected targets to identify targets that when silenced could restore trastuzumab sensitivity was also performed. Additionally Ca2+ signaling profiling and the quantitation of HER2, EGFR and IGF1R protein expression were conducted. All trastuzumab resistant cell lines maintained their overexpression of the HER2 receptor. Significantly increased mRNA levels of the voltage-gated calcium Ca2+ channel CaV3.2 was observed in both de novo resistant cell lines RV1 and RV2 compared to control cell lines SV1 and SV2. Acquired resistant cell lines RT1 and RT2 showed altered sensitivity to the purinergic receptor activator ATP, indicating a possible remodeling of Ca2+ signaling in these trastuzumab resistant cell lines. In the third part of this thesis, specific experiments were conducted to further evaluate two selected targets, the Ca2+ permeable ion channels CaV3.2 and TRPM7 channel. Pharmacological inhibition and silencing of CaV3.2 channel did not reverse trastuzumab resistance. However, CaV3.2 mRNA levels were higher in the basal HER2-positive trastuzumab resistant HCC1569 breast cancer cell line compared to the luminal HER2-positive trastuzumab sensitive SKBR3 cell line. Partial siRNA-mediated silencing of TRPM7 or pharmacological inhibition of TRPM7 channel activity did not reverse trastuzumab resistance in the trastuzumab resistant cell line RV1. However, the TRPM7 kinase inhibitor NH125 was able to promote trastuzumab activity in the trastuzumab resistant cell line RV1. Further studies are required to definitively associate TRPM7 kinase with trastuzumab resistance, given the reported sensitivity of other atypical α-kinases to NH125. In the last part of this thesis publically available data was mined to identify other potential calcium related proteins associated with trastuzumab resistance. These data sets included cDNA microarray analysis of trastuzumab resistant and sensitive SKBR3 cell lines and trastuzumab resistant breast cancer clinical samples and proteomic analysis of trastuzumab resistant and sensitive SKBR3 cell lines. These analyses indicated that the Ca2+ ATPase pump SERCA3 and galectin-3 may be associated with trastuzumab resistance. Results presented in this thesis suggest that the acquisition of trastuzumab resistance may be associated with the expression and/or activity of specific Ca2+ channels and pumps, including SERCA3, CaV3.2 channel and TRPM7 and the Ca2+-related protein galectin-3. Further studies of these proteins may help identify new approaches to reverse trastuzumab resistance and/or identify new biomarkers for predicting trastuzumab sensitivity in HER2-positive breast cancers.

We analyzed whether polypurine hairpins (PPRHs) had the ability to knock down gene expression. These hairpins are formed by two antiparallel purine domains linked by a loop that allows the formation of Hoogsteen bonds between both domains and Watson-Crick bonds with the target polypyrimidine sequence, forming triplex structures. To set up the experimental conditions, the human dhfr gene was used as a model. The PPRHs were designed toward the template strand of DNA. The transfection of the human breast cancer cell line SKBR3 with these template hairpins against the dhfr gene produced higher than 90% of cell mortality. Template PPRHs produced a decrease in DHFR mRNA, protein, and its corresponding enzymatic activity. In addition, the activity of DHFR PPRHs was tested against breast cancer cells resistant to methotrexate, observing high cell mortality. Given the difficulty in finding long polypyrimidine stretches, we studied how to compensate for the presence of purine interruptions in the polypyrimidine target sequence. The stability of PPRH was measured, resulting in a surprisingly long half-life of about 5 days. Finally, to test the generality of usage, template PPRHs were employed against two important genes involved in cell proliferation, telomerase and survivin, producing 80 and 95% of cell death, respectively. Taken together our results show the ability of antiparallel purine hairpins to bind the template strand of double strand DNA and to decrease gene transcription. Thus, PPRHs can be considered as a new type of molecules to modulate gene expression.

Co-amplification and co-overexpression of ErbB2 and Grb7 are frequently found in various cancers, including breast cancer. Biochemical and functional correlations of the two molecules have identified Grb7 to be a pivotal mediator downstream of ErbB2-mediated oncogenesis. However, it remains largely unknown how Grb7 is involve in the ErbB2-mediated tumorigenesis. In this study, we show that Grb7-mediated cell proliferation and growth are essential for the tumorigenesis that occurs in ErbB2-Grb7-overexpressing breast cancer cells. Intrinsically, EGF-induced de novo Grb7 tyrosine phosphorylation/activation recruits and activates Ras-GTPases and subsequently promotes the phosphorylation of ERK1/2, thereby stimulating tumor growth. Furthermore, we also found the anti-tumor effect could be synergized by co-treatment with Herceptin plus Grb7 knockdown in Sk-Br3 breast cancer cells. Our findings illustrate an underlying mechanism by which Grb7 promotes tumorigenesis through the formation of a novel EGFR-Grb7-Ras signaling complex, thereby highlighting the potential strategy of targeting Grb7 as an anti-breast cancer therapy.

ARX788, a novel anti-HER2 antibody-drug conjugate, shows anti-tumor effects in preclinical models of trastuzumab emtansine-resistant HER2-positive breast cancer and gastric cancer.

Human epidermal growth factor receptor 2 (HER2) positive breast cancer, characterized by overexpression of the HER2 protein, is an aggressive type of breast cancer comprising ~20% of diagnosed cases. Although targeted HER2 drugs, such as trastuzumab (TRZ), have reduced mortality by about one third, many patients still experience primary or acquired resistance. Grape seed/skin extracts are popular dietary supplements due to their potent antioxidant and anti-inflammatory properties. However, extracts from muscadine grapes are not extensively studied, and information regarding their therapeutic effects on cancer is limited. The objective of this study was to determine whether a muscadine grape skin and seed extract (MGE) inhibits the proliferation of TRZ-sensitive and -resistant HER2 positive breast cancer cells, and determine if combination treatment with MGE and TRZ has synergistic effects. Treatment with increasing concentrations of MGE for 48h significantly inhibited proliferation of TRZ-sensitive SKBR3 and -resistant HCC1954 human HER2 overexpressing breast cancer cells in a time and dose-dependent manner. HER2 heterodimerization and phosphorylation results in activation of the protein kinase B (AKT) pathway, which regulates breast cancer proliferation and cell survival. Treatment with 20 μg/mL MGE for 24h reduced HER2 protein expression by 52% (p≤0.01) in SKBR3 and 60% (p≤0.01) in HCC1954 cells. AKT activation was also reduced by 63% (p≤0.001) and 65% (p≤0.0001) in SKBR3 and HCC1954 cell lines, respectively. Active AKT stabilizes s-phase kinase-associated protein 2 (SKP2), ultimately leading to increased ubiquitination and degradation of cell-cycle inhibitor cyclin-dependent kinase inhibitor 1B (p27) and its transcription factor forkhead box protein 1 (FOXO1). Treatment with 20 μg/mL MGE for 24h increased total FOXO1 and p27 levels by 4-fold (p≤0.01) and 2.5-fold (p≤0.05), respectively, in SKBR3 cells. In addition, FOXO1 was increased 5-fold (p≤0.0001) and p27 4-fold (p≤0.05) in HCC1954 cells. Furthermore, SKP2 protein expression was reduced by 66% (p≤0.01) in SKBR3 and 73% (p≤0.01) in HCC1954 cells. MGE, in combination with TRZ, was synergistic at five different dose combinations (determined using Chou and Talalay method of synergy), and significantly inhibited proliferation to a greater extent than either agent alone in TRZ-sensitive SKBR3 cells. In summary, treatment with MGE reduced proliferation, inhibited components of the AKT pro-proliferation/survival pathway, and had synergistic effects when combined with TRZ in HER2 positive breast cancer cells. Thus, MGE may serve as an effective therapeutic either administered singly or in combination with targeted therapies for the treatment of HER2 positive breast cancer. Citation Format: Jessica Mackert, Patricia E. Gallagher, E. Ann Tallant. Muscadine grape extract prevents proliferation of trastuzumab-sensitive and -resistant HER2 positive breast cancer cells and has synergistic effects when combined with trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1860.

Abstract #1039 Introduction: The HER family of receptors HER1 (EGFR/ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) is critical in the regulation of cell growth, proliferation and survival. Several studies have linked over-expression of HER1 and HER2 with resistance to chemotherapy and radiation. Clinical trials combining the HER tyrosine kinase inhibitors (TKI) gefitinib (GEF), an inhibitor of HER1, and lapatinib (LAP), a dual inhibitor of HER1 and HER2, with chemotherapeutic agents have not shown synergy comparable to that found in preclinical models. We investigated the effects of chronic treatment of breast cancer cells with TKI inhibitors on DNA damage and repair resulting from chemotherapy and ionising radiation (IR).&amp;#x2028; Material and Methods: Western blotting was used to examine the expression of phosphorylated HER receptors, AKT and MAPK in the SKBR3 and BT474 human breast cancer cell lines. Etoposide (ET), doxorubicin (DOX) and IR induced DNA strand breaks (SB) were measured in the SKBR3 cell line using the single cell gel electrophoresis (comet) assay. Cisplatin-induced (PT) interstrand crosslinks (ICL) were measured with the modified alkaline comet assay. Cells were pre-treated with LAP or GEF for 1 hour or 48 hours, and TKI was replaced at 24-hourly intervals. Cell cycle analysis and intracellular DOX were measured by flow cytometry.&amp;#x2028; Results: In the SKBR3 and BT474 cell lines pHER1, pHER2, pHER3, pAKT and pMAPK were inhibited within 1 hour of exposure to LAP or GEF. However, after 48 hours, pHER3 and pAKT signalling was detected in cells treated with GEF, but not LAP. pHER1, pHER2 and pMAPK were inhibited for at least 72 hours with both drugs. Cell cycle analysis on the SKBR3 cell line showed that 48 hours exposure to either TKI reduced the number of cells in S (12.4±1.1% to 3.2±0.3%), and G2 (16.0±0.55% to 7.1±1.0%) phases of the cell cycle. There was no alteration in intracellular DOX levels at 1 hour or following 48 hours exposure to either TKI. SB were induced by ET, DOX and IR, and ICL by PT, in cells exposed to GEF or LAP for 1 hour. Exposure for 48 hours did not alter the number of ICL or SB induced by PT or IR. In contrast there was a 30-90% reduction in SB following ET treatment (depending on drug concentration) in cells treated with TKI for 48 hours compared with 1 hour treatment. The effect of 48 hours exposure to TKI on DOX-induced SB was more marked with &amp;gt;90% reduction, even at high DOX concentrations. Higher concentrations of ET resulted in increased SB, but increasing concentration of DOX did not alter the level of detectable SB.&amp;#x2028; Discussion: These results suggest why clinical trials of combinations of HER TKI with chemotherapy agents fail to show the magnitude of synergy predicted in preclinical studies. Whilst the cytostatic properties of both LAP and GEF may explain the reduction in DNA damage after 48 hours, this does explain why SB are absent in DOX-treated cells. Further studies investigating kinetics of DNA repair following chemotherapy or radiation in combination with TKI are on-going. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1039.

Background- GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and over-expressed with HER-2 in human breast cancer. Earlier studies found a functional role of GRB7 in breast cancer. The role of GRB7 in HER-2 positive human breast cancer resistant to HER-2 targeted therapy remains unexplored however. Materials and Methods- HCC-1954, 21MT1 and JimT1 are human HER-2 positive breast cancer cell lines that are resistant to trastuzumab and lapatinib treatment. Transient knock down of GRB7 protein expression was achieved with siRNA transfection and stable knock down with lentiviral vector mediated shRNA over-expression. Cell lines transfected with non-targeting siRNA or shRNA serve as negative controls. Knock down of GRB7 protein expression is verified by Western blotting. The growth of human breast cancer cell lines after GRB7 knock down in vitro is measured with the CellTiter Glo assay as well as the Incucyte live cell imaging. Activation status of specific signaling pathways was examined with phospho-specific antibody by immune-blotting and immune-precipitation. To assess the growth promoting function of GRB7 in human breast cancer cell lines in vivo, polyclonal HCC-1954, 21MT1 and JimT1 cells, with GRB7 knock down or their corresponding negative control, were orthotopically injected into the mammary fat pads of female immune-deficient NSG mice. The growth rates of these tumors, measured serially with caliper, and final tumor weights were compared between GRB7 knock down and the negative control. The proliferation rate and apoptosis of these tumors were studied with ki-67 staining and Tunel assay.The effects of GRB7 knock down on signaling were investigated with a proteome profiler receptor tyrosine kinase kit (R&amp;D). The role of signaling molecules differentially activated in the growth of breast cancer cells by GRB7 knock down was examined utilizing siRNA mediated knock down, and antibody and small molecule inhibitors. Results- GRB7 knock down decreased the growth of HCC-1954, 21MT1 and JimT1 cells in vitro and the growth of tumor xenograft these cells formed in animal models. When assayed by ki67 staining and Tunel assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC-1954 cells and increased apoptosis in JimT1 cells. Protein profiling found that tyrosine phosphorylation of candidate signaling molecules was reduced with GRB7 knock down in JimT1 cells. Immuno-blotting and immuno-precipitation experiments were performed to evaluate these effects in other cell lines. The effect of targeting these molecules in breast cancer cell growth by siRNA and inhibitors is being examined. Discussion- GRB7 has essential growth promoting function in therapy resistant HER-2 positive human breast cancer cells. GRB7 knock down has pleiotropic effects on signaling in various cellular contexts. The potential of targeting GRB7 signaling in treating therapy resistant HER-2 positive breast cancer merits further study. Citation Format: Luoh S-W, Wagoner W, Lai X, Hu Z, Chin K, Ramsey B. An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models [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 P2-08-03.

Background &amp; Goals: HER2 is an oncogenic receptor tyrosine kinase (RTK) implicated in several types of human cancer. It is strongly expressed in about 20% of breast cancer (BC), known as HER2-positive BC (HER2-BC), due to gene amplification. HER2 amplification or overexpression is a strong predictor of poor disease prognosis. Several HER2-targeting drugs are available for treating HER2-BC, including monoclonal antibodies trastuzumab (Ttzm) and pertuzumab, T-DM1 (Ttzm coupled to a microtubule inhibitor), and tyrosine kinase inhibitors (TKIs) lapatinib and neratinib. While these agents have greatly improved disease outcomes, primary and acquired drug resistance, including cross-resistance, is common. Overcoming drug resistance remains a major unmet medical need in HER2-BC. Notably, HER2 remains overexpressed in drug-resistant HER2-BC cells. We recently found that recombinant human peptidase D (PEPD), also known as prolidase, strongly downregulates HER2 and EGFR in cancer cells in vitro and in vivo via its binding to HER2 and EGFR. The enzymatic activity of PEPD plays no role in its modulation of HER2 and EGFR, and we subsequently focused on recombinant PEPDG278D, an enzymatically inactive mutant (point mutation at codon 278). Our objective in the present study is to determine whether PEPDG278D is active in preclinical models of HER2-BC resistant to clinically available HER2 inhibitors and if so, its mechanism of action. Results: PEPDG278D was exceedingly effective against HER2-BC cells that are resistant to Ttzm and other HER2 inhibitors. The cells carried activating PIK3CA mutations, low expression of PTEN, and/or cyclin E overexpression. All the cell lines also overexpressed MUC4 which shields HER2 from Ttzm and stabilizes HER2. However, PEPDG278D rapidly bound to and freed HER2 from MUC4, and rapidly disrupted the interaction of HER2 with other RTKs, including MET and IGF1R. PEPDG278D subsequently induced persistent downregulation of HER2 via internalization and lysosomal degradation. PEPDG278D also downregulated EGFR. Thus, PEPDG278D suppresses multiple RTKs either directly or indirectly in HER2-BC cells. In contrast, Ttzm cannot perform any of these functions of PEPDG278D. PEPDG278D was highly active in mouse tumor models of HER2-BC resistant to Ttzm and also enhanced the therapeutic efficacy of paclitaxel. However, adverse effects of PEPDG278D were not detected in the animal studies. Conclusions &amp; Therapeutic Relevance: The therapeutic activity of PEPDG278D highlights that HER2 remains a critical target in drug-resistant HER2-BC. The strong therapeutic activity of PEPDG278D in preclinical models of drug-resistant HER2-BC, which carry clinically relevant molecular changes that confer resistance to current HER2 inhibitors provides the scientific premise for potential evaluation of this recombinant human protein in patients with drug-resistant HER2-BC. Citation Format: Lu Yang, yun Li, Arup Bhattacharya, yuesheng Zhang. A recombinant human protein targeting HER2 overcomes drug resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 336.

Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of oncoprotein HER2, is widely used in standard therapy for women with HER2-positive breast cancer. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis, and apoptosis have been intensively investigated, the effect of trastuzumab on microRNA (miRNA) is unknown. Since miRNA provides a novel layer of gene regulation and may play role in trastuzumab action, we have consequently performed miRNA microarray profiling before and after trastuzumab treatment in both SKBr3 and BT474 breast cancer cells that overexpress HER2. Trastuzumab significantly decreased five human miRNAs and increased three others in SKBr3 cells, whereas in BT474 cells it significantly decreased two miRNAs and increased ten. The only miRNA that shared the same change in both cell lines was miRNA-194 (miR194), which was upregulated following trastuzumab treatment. Upregulation of miR194 by trastuzumab was further validated in vitro and in vivo by quantitative reverse transcription PCR (QRT-PCR) or Northern blotting. miR194 expression in 92 cases of breast cancer and 21 normal breast tissues (all were FFPE samples) was examined by QRT-PCR. miR194 levels were significantly higher in breast cancer samples versus normal tissues (p=0.0003), in estrogen-positive breast cancer samples (p=0.002), and in HER2-negative breast cancer samples (p=0.032). Importantly, expression of precursor miR194 specifically downregulated the protein levels of DNA methytransferase DNMT3A and actin cytoskeleton organizer profilin 2. miR-194 specifically inhibited the luciferase activity of wild type DNMT3A 3’-UTR construct, but not that of mutant vector, indicating that DNMT3A is a direct downstream target of miR-194. Both trastuzumab treatment and overexpression of miR-194 inhibited cell motility of breast cancer cells, which may result from the inhibition of DNMT3A and profilin 2. In summary, trastuzumab treatment upregulates miR194 expression and may exert its therapeutic effects through downregulation of miR194-downstream targets DNMT3A and profilin 2 in HER2-positive breast cancer cells. 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 4000. doi:10.1158/1538-7445.AM2011-4000

Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3'-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'-untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function.

Resistance to trastuzumab is a major issue in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Several potential resistance mechanisms have been investigated, but the results are controversial and no conclusion has been reached. Erythropoietin receptor (EPOR) may function in cell growth, and expressed in various cancer cells. Because the downstream signaling pathways for EPOR and HER2 partially overlapped, we hypothesized that EPOR may play a role in the inhibition effect of trastuzumab and resistance to trastuzumab. Here, we detected the expression of EPOR mRNA and protein in HER2-positive breast cancer cell lines and tissues. EPOR expressed in SKBR3, MDA-MB-453, and UACC-812 cell lines, but not in BT474. Of the 55 HER2-positive cancer tissues, EPOR was positive in 42 samples and highly expressed (H-score ≥ 25) in 24 by immunohistochemistry. The difference between EPOR expression and Ki67 index was significant (P = 0.033), and EPOR expression also positively correlated with higher pathological stage (Spearman correlation coefficient = 0.359; P = 0.007). Exogenous EPO antagonized trastuzumab-induced inhibition of cell proliferation in HER2/EPOR dual-positive breast cancer cells. We then exposed SKBR3 cells to trastuzumab for 4 months to obtain trastuzumab-resistant SKBR3 cell line, which demonstrated higher phosphorylated EPOR level, higher EPO expression and more extracellular secretion than non-resistant parental SKBR3 cells. Downregulation EPOR expression using short hairpin RNA resensitized trastuzumab-resistant cells to this drug, and SKBR3 cells with EPOR downregulation demonstrated attenuated trastuzumab resistance after the same resistance induction. EPOR downregulation plus trastuzumab produced a synergetic action in the inhibition of cell proliferation and invasion in SKBR3 and MDA-MB-453 cell lines. Therefore, EPOR expression may be involved in tumor progression and proliferation in HER2-positive breast cancer. EPO/EPOR contributes to the mechanism of trastuzumab resistance in SKBR3 cell lines, and EPOR downregulation can reverse the resistance to trastuzumab and increase the inhibition effect of this drug.

Background: The Src pathway is known to regulate tumor matastasis and plays a role in epithelial-mesenchymal transition(EMT). Lapatinib, although, has improved clinical outcome for HER2 positive breast cancer patients, acquired resistance to lapatinib remains an important reason influencing its clinical efficacy. Our previous research shows lapatinib acquired resistance HER2 positive breast cancer cell lines, SKBR3-R and BT474-R have EMT phenomenon and multiple pathway activated. Niclosamide is an FDA-approved antihelminthic agent, which is found has cytotoxicity effect on tumor stem cells recently. We wished to determine the effect of niclosamide on EMT and lapatinib resistance cells and investigate niclosamide as a potential therapeutic agent for HER2 positive breast cancer. Methods: SKBR3 and BT474, two HER2 positive breast cancer cell lines, were continuously exposed to increasing doses of lapatinib to establish two stable cell lines resistant to lapatinib, SKBR3-R and BT474-R. Cell proliferation was determined by CCK8 assay. Protein expression was determined by western-blotting. Invasion ability was analyzed by transwell assay. FITC staining flow cytometry (FCM) was conducted to observe the percentage of apoptosis. Results: Both two HER2 positive lapatinib resistant cell lines, SKBR3-R and BT474-R had EMT phenomenon. Niclosamide had a stronger inhibition effect on lapatinib resistant cell lines than parental cell lines, and induced more apoptosis by FCM. Western-blot showed niclosamide could reverse the EMT phenomenon of SKBR3-R and BT474-R with E-cadherin up-regulated and snail,vimentin down-regulated at the concentration of 0.5-1μM. A significant reduction of Src signaling was also confirmed, as well as the downstream Akt and Erk pathway. After adding niclosimide for 48 hours, SKBR3-R and BT474-R’s capability of invasion were inhibited. Conclusion: Our results suggested that niclosamide had a strong cytotoxic effect on HER2 positive lapatinib resistant breast cancer cell lines. The EMT induced by lapatinib resistance could be reversed by niclosamide, associated with the inhibition of Src pathway activation, as well as the downstream pathways. Niclosamide treatment produced reduced levels of invasion, serving as a novel therapeutic way for lapatinib-resistant breast cancer patients. Citation Format: Junjun Liu, Xiaosong Chen, Yan Mao, Kunwei Shen. Niclosamid overcomes epithelial-mesenchymal transition of lapatinib resistance through inhibiting Src activation in HER2 positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-06.

HER2 is overexpressed in approximately 20-25% of breast cancers and defines an aggressive sub-type of the disease. Fortunately for patients with HER2 positive breast cancer, the prognosis has improved in the last 10 years due to the development of HER2 targeted therapies, in particular trastuzumab, the HER2-directed monoclonal antibody. Recent data show that approximately 9.5% of patients with HER2 positive metastatic breast cancer achieve a durable complete response (&gt; 5 years) following trastuzumab-based therapy. However, 90% of these patients still continue to develop progressive disease. These data highlight the need to develop novel therapeutic strategies to overcome both innate and acquired resistance to HER2 directed therapeutics. Lapatinib, a small molecule tyrosine kinase inhibitor of HER2 and EGFR, has efficacy in trastuzumab resistant breast cancer and is approved for the treatment of patients with trastuzumab-refractory HER2 positive metastatic breast cancer. However, in metastatic disease, therapeutic action is often short-lived with most patients developing progressive disease. Thus, HER2 positive tumors can be innately resistant, or acquire resistance, to both trastuzumab and lapatinib. Over the past 2 years, two new HER2 targeted therapies, pertuzumab and T-DM1 have received approval for the treatment of HER2 positive metastatic breast cancer. The pertuzumab monoclonal antibody blocks dimerization of HER2 with other members of the HER family and clinically has been shown to improve overall survival when used in combination with trastuzumab and docetaxel. The novel antibody drug conjugate, T-DM1, which links trastuzumab to the cytotoxic agent emtansine, improves the response of patients with trastuzumab-refractory metastatic breast cancer, compared to the standard of care lapatinib plus capecitabine regimen. A number of second generation irreversible pan-HER tyrosine kinase inhibitors are currently in clinical development, including neratinib, afatinib and dacomitinib. More recently, the utility of HER2-directly therapeutics has been expanded into nonbreast tumors such as gastric cancers that carry the HER2-alteration. Data from ongoing clinical trials will determine if targeting HER2 can also improve the prognosis of these cancer patients.