HER2 In Breast Cancer Cells Research Articles

Red-Fluorescent Pt Nanoclusters for Detecting and Imaging HER2 in Breast Cancer Cells.

Overexpression of human epidermal growth factor receptor 2 (HER2) is associated with more frequent cancer recurrence and metastasis. Sensitive sensing of HER2 in living breast cancer cells is crucial in the early stages of cancer and to further understand its role in cells. Biomedical imaging has become an indispensable tool in the fields of early cancer diagnosis and therapy. In this study, we designed and synthesized platinum (Pt) nanocluster bionanoprobes with red emission (Ex/Em = 535/630 nm) for fluorescence imaging of HER2. Our Pt nanoclusters, which were synthesized using polyamidoamine (PAMAM) dendrimer and preequilibration, exhibited approximately 1% quantum yield and possessed low cytotoxicity, ultrasmall size, and excellent photostability. Furthermore, combined with ProteinA as an adapter protein, we developed Pt bionanoprobes with minimal nonspecific binding and utilized them as fluorescent probes for highly sensitive optical imaging of HER2 at the cellular level. More importantly, molecular probes with long-wavelength emission have allowed visualization of deep anatomical features because of enhanced tissue penetration and a decrease in background noise from tissue scattering. Our Pt nanoclusters are promising fluorescent probes for biomedical applications.

Dose-dependent cell cycle arrest and apoptosis in HER2 breast cancer cells by177Lu-CHX-A"-DTPA-Trastuzumab.

Trastuzumab is a Food and Drug Administration-approved humanized monoclonal antibody which targets the extracellular domain of human epidermal growth factor receptor 2 (HER2) receptor overexpressed on HER2-positive breast cancer cells. The combination of Lutetium-177 (177 Lu) (t½= 6.7 days, Eβmax497 keV (78.6%) and trastuzumab makes it a suitable targeting agent for radioimmunotherapy. In preclinical and clinical studies,177 Lu-Trastuzumab has proven to be effective for the treatment of HER2-positive malignancies such as breast and ovarian cancer. In this study, we report the mechanism of action of177 Lu-CHX-A"-diethylenetriaminepentaacetic acid (DTPA)-trastuzumab at the cellular and molecular level by performing various in vitro assays in HER2-positive MDA-MB-453 breast cancer cells. Trastuzumab was conjugated to the bifunctional chelating agent (BFCA) para-isothiocyanatobenzyl-DTPA and radiolabeled with177 Lu. In vitro cell binding studies were carried out in MDA-MB-453 cells to confirm the specificity of the complex toward the receptor. Cellular toxicity, cell cycle, and cell death analysis were also performed for exploring the potential of the radioimmunoconjugate at cellular and molecular level. In vitro cell binding studies showed a maximum binding of 10.7 ± 0.1% which reduced to 2.9 ± 0.1% on coincubation with unlabeled antibody. Our study revealed that the cellular toxicity was dose dependent, and mode of cell death was predominantly by apoptosis. The radioimmunoconjugate retarded the cell in the S phase of cell cycle with two-fold increase in G2/M arrest which justifies the enhanced apoptosis at higher doses. The study revealed that the formulation can execute a dose-dependent cellular toxicity through induction of apoptosis.

Intracellular Delivery of an Antibody Targeting Gasdermin-B Reduces HER2 Breast Cancer Aggressiveness.

Gasdermin B (GSDMB) overexpression/amplification occurs in about 60% of HER2 breast cancers, where it promotes cell migration, resistance to anti-HER2 therapies, and poor clinical outcome. Thus, we tackle GSDMB cytoplasmic overexpression as a new therapeutic target in HER2 breast cancers. We have developed a new targeted nanomedicine based on hyaluronic acid-biocompatible nanocapsules, which allow the intracellular delivery of a specific anti-GSDMB antibody into HER2 breast cancer cells both in vitro and in vivo. Using different models of HER2 breast cancer cells, we show that anti-GSDMB antibody loaded to nanocapsules has significant and specific effects on GSDMB-overexpressing cancer cells' behavior in ways such as (i) lowering the in vitro cell migration induced by GSDMB; (ii) enhancing the sensitivity to trastuzumab; (iii) reducing tumor growth by increasing apoptotic rate in orthotopic breast cancer xenografts; and (iv) diminishing lung metastasis in MDA-MB-231-HER2 cells in vivo. Moreover, at a mechanistic level, we have shown that AbGB increases GSDMB binding to sulfatides and consequently decreases migratory cell behavior and may upregulate the potential intrinsic procell death activity of GSDMB. Our findings portray the first evidence of the effectiveness and specificity of an antibody-based nanomedicine that targets an intracellular oncoprotein. We have proved that intracellular-delivered anti-GSDMB reduces diverse protumor GSDMB functions (migration, metastasis, and resistance to therapy) in an efficient and specific way, thus providing a new targeted therapeutic strategy in aggressive HER2 cancers with poor prognosis.

Abstract 4268: Protein folding pathway modulation upon Hsp70 inhibition in cancer cells

Abstract Cancer cells experience acute stress conditions such as low oxygen and energy, and exposure to toxic agents. To survive proliferate without accumulating toxic misfolded proteins, cancer cells constantly modulate protein homeostasis. Thus, it is not surprising that molecular chaperones, like Hsp70, as well as protein degradation pathways are upregulated in cancer cells compared to their normal counterparts. These data suggest that chaperones are potential targets for cancer therapy. We previously demonstrated the dependence of patient-driven rhabdomyosarcoma cell survival on cytoplasmic Hsp70 activity, thanks to the use of a specific Hsp70 inhibitor, MAL3-101. In particular, we discovered that MAL3-101-mediated Hsp70 inhibition activates the PERK arm of the unfolded protein response (UPR) that results in CHOP-dependent cell death (Sabinis et al., 2016). Moreover, by taking advantage of a MAL3-101-resistant cell line (RMS13-R), we recently determined which compensatory mechanism alters MAL3-101-driven cell death. We found that both endoplasmic reticulum-associated degradation (ERAD) and autophagy are upregulated in RMS13-R cells, underlying increased demand on two protein degradation pathways upon inhibition of Hsp70. However, only autophagy inhibition—but not inhibition of ERAD—re-sensitized RMS13-R cells to Hsp70 inhibition, suggesting that autophagy was the key compensatory mechanism for Hsp70 inhibition. Autophagy was further induced by MAL3-101 treatment in RMS13-R cells, as evidenced by an increase in the messages and proteins corresponding to key autophagy components as well as to the accumulation of autophagic-like structures detected by electron microscopy (Sannino et al., 2018). These data highlight a pro-survival role for autophagy induction upon exposure to an Hsp70 inhibitor in cancer, and provide a link between Hsp70, proteasomal degradation, UPR, and autophagy in rhabdomyosarcoma. We next asked if other cancer types might be sensitive to Hsp70 inhibition, and we investigated the potential benefit of combined treatment with autophagy and/or proteasome inhibitors together with MAL3-101. Specifically, we are investigating the effects of Hsp70 inhibition in breast cancer cells, a cancer type in which higher levels of Hsp70 correlate to increased metastasis and poor prognosis in patients. Our preliminary data suggest that HER2-expressing cells are less sensitive to MAL3-101-mediated Hsp70 inhibition and combinatory treatments including, MAL3-101 and autophagy inhibitors promoted HER2-breast cancer cell death. Further investigations will reveal the potential carcinogenic role of Hsp70 inhibitors in breast cancer treatment and highlight which pathways reduce proteotoxicity in different breast cancer subtypes. Citation Format: Sara Sannino, Christopher J. Guerriero, Amit J. Sabnis, Jeffrey J. Bridsky. Protein folding pathway modulation upon Hsp70 inhibition in cancer cells [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 4268.

Crumbs protein homolog 3 (CRB3) expression is associated with oestrogen and progesterone receptor positivity in breast cancer.

The crumbs protein homolog 3 (CRB3) regulates the tight junction to help maintain epithelial polarity. Altered CRB3 expression was associated with carcinogenesis of epithelial cells. This study detected CRB3 expression in 192 cases of breast cancer tissues and in the Molecular Taxonomy of Breast Cancer International Consortium (Metabric) and The Cancer Genome Atlas (TCGA) datasets for association with triple negative breast cancer (TNBC) phenotypes. The in vitro experiments confirm the ex vivo data. The data showed that levels of both CRB3 mRNA and protein were associated with TNBC phenotypes, ie, 41.1% (39/95) of ER+ breast cancer was CRB3‐positive, whereas 26.9% (25/93) ER– tumour was CRB3‐positive (P = 0.046). Moreover, 47.6% (30/63) of PR+ breast cancer was CRB3‐positive vs 28.4% (33/116) PR‐ tumours positive for CRB3 (P = 0.013). In addition, 40.1% (27/66) of ER+/PR+ tumour was CRB3‐positive, but only 22.4% (19/85) of TNBC showed CRB3 expression (P = 0.048). Indeed, levels of CRB3 mRNA were higher in non‐TNBC than TNBC in both Metabric (P = 3.682e‐10) and TCGA datasets (P = 2.501e‐07). The in vitro data showed that CRB3 expression was higher in luminal (MCF7 and T47D) than in HER2 (MDA‐MB‐453 and SK‐BR‐3) and basal (MDA‐MB‐231 and BT‐549) breast cancer cell lines. More interestingly, ERα regulated expression of CRB3 protein in MCF7 and BT‐549 cells and ERα expression was associated with CRB3 expression in breast cancer tissues specimens. This study demonstrated that ERα could be a novel regulator for CRB3 expression in breast cancer.

PYK2 promotes HER2-positive breast cancer invasion

BackgroundMetformin, a biguanide, is one of the most commonly prescribed treatments for type 2 diabetes and has recently been recommended as a potential drug candidate for advanced cancer therapy. Although Metformin has antiproliferative and proapoptotic effects on breast cancer, the heterogenous nature of this disease affects the response to metformin leading to the activation of pro-invasive signalling pathways that are mediated by the focal adhesion kinase PYK2 in pure HER2 phenotype breast cancer.MethodsThe effect of metformin on different breast cancer cell lines, representing the molecular heterogenicity of the disease was investigated using in vitro proliferation and apoptosis assays. The activation of PYK2 by metformin in pure HER2 phenotype (HER2+/ER−/PR-) cell lines was investigated by microarrays, quantitative real time PCR and immunoblotting. Cell migration and invasion PYK2-mediated and in response to metformin were determined by wound healing and invasion assays using HER2+/ER−/PR- PYK2 knockdown cell lines. Proteomic analyses were used to determine the role of PYK2 in HER2+/ER−/PR- proliferative, migratory and invasive cellular pathways and in response to metformin. The association between PYK2 expression and HER2+/ER−/PR- patients’ cancer-specific survival was investigated using bioinformatic analysis of PYK2 expression from patient gene expression profiles generated by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) study. The effect of PYK2 and metformin on tumour initiation and invasion of HER2+/ER−/PR- breast cancer stem-like cells was performed using the in vitro stem cell proliferation and invasion assays.ResultsOur study showed for the first time that pure HER2 breast cancer cells are more resistant to metformin treatment when compared with the other breast cancer phenotypes. This drug resistance was associated with the activation of PTK2B/PYK2, a well-known mediator of signalling pathways involved in cell proliferation, migration and invasion. The role of PYK2 in promoting invasion of metformin resistant HER2 breast cancer cells was confirmed through investigating the effect of PYK2 knockdown and metformin on cell invasion and by proteomic analysis of associated cellular pathways. We also reveal a correlation between high level of expression of PYK2 and reduced survival in pure HER2 breast cancer patients. Moreover, we also report a role of PYK2 in tumour initiation and invasion-mediated by pure HER2 breast cancer stem-like cells. This was further confirmed by demonstrating a correlation between reduced survival in pure HER2 breast cancer patients and expression of PYK2 and the stem cell marker CD44.ConclusionsWe provide evidence of a PYK2-driven pro-invasive potential of metformin in pure HER2 cancer therapy and propose that metformin-based therapy should consider the molecular heterogeneity of breast cancer to prevent complications associated with cancer chemoresistance, invasion and recurrence in treated patients.

PO-1076 Concomitant association of T-DM1 and radiation: An in vitro study on HER2 breast cancer cells

PO-1076 Concomitant association of T-DM1 and radiation: An in vitro study on HER2 breast cancer cells

Anticancer effects of curcumin in luminal B and HER2 breast cancer cell line models

Anticancer effects of curcumin in luminal B and HER2 breast cancer cell line models

MiR-342-5p as a Potential Regulator of HER2 Breast Cancer Cell Growth

HER2 positive Breast Cancers (BC) have aggressive behavior and poor prognosis. Previously, we have identified miR-342-5p as an upstream regulator of HER2 signaling, as well as inhibitor of HER2 positive BC cell line growth. Here, we aimed to further investigate the molecular mechanisms behind miR-342-5pinduced HER2 pathway deregulation. Two HER2 amplified breast cancer cell lines were transiently transfected with miR-342-5p mimic or negative control, and gene expression was analyzed by Agilent microarrays. Three clinical datasets with BC patients were used to identify correlations between candidate genes and miR-342- 5p, and associations with survival. Pathway analyses of all deregulated genes revealed a significant suppression of the HER2 downstream pathways ERK/MAPK and SAPK/JNK, whereas the miR-342-5p predicted target genes were enriched for pathways associated with cell motility.Biological functions linked to mitochondrial stability were ranked among the top toxicological functions in both gene lists. Among the most deregulated genes, Cytochrome B5 Reductase 3 (CYB5R3) and Rap Guanine Nucleotide Exchange Factor 6 (RAPGEF6) significantly anticorrelated and correlated, respectively, with miR-342-5p in all three clinical BC datasets. Low CYB5R3 levels and high RAPGEF6 levels were significantly associated with survival, although this was not directly associated with HER2 expression. Our data suggest that miR-342-5p overexpression in HER2 positive BC cell lines elicits broad effects on HER2 downstream signaling, cell motility and mitochondrial stability. Together these effects may render cells less proliferative and more sensitive to cellular stress.

Abstract P6-21-14: Targeting Rac/Cdc42 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer

Abstract Breast cancer metastasis, when cancer cells move and establish tumors in distant organs, confounds treatment options. Therefore, there is an unmet need for targeted therapeutics to address metastasis, especially in the intractable HER2 positive (+) breast cancer type. HER2 (+) breast cancer is treated with the monoclonal HER2 antibody trastuzumab and the HER2/EGFR targeting small molecule lapatinib. However, ˜20% of early stage breast cancer patients and ˜70% of patients with metastatic disease are resistant to trastuzumab. Trastuzumab-resistant breast cancers circumvent HER2 inhibition via bypass signaling, which includes the PI3-K/Akt/mTOR pathway. HER2/EGFR and PI-3K/Akt signaling activate the Rho GTPases Rac and Cdc42, and overexpression of Rac or its downstream effector p21-activated kinase (PAK) significantly diminishes response to anti HER2 therapy and patient survival. Therefore, targeting Rac and Cdc42, pivotal regulators of cancer cell migration/invasion, and thus, metastasis, is a viable option to overcome HER2/EGFR therapy resistance. First, we designed and developed the Rac inhibitor Ehop-016 (US patents 8,884,006 B2, 9,278,956B1), which inhibits Rac activation with an IC50 of 1 μM and reduces breast cancer growth and metastasis in mouse models of trastuzumab resistant HER2 (+) breast cancer. A more potent dual Rac and Cdc42 inhibitor, MBQ-167 (US Patent 9,981,980 B2), inhibits Rac and Cdc42 activation with IC50s of 103 nM and 78 nM, respectively. In vivo, MBQ-167 inhibits trastuzumab resistant HER2 (+) mammary tumor growth and metastasis by ˜90-100% (Humphries-Bickley et al. 2017, Mol Cancer Therap). Therefore, Rac/Cdc42 inhibition blocks cell proliferation, cell cycle progression, migration, and induces apoptosis to ultimately impede tumor growth and metastasis in trastuzumab resistant HER2 (+) breast cancer models. To further investigate the role of Rac/Cdc42 inhibitors in overcoming HER2 therapy resistance, we developed a lapatinib resistant variant of the SKBR3 HER2 (+) breast cancer cell line and found that Rac was overexpressed and over activated in the therapy resistant variant compared to the parental cells. Accordingly, the Rac/Cdc42 inhibitors overcame lapatinib resistance by decreasing cell viability and inducing apoptosis in parental and SKBR3 therapy resistant variants. Pharmacokinetic analysis in mouse plasma demonstrated that the bioavailability of EHop-016 and MBQ-167 is ˜30% with a half-life of 3-4 h. Therefore, to increase the bioavailability of Rac/Cdc42 inhibitors and to facilitate sensitization of trastuzumab therapy, we designed and developed novel nanoliposomal formulations containing Rac/Cdc42 inhibitors with trastuzumab on the outer surface to target HER2 (+) breast cancer cells. This data demonstrates the utility of developing Rac/Cdc42 inhibitors as mono or combined therapy with current targeted therapeutics for HER2 (+) breast cancer. Citation Format: Rivera-Robles MJ, Medina-Vlazquez JI, Borrero-Garcia LD, Velazquez-Vega LE, Maldonado MdM, Ruiz-Calderon J, Castillo-Pichardo L, Vivas-Mejia P, Dharmawardhane S. Targeting Rac/Cdc42 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-21-14.

Effectiveness of EGFR/HER2-targeted drugs is influenced by the downstream interaction shifts of PTPIP51 in HER2-amplified breast cancer cells

Breast cancer is the most common female cancerous disease and the second most cause of cancer death in women. About 20–30% of these tumors exhibit an amplification of the HER2/ErbB2 receptor, which is coupled to a more aggressive and invasive growth of the cancer cells. Recently developed tyrosine kinase inhibitors and therapeutic antibodies targeting the HER2 receptor improved the overall survival time compared with sole radio- and chemotherapy. Upcoming resistances against the HER2-targeted therapy make a better understanding of the receptor associated downstream pathways an absolute need. In earlier studies, we showed the involvement of Protein Tyrosine Phosphatase Interacting Protein 51 (PTPIP51) in the mitogen-activated protein kinase (MAPK) pathway. The MAPK pathway is one of the most frequently overactivated pathways in HER2-amplified breast cancer cells. This study is aimed to elucidate the effects of four different TKIs on the interactome of PTPIP51, namely with the receptors EGFR and HER2, 14-3-3/Raf1 (MAPK pathway), its regulating enzymes, and the mitochondria-associated interaction partners in HER2 breast cancer cell lines (SK-BR3 and BT474) by using the Duolink proximity ligation assay, immunoblotting and knockdown of PTPIP51. Inhibition of both EGFR and HER2/ErbB2R shifted PTPIP51 into the MAPK pathway, but left the mitochondria-associated interactome of PTPIP51 unattended. Exclusively inhibiting HER2/ErbB2 by Mubritinib did not affect the interaction of PTPIP51 with the MAPK signaling. Selective inhibition of HER2 induced great alterations of mitochondria-associated interactions of PTPIP51, which ultimately led to the most-effective reduction of cell viability of SK-BR3 cells of all tested TKIs. The results clearly reveal the importance of knowing the exact mechanisms of the inhibitors affecting receptor tyrosine kinases in order to develop more efficient anti-HER2-targeted therapies.

PO-355 Antitumor activity of HSA-miR-X in HER2 positive cells is mediated by the regulation of AKT and ERK

IntroductionHER2 overexpression has been reported in approximately 30% of human breast tumours, and is associated with poor clinical outcome and recurrent disease. Herceptin is a humanised monoclonal antibody targeting HER2. PI3K-AKT is a downstream effector of the HER2 signalling pathway. microRNAs (miRNA) are small non-coding RNAs of 19–22 nucleotides that can negatively regulate gene expression at post-transcriptional levels. Herceptin plays an important role in treating metastatic breast cancer by targeting Erbb2 (HER2), therefore, combining Herceptin with miRNAs might enhance its antitumor activity. In this study the antitumor activity of hsa-miR-X, which was found to be Herceptin–responsive by our group, with Herceptin in HER2 positive breast cancer cell lines was identified in SK-Br-3 and BT-474 cells.Material and methodsWe investigated the synergistic effect of Herceptin and miRNA mimic combination on tumorigenic activities of the cells. To check its role in migration and invasion, wound healing and invasion assays were performed respectively. Cell invasion was monitored over a period of 24 hours by xCELLigence real-time cell analyzer measuring impedance-based signals. Furthermore, viability of breast cancer cells was measured by the WST1 assay after transfection with hsa-miR-X miRNA mimic. We measured the expression of AKT and ERK in HER2 positive cells treated with miR-X mimic plus Herceptin by Western blot analysis.Results and discussionsWe showed that increased expression of hsa-miR-X significantly inhibits migration and invasion of HER2 (+) breast cancer cells. Additionally, western blot experiments demonstrated that transfection of hsa-miR-X with Herceptin treatment decreased the protein level of AKT and ERK in these cells.ConclusionIt may be concluded that hsa-miR-X may increase the activity of the drug by pulling down its effective concentration and regulating PI3K-AKT pathway in HER2 positive cells. hsa-miR-X could decrease proliferation, motility and invasion via inhibiting Akt phosphorylation and may represent a new strategy for treating HER2 positive breast cancer.

Abstract 825: Identification of molecular therapeutic targets that enhance the antitumor activity of neratinib in breast cancer cells

Abstract Background: Aberrant expression of members of the ErbB family of receptor tyrosine kinases and its downstream pathways is known to play pivotal roles in breast cancer malignancy. HER2-positive breast cancer has an aggressive nature that is driven by HER2 gene amplification. Inflammatory breast cancer is a rare but the most aggressive clinical subtype of breast cancer. Triple-negative breast cancer is an aggressive receptor subtype of breast cancer. More than 30% of cases of each of these groups express EGFR. Emerging therapeutic resistance continues to be a challenge in recurrent and/or metastatic disease. Neratinib is a potent irreversible EGFR, HER2, and HER4 tyrosine kinase inhibitor that blocks signal transduction generated via these receptors. In this study, we sought to identify the optimal targets in different subtypes of breast cancer and synergistic partners to maximize the antitumor effect of neratinib against breast cancer cells in vitro and in vivo. Methods: Expression of EGFR and HER2 was assessed via Western blot in 31 breast cancer cell lines, including HER2-targeted drug-resistant cell lines (14 triple-negative breast cancer and 10 HER2-positive). The antiproliferative effects of neratinib were measured using cell proliferation and anchorage-independent colony-formation (soft agar) assays. A reverse-phase protein array was used to profile and validate the signaling networks induced by neratinib-based treatment. To identify the synergistic targets, RNA interference screening of a 779-kinome library was performed under multicellular tumor spheroid culture conditions. Results: In vitro proliferation data demonstrated that neratinib had a nanomolar range of half-maximal inhibitory concentrations in most of the tested cell lines, including T-DM1-resistant HER-2 breast cancer cell lines. Neratinib inhibited colony formation in a dose-dependent manner (P = 0.0001). Reverse-phase protein array data revealed that the efficacy of neratinib correlated with phosphorylated EGFR (R2 = 0.6055) or phosphorylated HER2 (R2 = 0.6734) expression level. Statistical analysis identified the 25 most relevant targets in the RNA interference screening, which we will validate for combinational antitumor effects with neratinib in in vitro and in vivo studies. Conclusion: We determined the efficacy of neratinib and identified target molecules to enhance neratinib efficacy in different breast cancer subtype cell lines with ErbB alterations. Our data inform new combined treatment strategies with neratinib for breast cancer that will lead to innovative designs using optimized neratinib combinations in clinical trials. Citation Format: Bora Lim, Jangsoon Lee, Troy Pearson, Liu Huey, Minha Hwang, Kuicheon Choi, Francesca Avogadri-Connors, Richard E. Cutler, Debu Tripathy, Naoto T. Ueno. Identification of molecular therapeutic targets that enhance the antitumor activity of neratinib in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 825.

Liposomal nanoparticle armed with bivalent bispecific single-domain antibodies, novel weapon in HER2 positive cancerous cell lines targeting

Breast cancer is the leading cause of mortality among all cancers. HER2, human epidermal growth factor receptors type 2, a receptor tyrosine kinase that induces interminable cell proliferation, is overexpressed in 20–25 percent of breast cancers. In spite of significant progress in nanomedicine in the past decade, being subjected to genetic drift that hides many paramount epitopes has rendered targeting HER2 as a big challenge. In the present study, we developed monovalent and bivalent monospecific along with bivalent bispecific VHH targeting different epitopes on HER2, and showed that bivalent bispecific VHH has the highest affinity among other tested modalities. Then we covalently coupled VHHs to the fluorescent labeled liposomal nanoparticle to produce targeted liposomes. Based on flow cytometry results, bivalent bispecific VHH targeted liposomes showed the highest fluorescent intensity, on HER2 breast cancer cells. Liposomes conjugated to bivalent monospecific VHH exhibited enhanced affinity toward HER2 positive cell lines compared to monovalent targeted liposomes, with bivalent bispecific liposomes appearing as the most robust probe.

Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer

Elevated expression of HER3, which interacts with HER2 in breast cancer cells, confers chemoresistance via phosphoinositide 3-kinase (PI-3K)/Akt-dependent upregulation of Survivin. However, the underlying mechanism is not clear. Ectopic expression or specific knockdown of HER3 in HER2-overexpressing breast cancer cells did not alter Survivin mRNA levels and Survivin protein stability, supporting the notion that HER3 signaling may regulate specific miRNAs that target Survivin to alter its protein translation. Here we showed that overexpression and specific knockdown of HER3 reduced and enhanced expression of two Survivin-targeting miRNAs, miR-203 and miR-542-3p, in breast cancer cells, respectively. While the specific inhibitor of either miR-203 or miR-542-3p attenuated an anti-HER3 antibody-induced downregulation of Survivin, inhibition of miR-542-3p exhibited a better efficacy than miR-203 inhibition did. Consistently, miR-542-3p mimic was much more effective than miR-203 mimic not only in inhibition of Survivin, but also in enhancement of paclitaxel-induced apoptosis in HER2-overexpressing breast cancer cells. Moreover, the combination of miR-542-3p mimic and paclitaxel, as compared with either agent alone, significantly inhibited in vivo tumor growth of HER2-overexpressing breast cancer cells. Collectively, our data indicated that the HER3/PI-3K/Akt signaling upregulates Survivin via suppression of miR-203 and miR-542-3p. Because miR-542-3p has three binding sites on the 3′-UTR of Survivin mRNA, its mimic was able to effectively downregulate Survivin in vitro and in vivo. Thus, miR-542-3p-replacement therapy is an excellent approach to overcome HER3-mediated paclitaxel resistance and significantly enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer.

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.

Specific cytotoxicity of a novel HER2-based chimeric antigen receptor modified T lymphocytes against HER2-positive tumor cells

Objective: To construct the third generation chimeric antigen receptor based on a novel humanized anti-HER2 H1-2 scFv, and to investigate the specific cytotoxicity of H1-2 CAR modified T lymphocytes(CAR-T) against HER2(+) tumor cells. Method: The expression cassette of the third generation CAR gene and anti-HER2 H1-2 scFv were constructed and cloned into lentivirus transfer plasmid, and then the third generation H1-2 CAR was transduced into human T lymphocytes using lentivirus.Enzyme linked immunosorbent assay was used to detect the expression of cytokines IL2, and LDH release assay was used to detect the cytotoxic effect of the H1-2 CAR-T.Finally, NOD/SCID mice and HER2(+) breast cancer cell line SKBR3 were used to detect the anti-tumor effect of H1-2 CAR-T in vivo. Results: The third generation H1-2 CAR was successfully constructed.H1-2 CAR-T secreted high dose of IL2 after confrontation with HER2(+) breast cancer cells.In vitro, the cytolytic rate of H1-2 CAR-T on high expression HER2(+) tumor cells was significantly higher than that in low expression HER2 or non-expression HER2 tumor cells. At the efficacy to target ratio of 20, the cytolytic rate of H1-2 CAR-T against breast cancer cell SK-BR-3 could reach (90.1±2.8)%, while the cytolytic rate of H1-2 CAR-T against HER2(-) breast cancer cell MDA-MB-231 was only (13.5±4.7)%. In the mouse xenograft tumor model, H1-2 CAR-T cells inhibited breast cancer growth in vivo.At the end of the experiments, the average tumor weight in the H1-2 CAR-T cell treatment group was (0.7±0.1) g, the non-transfected T cell therapeutic group was (1.2±0.2) g, and the PBS group was (1.2±0.2) g. There was significant difference between the H1-2 CAR-T therapeutic group and the non-transfected T cell therapeutic group (P<0.05). However, there was no significant difference between the non-transfected T cell therapeutic group and the PBS treatment group (P>0.05). Conclusion: The HER2-sepcific H1-2 CAR-T cells specifically kill HER2 positive cells, and further studies on CAR-T cells for the treatment of HER2(+) cancers are useful.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 33: The binding mode of the bispecific anti-HER2xHER3 antibody MCLA-128 is responsible for its potent inhibition of HRG-driven tumorigenesis

Abstract Introduction: MCLA-128 is as an ADCC-enhanced IgG1 bispecific antibody that targets the HER2:HER3 dimer and is currently being tested in Phase I/II clinical trials. MCLA-128 demonstrates an in vitro potency superior to other anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of heregulin (HRG) thereby overcoming one of the resistance mechanisms of current HER2 therapies. This study investigates the binding mode of MCLA-128 and proof of concept studies in HRG-driven tumor models. Methods: Alanine scanning shotgun mutagenesis was used to map the epitopes of MCLA-128 to HER2 and HER3. Fab fragments of MCLA-128 were crystallized with the soluble extracellular domains of HER2 and HER3. SAXS analysis on the HER2-HER3-MCLA-128 complex was performed to investigate the binding mode of the bispecific antibody in solution. Ligand-induced dimer specificity was investigated with PathHunter® heterodimerization assays. Bispecific anti-HER2xHER3 antibody and its parental anti-HER3 monoclonal antibody were labelled with 64Cu to compare their biodistribution profiles. The efficacy of MCLA-128 in HRG-driven systems was shown in vitro in MDA-MB-175 cells and in vivo in an orthotopic intracranial patient-derived xenograft (PDX) model originating from a breast cancer brain metastasis Results: The shotgun mutagenesis study identified that the bispecific antibody MCLA-128 binds amino acids T144, R166, R181 in HER2 domain I and R426 in HER3 domain III. Crystallographic studies confirmed the involvement of these critical residues and suggested that MCLA-128 locks the HER3 receptor in its ligand-unbound inactive confirmation. SAXS analysis suggests that the bispecific antibody MCLA-128 forms inter-dimer rather than intra-dimer interactions. In vitro, MCLA-128 specifically blocked HRG-induced signaling of HER2:HER3 but not HER2:HER4 heterodimers. Biodistribution of MCLA-128 in a xenograft model of breast cancer showed that the penetration of MCLA-128 in JIMT-1 HER2-amplified tumors is HER2-dependent despite the high affinity of the HER3 Fab arm for its receptor. MCLA-128 efficiently blocked tumor growth of the HRG-driven HER2 (1+) breast cancer cell line MDA-MB-175 in 3D in vitro. Treatment of orthotopically transplanted HER2-amplified breast cancer brain tumors in mice led to 100% survival with MCLA-128, in contrast to 38% and 0% survival in T-DM1 and vehicle treated mice respectively. Conclusion: MCLA-128 targets HER2-positive tumors via its HER2 arm and locks HER3 in an inactive confirmation. The potent anti-proliferative activity of MCLA-128 in vitro and in vivo supports the clinical development of this bispecific HER2xHER3 antibody in HRG-driven tumors. Citation Format: David Maussang-Detaille, Camilla de Nardis, Linda Hendriks, Carina Bartelink-Clements, Eric Rovers, Tristan Gallenne, Robert Doornbos, Lex Bakker, John de Kruif, Ton Logtenberg, Piet Gros, Cecile Geuijen, Mark Throsby. The binding mode of the bispecific anti-HER2xHER3 antibody MCLA-128 is responsible for its potent inhibition of HRG-driven tumorigenesis [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 33. doi:10.1158/1538-7445.AM2017-33

Abstract 623: Identifying candidate antigens for a ductal carcinoma in situ vaccine that are essential to breast cancer survival across multiple subtypes

Abstract Vaccine therapy may be ideal to destroy ductal carcinoma in situ (DCIS) and prevent recurrence. A vaccine can induce type 1 T-cells against DCIS antigens that could migrate from the circulation to invade and destroy the tumor as well as generate immunologic memory to provide long lasting protective immunity. One of the limitations to developing a DCIS vaccine is identifying antigens that target all subtypes of DCIS. DCIS has many of the same molecular abnormalities as invasive breast cancer (IBC), including overexpressed proteins that may be drivers of progression to invasive disease. Eliminating cells that are overexpressing driver proteins by vaccination may prevent progression to IBC if such proteins could be determined. In this study, we identified proteins that were overexpressed from normal breast in both DCIS and IBC across fifteen Geo and Array Express data sets. From 68 candidate proteins, we identified twelve whose expression was necessary for cancer cell survival across breast cancer subtypes. We selected overexpressed proteins necessary for cancer survival using a high throughput siRNA screen and chose candidates that increased apoptosis and decreased cell survival in HER2 positive (HER2), triple negative (TN), and hormone receptor positive HER2 negative (HR) human breast cancer cell lines with decreased expression of the target protein. For example, decreased expression of NDC80 caused decreased cell survival to 56±3% in HR, 46±3% in TN, and 77±2% in HER2 breast cancer cell lines and increased apoptosis by 1.4±0.03 fold in HR and 1.2±0.03 fold in TN breast cancer cell lines. Decreased expression of RRM2 caused decreased cell survival to 66±3% in HR and 89±2% in HER2 breast cancer cell lines and increased apoptosis by 1.6±0.08 fold in HR, 1.4±0.07 fold in TN, and 2.4±0.3 fold in HER2 breast cancer cell lines. Twelve proteins (AURKA, KIF11, NDC80, RRM2, SDC1, UBE2C, HJURP, CENPA, CENPF, HIST2H2AA3, HIST1H2BD, and TOP2A) were essential for cancer cell survival in at least 2 breast cancer subtypes. These protein targets are immunogenic in patients with DCIS. In the sera of women without breast atypia (n=36), autoantibodies to NDC80 were detected with a mean of 5.3±1.8 ng/mL while in women with fibroadenoma (n=36) autoantibodies were detected with a mean of 8.3±1.5 (p=0.05). In the sera of women without breast atypia (n=36), autoantibodies to RRM2 were detected with a mean of 0.16±0.7 ng/mL while in women with fibroadenoma (n=36) autoantibodies were detected with a mean of 7.2±2.1 (p=0.05) and in women with DCIS (n=59) autoantibodies were detected with a mean of 2.6±0.6 ng/mL (p=0.0003). These proteins represent DCIS antigens of biologic importance in tumor growth and, potentially, progression to IBC and are candidate immunogens for a vaccine to treat DCIS. Citation Format: Sasha Elizabeth Stanton, Erik Ramos, James Annis, Andrew Timms, Tessa Rue, Mary L. Disis. Identifying candidate antigens for a ductal carcinoma in situ vaccine that are essential to breast cancer survival across multiple subtypes [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 623. doi:10.1158/1538-7445.AM2017-623