HER2-overexpressing Breast Cancer Cell Lines Research Articles

Targeted therapies in HER2-positive breast cancer with receptor-redirected Arazyme-linker-Herceptin as a novel fusion protein.

Targeted treatment of different types of cancers through highly expressed cancer cell surface receptors by fusion proteins is an efficient method for cancer therapy. The HER2 receptor is a member of the tyrosine kinase receptors family, which plays a notable role in breast cancer tumor development. About 25-30% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2). In this study, we evaluated the particulars of a designed recombinant protein formed by HER2-specific Mab Herceptin linked with Arazyme on a HER2-overexpressing breast cancer cell line (SKBR3). Arazyme, a metalloprotease produced by Serratia proteamaculans was fused to the variable area of light and heavy chains of the Herceptin. The cytotoxic assay of the Arazyme-linker-Herceptin in the SKBR3 and MDA-MB-468 cells was evaluated by the MTT and flow cytometry techniques. The Caspase‑3 activity determination and adhesion assay were performed to evaluate the antitumor activity of the Arazyme-linker-Herceptin against SKBR3 cells. Furthermore, RT-PCR was used to measure the expression levels of the Bcl-2, Bax, MMP2, MMP9, and RIP3 genes. The Arazyme-linker-Herceptin showed higher cytotoxicity in SKBR3 cells compared to MDA-MB-468 cells. In addition, flow cytometry results revealed that the Arazyme-linker-Herceptin can significantly induce apoptosis in the HER2-overexpressing breast cancer cell line (SKBR3), which was confirmed by Bax upregulation and the decrease in adhesion of tumor cells and MMP2/MMP9. The findings of this study demonstrated that the Arazyme-linker-Herceptin induced apoptosis and decreased metastatic genes in SKBR3 cells; however, further research is required to confirm the effectiveness of the fusion protein.

Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

BackgroundBreast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy.ResultsIn this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression.ConclusionsOur results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients’ outcome.

AMPK activation attenuates HER3 upregulation and Neuregulin-Mediated rescue of cell proliferation in HER2-Overexpressing breast cancer cell lines exposed to lapatinib

Lapatinib is a highly selective reversible inhibitor of the tyrosine kinase domains of HER2 and EGFR, approved for the treatment of advanced stage HER2-overexpressing breast cancers. Although targeted therapy with lapatinib provides initial clinical advantage, cancer cells' adaptive responses can overcome the inhibitory effects of lapatinib. HER3 upregulation and autocrine induction of HER3 ligand neuregulin-1 (NRG), have been implicated in the restoration of AKT and ERK1/2 activity and rescue of cell proliferation. In this study we evaluated the effects of lapatinib alone and in combination with AMPK activator GSK-621 in HER2-overexpressing breast cancer cell lines SKBR3 and BT474. Our results show that in cells exposed to lapatinib and GSK-621 in combination, lapatinib-mediated HER3 upregulation was reduced and reactivation of AKT and ERK1/2 kinases was prevented. The two drugs in combination decreased cell viability in a synergistic manner and greatly reduced the ability of NRG to rescue cell proliferation. Finally, we provide evidence that in cells exposed to lapatinib and GSK-621 in combination the establishment of a transcriptionally permissive chromatin structure at the HER3 promoter is hampered. The results of this study highlight a potential role for AMPK activation in counteracting lapatinib-induced adaptive responses of HER2-overexpressing breast cancer cells.

Newly identified breast luminal progenitor and gestational stem cell populations likely give rise to HER2-overexpressing and basal-like breast cancers

Breast Cancer (BrC) is a common malignancy with genetically diverse subtypes. There is evidence that specific BrC subtypes originate from particular normal mammary cell populations. However, the cell populations that give rise to most BrC subtypes are unidentified. Several human breast scRNAseq datasets are available. In this research, we utilized a robust human scRNAseq dataset to identify population-specific marker genes and then identified the expression of these marker genes in specific BrC subtypes. In humans, several BrC subtypes, HER2-enriched, basal-like, and triple-negative (TN), are more common in women who have had children. This observation suggests that cell populations that originate during pregnancy give rise to these BrCs. The current human datasets have few normal parous samples, so we supplemented this research with mouse datasets, which contain mammary cells from various developmental stages. This research identified two novel normal breast cell populations that may be the origin of the basal-like and HER2-overexpressing subtypes, respectively. A stem cell-like population, SC, that expresses gestation-specific genes has similar gene expression patterns to basal-like BrCs. A novel luminal progenitor cell population and HER2-overexpressing BrCs are marked by S100A7, S100A8, and S100A9 expression. We bolstered our findings by examining SC gene expression in TN BrC scRNAseq datasets and S100A7-A9 gene expression in BrC cell lines. We discovered that several potential cancer stem cell populations highly express most of the SC genes in TN BrCs and confirmed S100A8 and A9 overexpression in a HER2-overexpressing BrC cell line. In summary, normal SC and the novel luminal progenitor cell population likely give rise to basal-like and HER2-overexpressing BrCs, respectively. Characterizing these normal cell populations may facilitate a better understanding of specific BrCs subtypes.

Strategy for Avoiding Protein Corona Inhibition of Targeted Drug Delivery by Linking Recombinant Affibody Scaffold to Magnetosomes.

PurposeNanoparticles (NPs) decorated with functional ligands are promising candidates for cancer diagnosis and treatment. However, numerous studies have shown that chemically coupled targeting moieties on NPs lose their targeting capability in the biological milieu because they are shielded or covered by a “protein corona”. Herein, we construct a functional magnetosome that recognizes and targets cancer cells even in the presence of protein corona.MethodsMagnetosomes (BMPs) were extracted from magnetotactic bacteria, M. gryphiswaldense (MSR-1), and decorated with trastuzumab (TZ) via affibody (RA) and glutaraldehyde (GA). The engineered BMPs are referred to as BMP-RA-TZ and BMP-GA-TZ. Their capacities to combine HER2 were detected by ELISA, the quantity of plasma corona proteins was analyzed using LC-MS. The efficiencies of targeting SK-BR-3 were demonstrated by confocal laser scanning microscopy and flow cytometry.ResultsBoth engineered BMPs contain up to ~0.2 mg TZ per mg of BMP, while the quantity of HER2 binding to BMP-RA-TZ is three times higher than that binding to BMP-GA-TZ. After incubation with normal human plasma or IgG-supplemented plasma, GA-TZ-containing BMPs have larger hydrated radii and more surface proteins in comparison with RA-TZ-containing BMPs. The TZ-containing BMPs all can be targeted to and internalized in the HER2-overexpressing breast cancer cell line SK-BR-3; however, their targeting efficiencies vary considerably: 50–75% for RA-TZ-containing BMPs and 9–19% for GA-TZ-containing BMPs. BMPs were incubated with plasma (100%) and cancer cells to simulate human in vivo environment. In this milieu, BMP-RA-TZ uptake efficiency of SK-BR-3 reaches nearly 80% (slightly lower than for direct interaction with BMP-RA-TZ), whereas the BMP-GA-TZ uptake efficiency is <17%.ConclusionApplication of the RA scaffold promotes and orients the arrangement of targeting ligands and reduces the shielding effect of corona proteins. This strategy improves the targeting capability and drug delivery of NP in a simulated in vivo milieu.

Herceptin-conjugated magnetic polystyrene-Agsbox nanoparticles as a theranostic agent for breast cancer

Breast cancer is a malignant tumor, which has derived from cells of the breast. Further, a relatively rapid metastasis, and resistance development against all the conventional drug combinations are major clinical issues in breast cancer patients as well as limitations like toxicity, genetic mutation, and metastasis make difficult the use of conventional therapy methods such as chemotherapy, radiotherapy, and local surgery. Therefore, considering the urgent needs, and high death rate in breast cancer cases, the development of new diagnosis and treatment regimens which diagnosed at the early stage and protected normal tissues required for clinical applications. Recently, the combination of tumor diagnosis and treatment within a single platform is a novel perspective, and magnetic nanoparticles are potential candidate owing to their low toxic effect, biocompatibility, biological degradability, superior magnetic properties, and targeting ability to overcome the problems of conventional diagnosis and therapy techniques. Considering these restrictions and requirements, the goal of this research was to investigate the potential of an innovative theranostic agent, which is soybean oil-based polystyrene (PS)-g-soybean oil graft copolymer containing AgNPs (PS-Agsbox) for treatment and MRI-based diagnosis of cancer. Herein, we designed targeted magnetic PS-Agsbox nanoparticles carrying thymoquinone (TQ) that is known for its anticancer potential against breast cancer, and herceptin (HER), which is to bind to the HER2 receptor protein on the surface of HER2-positive tumor cells, and acts by blocking the effects of it. We have successfully demonstrated selective binding, effective uptake of HER-conjugated magnetic PS-Agsbox nanoparticles into MDA-MB-231 (human breast carcinoma cells, a HER2-underexpressing cell line) and SKBR-3 (human breast cancer cells, a HER2-overexpressing breast cancer cell line) cell lines while no effect against L929 (mouse fibroblast cell line). Moreover, the magnetic resonance (MRI) properties of HER-conjugated magnetic PS-Agsbox nanoparticles were also confirmed.

Western Blot Analysis of Lapatinib-Mediated Inhibition of the Epidermal Growth Factor Receptor 2 (HER2) Pathway in Breast Cancer Cells.

Western blotting is an excellent technique to investigate aberrations and/or therapy-induced changes in signaling proteins in cancer. Using an in vitro system, we prepared whole cell lysates from HER2-overexpressing breast cancer cell lines, treated or not with the tyrosine kinase inhibitor, lapatinib, in the presence and absence of IFN-γ. Here we describe the protocol whereby proteins in the lysates were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes followed by an enzyme-linked immunoassay and chemiluminescence to reveal the relevant phosphorylated and dephosphorylated proteins. Herein, Western blot analysis confirmed lapatinib dephosphorylated HER2 and downstream signaling proteins and IFN-γ induced phosphorylation of STAT1.

Abstract 1078: Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features

Abstract Trastuzumab and trastuzumab emtansine (T-DM1) targeted therapies are the main choice for the treatment of HER2-overexpressing breast cancer patients. However, de novo or acquired resistance is still the major obstacle in clinical practice. It has been shown that several pathways, including HER2, can lead to HIF-α stabilization in breast cancer cells. Thus, the purpose of our study was to analyse the effect of hypoxia in acquired resistance to anti-HER2 therapies. We used HER2-overexpressing BT-474 and non-overexpressing MCF-7 human breast cancer cell lines. As an acute hypoxia model, we added CoCl2 (100 µM) to cell culture medium. The hypoxic status of the cells was evaluated by a Western blot analysis, showing a peak of HIF-1α expression after 6 hours of CoCl2 exposure. This result correlated with VEGF induction, as measured by RT-qPCR (p&amp;lt;0.05). Interestingly, under hypoxia, BT-474 cells treated with increasing concentrations of trastuzumab and T-DM1 for 72 hours presented a significantly higher viability compared with non-hypoxic cells (p&amp;lt;0.05). However, although the drugs decreased membrane HER2 protein expression (p&amp;lt;0.01), HER2 levels did not change under hypoxic conditions, as shown by flow cytometry and Western blot assays. Moreover, drug effects on MCF-7 cells did not change under hypoxic conditions. To further analyse this cytoprotective effect of hypoxia, proliferation-associated markers such as PCNA expression and AKT activation were evaluated by Western blot, while apoptosis frequency was measured by flow cytometry in BT-474 cells. PCNA and pAKT expression remained unaltered, while T-DM1-mediated apoptosis was significantly reduced under hypoxia (p&amp;lt;0.05). Then, using different open gene expression datasets, we identified a group of four genes (COL12A1, DKK1, CEACAM5 and ANTXR1) differentially expressed in T-DM1 resistant HER2-overexpressing breast cancer BT-474 and KPL-4 cell lines (p&amp;lt;0.05), which were coexpressed with hypoxia-related genes (HIF1A, EPAS1 and VEGFA) in HER2-overexpressing breast cancer tumours and cell lines from TCGA (p&amp;lt;0.01). Further in silico analyses revealed a direct association between those genes and the fibronectin gene (FN1, p&amp;lt;0.05). Remarkably, they were involved in TGF-β and angiogenesis pathways, as shown by a functional enrichment analysis (p&amp;lt;0.01). In addition, we found that BT-474 cells under hypoxia presented a higher mammosphere formation efficiency than non-hypoxic cells (p&amp;lt;0.05), showing a tendency towards a cancer stem cell phenotype. It is known that hypoxia represents a common feature of metastatic tumours and has a role in regulating stem cell behaviour. According to this, our results highlight a link between anti-HER2 therapy resistance and epithelial-to-mesenchymal transition characteristics, where hypoxia plays a key role which deserves further study. Citation Format: Virginia Judith Wolos, Ivana Jaqueline Tapia, Marianela Abrigo, Ezequiel Lacunza, Elisa Dora Bal de Kier Joffé, Gabriel León Fiszman. Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1078.

HSP90 Inhibitor, 17-DMAG, Alone and in Combination with Lapatinib Attenuates Acquired Lapatinib-Resistance in ER-positive, HER2-Overexpressing Breast Cancer Cell Line.

Simple SummaryLapatinib is a tyrosine kinase inhibitor widely used as a treatment for a Human Epidermal growth factor Receptor 2 (HER2) (+) breast cancer patients. However, when resistance is acquired through continued exposure, and it is associated with a poor prognosis for patients. In this study, we identified HSP90 as a common node for acquired resistance to lapatinib in two lapatinib resistant cell lines using proteomic analysis. Notably, in vitro and in vivo studies demonstrated synergy effect between lapatinib and an HSP90 inhibitor were observed in the estrogen receptor (+) HER2 (+) breast cancer cell only. These results could be a potential strategy for future clinical trials for HSP90 inhibitors in treatment—refractory HER2 (+) metastatic cancer patientsLapatinib, a Human Epidermal growth factor Receptor 2 (HER2)-targeting therapy in HER2-overexpressing breast cancer, has been widely used clinically, but the prognosis is still poor because most patients acquire resistance. Therefore, we investigated mechanisms related to lapatinib resistance to evaluate new therapeutic targets that may overcome resistance. Lapatinib-resistant cell lines were established using SKBR3 and BT474 cells. We evaluated cell viability and cell signal changes, gene expression and protein changes. In the xenograft mouse model, anti-tumor effects were evaluated using drugs. Analysis of the protein interaction network in two resistant cell lines with different lapatinib resistance mechanisms showed that HSP90 protein was commonly increased. When Heat Shock Protein 90 (HSP90) inhibitors were administered alone to both resistant cell lines, cell proliferation and protein expression were effectively inhibited. However, inhibition of cell proliferation and protein expression with a combination of lapatinib and HSP90 inhibitors showed a more synergistic effect in the LR-BT474 cell line than the LR-SKBR3 cell line, and the same result was exhibited with the xenograft model. These results suggest that HSP90 inhibitors in patients with lapatinib-resistant Estrogen Receptor (ER) (+) HER2 (+) breast cancer are promising therapeutics for future clinical trials.

Abstract 6352: Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells

Abstract Background/Significance: Exosomes are 30-100 nm membrane vesicles secreted by many cell types. Tumors secrete large numbers of exosomes that transport oncoproteins and immune suppressive molecules, supporting tumor growth and metastasis. The role of exosomes in intercellular signaling is shown by human epidermal growth factor receptor type 2 (HER2)-expressing breast tumors, where exosomes with HER2 stimulate tumor growth and interfere with activity of the therapeutic antibody Herceptin, which binds to the extracellular domain of HER2 and prolongs the overall survival of HER2-positive breast cancer patients. Exosome release is modulated by growth factors EGF and heregulin, HER2 receptor-activating ligands naturally present in the surrounding tumor microenvironment. The SMRwt peptide reduces secretion of HER2 exosomes; thus, it can potentially be exploited as an anticancer agent. Methods: BT-474 and HCC1569 HER2-positive breast cancer cells overexpressing HER2 and mortalin were used in these experiments. Exosome size and concentration were measured by NanoSight. Purified tumor exosomes were prepared using paramagnetic beads coated with antibodies against tumor specific proteins and confirmed by flow cytometry and electron microscopy. To identify biological functions, we cultured tumor exosomes with breast cancer cells, which increased tumor cell proliferation. To determine SMRwt peptide inhibition effects, we assayed tumor exosome secretion and cell proliferation by NanoSight, Western blot and MTT. Results: We characterized exosomes secreted by mortalin and HER2-overexpressing breast cancer cell lines and analyzed SMRwt peptide's role in reducing tumor exosome release and cell proliferation. Exosome secretion increased in HER2-overexpressing tumor cell lines HCC1596 and BT474. Exosomes from HER2-positive tumor cell-conditioned supernatants stimulated tumor cell growth and tumor exosome release and inhibited Herceptin activity on HCC1569 and BT474 cell proliferation. Addition of SMRwt peptide reduced tumor cell proliferation and tumor exosome release. Conclusions: These data show the role of HER2-positive exosomes in modulating sensitivity to Herceptin and thus, to HER2-driven tumor aggressiveness; and indicate that the SMRwt peptide has potential anticancer functions in HER2-positive HCC1569 and BT474 human breast cancer cells. The data also indicate tumor exosomes affect the proliferation of parental cells. Tumor exosomes derived from BT-474 cells and HCC1569 cells stimulated the proliferation of BT-474 cells and HCC1569 cells, suggesting a biological function for tumor exosomes. Citation Format: Ming Bo Huang. Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6352.

Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

Human surfactant protein D (SP-D) belongs to the family of collectins that is composed of a characteristic amino-terminal collagenous region and a carboxy-terminal C-type lectin domain. Being present at the mucosal surfaces, SP-D acts as a potent innate immune molecule and offers protection against non-self and altered self, such as pathogens, allergens, and tumor. Here, we examined the effect of a recombinant fragment of human SP-D (rfhSP-D) on a range of breast cancer lines. Breast cancer has four molecular subtypes characterized by varied expressions of estrogen (ER), progesterone (PR), and epidermal growth factor (EGF) receptors (HER2). The cell viability of HER2-overexpressing (SKBR3) and triple-positive (BT474) breast cancer cell lines [but not of a triple-negative cell line (BT20)] was reduced following rfhSP-D treatment at 24 h. Upregulation of p21/p27 cell cycle inhibitors and p53 phosphorylation (Ser15) in rfhSP-D-treated BT474 and SKBR3 cell lines signified G2/M cell cycle arrest. Cleaved caspases 9 and 3 were detected in rfhSP-D-treated BT474 and SKBR3 cells, suggesting an involvement of the intrinsic apoptosis pathway. However, rfhSP-D-induced apoptosis was nullified in the presence of hyaluronic acid (HA) whose increased level in breast tumor microenvironment is associated with malignant tumor progression and invasion. rfhSP-D bound to solid-phase HA and promoted tumor cell proliferation. rfhSP-D-treated SKBR3 cells in the presence of HA showed decreased transcriptional levels of p53 when compared to cells treated with rfhSP-D only. Thus, HA appears to negate the anti-tumorigenic properties of rfhSP-D against HER2-overexpressing and triple-positive breast cancer cells.

Mathematical modeling of drug-induced receptor internalization in the HER2-positive SKBR3 breast cancer cell-line

About 20% of breast cancer tumors over-express the HER2 receptor. Trastuzumab, an approved drug to treat this type of breast cancer, is a monoclonal antibody directly binding at the HER2 receptor and ultimately inhibiting cancer cell growth. The goal of our study was to understand the early impact of trastuzumab on HER2 internalization and recycling in the HER2-overexpressing breast cancer cell line SKBR3. To this end, fluorescence microscopy, monitoring the amount of HER2 expression in the plasma membrane, was combined with mathematical modeling to derive the flux of HER2 receptors from and to the membrane. We constructed a dynamic multi-compartment model based on ordinary differential equations. To account for cancer cell heterogeneity, a first, dynamic model was expanded to a second model including two distinct cell phenotypes, with implications for different conformational states of HER2, i.e. monomeric or homodimeric. Our mathematical model shows that the hypothesis of fast constitutive HER2 recycling back to the plasma membrane does not match the experimental data. It conclusively describes the experimental observation that trastuzumab induces sustained receptor internalization in cells with membrane ruffles. It is also concluded that for rare, non-ruffled (flat) cells, HER2 internalization occurs three orders of magnitude slower than for the bulk, ruffled cell population.

Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer

BackgroundCurrent prognostic tools and targeted therapeutic approaches have limited value for metastatic triple negative breast cancer (TNBC). Building upon current knowledge, we hypothesized that epoxyeicosatrienoic acids (EETs) and related CYP450 epoxygenases may have differential roles in breast cancer signaling, and better understanding of which may uncover potential directions for molecular stratification and personalized therapy for TNBC patients.MethodsWe analyzed the oxylipin metabolome of paired tumors and adjacent normal mammary tissues from patients with pathologically confirmed breast cancer (N = 62). We used multivariate statistical analysis to identify important metabolite contributors and to determine the predictive power of tumor tissue metabolite clustering. In vitro functional assays using a panel of breast cancer cell lines were carried out to further confirm the crucial roles of endogenous and exogenous EETs in the metastasis transformation of TNBC cells. Deregulation of associated downstream signaling networks associated with EETs/CYPs was established using transcriptomics datasets from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Comparative TNBC proteomics using the same tissue specimens subjected to oxylipin metabolomics analysis was used as validation set.ResultsMetabolite-by-metabolite comparison, tumor immunoreactivity, and gene expression analyses showed that CYP epoxygenases and arachidonic acid-epoxygenation products, EET metabolites, are strongly associated with TNBC metastasis. Notably, all the 4 EET isomers (5,6-, 8,9-, 11,12-, and 14,15-EET) was observed to profoundly drive the metastasis transformation of mesenchymal-like TNBC cells among the TNBC (basal- and mesenchymal-like), HER2-overexpressing and luminal breast cancer cell lines examined. Our pathway analysis revealed that, in hormone-positive breast cancer subtype, CYP epoxygenase overexpression is more related to immune cell-associated signaling, while EET-mediated Myc, Ras, MAPK, EGFR, HIF-1α, and NOD1/2 signaling are the molecular vulnerabilities of metastatic CYP epoxygenase-overexpressing TNBC tumors.ConclusionsThis study suggests that categorizing breast tumors according to their EET metabolite ratio classifiers and CYP epoxygenase profiles may be useful for prognostic and therapeutic assessment. Modulation of CYP epoxygenase and EET-mediated signaling networks may offer an effective approach for personalized treatment of breast cancer, and may be an effective intervention option for metastatic TNBC patients.

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets MET and AXL in triple-negative breast cancer

BackgroundBreast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.MethodsBioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.ResultsBy applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line. ConclusionsThese findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5 h in human serum compared to the half-life of 2 h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.

Targeting of HER3 with Functional Cooperative miRNAs Enhances Therapeutic Activity in HER2-Overexpressing Breast Cancer Cells

BackgroundThe HER3 receptor functions as a major cause of drug resistance in cancer treatment. It is believed that therapeutic targeting of HER3 is required to improve patient outcomes. It is not clear whether a novel strategy with two functional cooperative miRNAs would effectively inhibit erbB3 expression and potentiate the anti-proliferative/anti-survival effects of a HER2-targeted therapy (trastuzumab) and chemotherapy (paclitaxel) on HER2-overexpressing breast cancer cells.ResultsCombination of miR-125a and miR-205, as compared to either miRNA alone, potently inhibited expression of HER3 in HER2-overexpressing breast cancer BT474 cells. Co-expression of the two miRNAs not only reduced the levels of phosphorylated erbB3 (P-erbB3), Akt (P-Akt), and Src (P-Src), it also inhibited cell proliferation and increased cells at G1 phase. A multi-miRNA lentiviral vector - the cluster of miR-125a and miR-205 - was constructed to simultaneously express the two miRNAs in HER2-overexpressing breast cancer cells. Concurrent expression of miR-125a and miR-205 via the miRNA cluster transfection significantly enhanced trastuzumab-mediated growth inhibition and cell cycle G1 arrest in BT474 cells and markedly increased paclitaxel-induced apoptosis in another HER2-overexpressing breast cancer cell line HCC1954.ConclusionsHere, we showed that functional cooperative miRNAs effectively suppressed erbB3 expression. This novel approach targeting of HER3 was able to enhance the therapeutic efficacy of trastuzumab and paclitaxel against HER2-overexpressing breast cancer.

Micromolecular methods for diagnosis and therapeutic strategy: a case study.

An intraductal carcinoma, 55 mm across, was diagnosed on a total mastectomy in a 45-year-old woman. The 2 micro-invasive areas found were too small for reliable immunostainings for estrogen, progesterone, and HER2 receptors. In the sentinel lymph-node, a subcapsular tumor embole of about 50 cancer cells was identified on the extemporaneous cryo-cut section, but not on further sections after paraffin-embedding of the sample.Considering this tumor metastatic potential, we decided to assess HER2 status on the metastatic embole using pathological and molecular micro-methods. We laser-microdissected the tumor cells, extracted their DNA, and performed droplet-digital-PCR (ddPCR) for HER2 gene copy number variation. The HER2/RNaseP allele ratio was 5.2 in the laser-microdissected tumor cells, similar to the 5.3 ratio in the HER2-overexpressing breast cancer cell line BT-474.We thus optimized the adjuvant treatment of our patient and she received a trastuzumab-based adjuvant chemotherapy.

HER2-Targeted Multifunctional Silica Nanoparticles Specifically Enhance the Radiosensitivity of HER2-Overexpressing Breast Cancer Cells.

We investigated the effects of targeted functionalized silica nanoparticles on the radiosensitivity of cancer cells. Better control of the local concentration of silica nanoparticles may facilitate their use as an adjuvant in conjunction with ionizing radiation to target cancer cells while preventing damage to normal cells. Hyperbranched polyamidoamine (PAMAM) was grafted onto the surface of amorphous silica nanoparticles to functionalize them. The PAMAM-coated silica nanoparticles (PCSNs) were then conjugated with fluorescent dyes. Anti-HER2 antibodies were covalently attached to the labeled PCSNs. The HER2-overexpressing SK-BR3 breast cancer cell line was incubated in medium containing the PCSN probes. After incubation; the cells were exposed to X-ray radiation. Cells were counted in all samples using cell proliferation assays; and apoptotic cells were detected. The cell survival results showed that the combination of the targeted PCSN probes and radiation reduced the survival rate of SK-BR3 cells to a greater extent than when either PCSN probes, PCSNs or radiation were applied individually. The results also showed an increase in apoptosis in the SK-BR3 cells that internalized the PCSN probes and were then irradiated. Based on these data, PCSN probes act as specific radiosensitizing agents for HER2-overexpressing cells.

Heregulin-induced cell migration is promoted by aryl hydrocarbon receptor in HER2-overexpressing breast cancer cells

HER2 overexpression accounts for approximately 15–20% of all breast cancers. We have shown that HER2 overexpression leads to elevated expression of the aryl hydrocarbon receptor (AhR) in breast cancer cells. In this study, firstly, we showed that AhR expression was up-regulated by treatment with the HER3 ligand heregulin (HRG) in HER2-overexpressing breast cancer cell lines. Induction of AhR was mediated by transcriptional activation of the region of AhR promoter corresponding to − 190 to − 100 bp. In addition, HRG treatment elicited nuclear translocation of AhR. To investigate the role of AhR in HRG-HER2/HER3 signaling in HER2-overexpressing cells, we established AhR knockout (KO) HER2-overexpressing cells to perform wound-healing assays. HRG-induced cell migration was markedly attenuated by AhR KO. HRG-induced cell migration was associated with increased expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 in wild type cells, but not in AhR KO cells. These results elucidate that AhR is an important factor for the malignancy in HER2 overexpressing breast cancers.

Abstract P5-01-03: HER2 imaging by SPECT-CT using 111In radiolabeled pertuzumab-fab DOTAGA-conjugate: A proof of concept study in a preclinical model of breast cancer

Abstract Introduction: HER2 is positive in approximately 20-30% of all breast cancer and is associated with poor prognosis, higher mortality and higher metastatic incidence. Current diagnosis of HER2 expression relies on invasive methods requiring tissue biopsy which can lead to variable results due to inter/intra-metastatic and intratumoral heterogeneity in breast cancers. It has been recently demonstrated that HER2 molecular imaging based on pertuzumab, an antibody targeting HER2, could represent a more accurate non-invasive method to assess HER2 expression and evaluate its spatial and temporal heterogeneity. Aim: In the present study, we aimed at developing radiolabeled pertuzumab Fab fragments for HER2 imaging. Tumor uptake of radiolabeled Fab was evaluated in an animal model of HER2 breast cancer by SPECT-CT, and the impact of trastuzumab on HER2 imaging was assessed. The objective of this study was to validate the feasibility of HER2 imaging with a pertuzumab-derived probe and to evaluate the possible translation of such a probe for clinical use in patients treated or not with anti-HER2 therapy. Methods: Fab fragments of pertuzumab have been generated by papain digestion and bioconjugated with the bifunctional chelating agent DOTAGA for incorporation of Indium 111 to generate an HER2-specific probe for SPECT (111In-DOTAGA-pertuzumab-Fab). The functionality of both radiolabeled Fab and whole pertuzumab was evaluated by in vitro assays using HER2-overexpressing human breast cancer cell line (HCC1954). Tumor uptake of pertuzumab-Fab and whole pertuzumab (111In-DOTAGA-Pertuzumab) has been evaluated in Balb/c nude mice bearing BT-474 tumors with or without pre-treatment with trastuzumab for 24h. Results: In the current study we demonstrate that Fab fragments of pertuzumab keep similar HER2 binding properties than whole pertuzumab and can therefore be a suitable HER2-targeted probe. In vivo, 111In-DOTAGA-pertuzumab-Fab showed better pharmacokinetics than whole pertuzumab with faster tumor uptake and blood clearance allowing faster imaging with better tumor/blood ratio. In addition, tumor uptake of 111In-DOTAGA-pertuzumab-Fab is not modified by pre-treatment with trastuzumab. Conclusion: We assume that radiolabeled pertuzumab-Fab should be of great interest if the intention to treat is based on anti-HER2 monoclonal antibodies-based therapies rather than small molecules (e.g. lapatinib). Interestingly, pertuzumab has been shown to be also beneficial in combination with trastuzumab and chemotherapy in non-metastatic patients through its approval for the neoadjuvant treatment and very recently in the APHINITY clinical trial aiming for an approval in the adjuvant treatment of early breast cancer. These data suggest that pertuzumab will be extensively used in various HER-2 positive breast cancers underlining the interest to perform molecular imaging of HER2 expression as a predictive biomarker of efficacy. Citation Format: Oudot A, Bellaye P-S, Vrigneaud J-M, Raguin O, Bernhard C, Dumont L, Brunotte F, Savina A, Bouquet F, Fumoleau P, Collin B. HER2 imaging by SPECT-CT using 111In radiolabeled pertuzumab-fab DOTAGA-conjugate: A proof of concept study in a preclinical model of breast cancer [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 P5-01-03.