HER2-overexpressing Human Breast Cancer Cells Research Articles

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<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<0.05). However, although the drugs decreased membrane HER2 protein expression (p<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<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<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<0.01). Further in silico analyses revealed a direct association between those genes and the fibronectin gene (FN1, p<0.05). Remarkably, they were involved in TGF-β and angiogenesis pathways, as shown by a functional enrichment analysis (p<0.01). In addition, we found that BT-474 cells under hypoxia presented a higher mammosphere formation efficiency than non-hypoxic cells (p<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.

Arctigenin-mediated cell death of SK-BR-3 cells is caused by HER2 inhibition and autophagy-linked apoptosis.

Human epidermal growth factor receptor 2 (HER2) is well-known as the therapeutic marker in breast cancer. Therefore, we evaluated anti-cancer activity of arctigenin (ATG) on in SK-BR-3 HER2-overexpressing human breast cancer cells. Cell viability and cytotoxicity were analyzed with MTT and colony-forming assay and cell cycle analysis was performed by flow cytometry. The expression and/or phosphorylation of proteins in whole cell lysate and mitochondrial fraction were analyzed by Western blotting. Cellular levels of LC3 and sequestosome 1 (SQSTM1/P62) were observed by immunofluorescence analysis. The result showed that ATG decreased cell viability of SK-BR-3 cells in a concentration-dependent manner. Moreover, ATG increased the sub G1 population linked to the suppression of HER2/EGFR1 signaling pathway. Furthermore, ATG increased the phosphorylation of H2AX and down-regulated RAD51 and survivin expressions, indicating that ATG induced DNA damage and inhibited the DNA repair system. We also found that cleavages of caspase-7 and PARP by releasing mitochondrial cytochrome c into the cytoplasm were induced by ATG treatment for 72h through the reduction of Bcl-2 and Bcl-xL levels in mitochondria. In contrast, the levels of LC-3 and SQSTM1/P62 were increased by ATG for 24h through the Akt/mTOR and AMPK signaling pathway. Taken together, this study indicates that autophagy-linked apoptosis is responsible for the anti-cancer activity of ATG in SK-BR-3 cells, and suggests that ATG is considered a potential therapeutic for the treatment of HER2-overexpressing breast cancer.

Human Domain Antibodies to Conserved Epitopes on HER2 Potently Inhibit Growth of HER2-Overexpressing Human Breast Cancer Cells In Vitro.

The FDA approval of two anti-HER2 monoclonal antibodies, trastuzumab and pertuzumab, and an antibody-drug conjugate, trastuzumab emtansine, has transformed clinical practice for HER2-positive cancers. However, not all patients respond to therapy, and the majority of responders eventually develop resistance. In addition, cardiotoxicity is a major safety concern for their clinical use. Thus, there remains a need for the discovery and development of novel classes of HER2-targeted therapeutics with high efficacy and specificity. In this study, we report the identification and characterization of fully human single-domain antibodies (dAbs) targeting HER2 epitopes that are highly conserved among various species and non-overlapping with those of trastuzumab and pertuzumab. An Fc-fusion protein of the best binder demonstrated much higher inhibitory activity against HER2-amplified human breast cancer cell lines than trastuzumab and pertuzumab. Unlike the latter, however, it did not have an effect on gastric and ovarian cancer cell lines with HER2 overexpression. The dAb-Fc fusion protein showed poor pharmacokinetics in mice, thus limiting its potential for therapeutic use. It could be useful as an agent for the exploration of functionally important conserved structures on HER2 with implications for the design of novel therapeutics and elucidation of mechanisms of HER2-mediated tumorigenesis.

Abstract A36: Chemosensitivity to trastuzumab emtansine (T-DM1) differs in naturally or transduced HER2-overexpressing human breast cancer cells

Abstract Human epidermal growth factor receptor-2 (HER2) is overexpressed in 25-30% of breast cancers and is associated with distant metastasis and poor survival. Trastuzumab emtansine (T-DM1), an antibody-drug conjugate of anti-HER2 mAb trastuzumab linked to microtubule-targeting agent mertansine, has been approved for the treatment of HER2-positive metastatic breast cancer. Chemoresistance has been a major obstacle to T-DM1 treatment, and mechanisms remain incompletely understood. We tested T-DM1 chemosensitivity in vitro and in vivo using two human breast cancer cell lines, MDA-MB-231BR-HER2 (triple-negative cells stably transfected with HER2 plasmid) and HCC1954 (natural HER2 overexpression), which show equivalent HER2 and αv integrin protein levels. MDA-MB-231BR-HER2 was strongly resistant to T-DM1, with doses as high as 1 µg/ml failing to induce apoptosis indicated by the presence of cleaved PAPR protein. In contrast, HCC1954 cells are sensitive to T-DM1, with doses as low as 10 ng/mL causing cytotoxicity in a dose-dependent manner. Cellular internalization of trastuzumab-pHrodo conjugates was significantly higher in HCC1954 than MDA-MB-231BR-HER2, but no difference was found in anti-EGFR-Ab-pHrodo conjugates internalization. Endocytosis-related proteins caveolin-1 and rab5, but not early endosome antigen, were diminished in HCC1954 cells. Consistently, single-dose treatment with T-DM1 (3.6 mg/kg, IV) significantly reduced brain tumor volume in rats inoculated with HCC1954 but not MDA-MB-231BR-HER2 cells in an intracerebral xenograft breast cancer brain metastasis model. In conclusion, our findings suggest that rab5 and caveolin-1 proteins mediating antibody internalization and/or natural vs transduced HER2 overexpression may provide possible mechanisms in resistance of T-DM1 and should be investigated when HER2-positive cancer patients fail T-DM1 HER2-targeted therapy in clinical settings. Citation Format: Jeffrey Wu, Leslie Muldoon, Edward Neuwelt. Chemosensitivity to trastuzumab emtansine (T-DM1) differs in naturally or transduced HER2-overexpressing human breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A36.

Trastuzumab upregulates PD-L1 as a potential mechanism of trastuzumab resistance through engagement of immune effector cells and stimulation of IFNγ secretion

Here, we report that treatment of syngeneic mouse tumors transduced to overexpress human epidermal growth factor receptor-2 (HER2) with the anti-human HER2 antibody trastuzumab upregulated the level of programmed death-ligand 1 (PD-L1), an important negative regulator of T-cell response, in a transgenic mouse model immune-tolerant to human HER2. We further found that trastuzumab alone had no detectable effect on the level of PD-L1 expression in monocultures of HER2-overexpressing human breast cancer cells but upregulated PD-L1 in the same panel of HER2-overexpressing breast cancer cells when they were co-cultured with human peripheral blood mononuclear cells, and the upregulation of PD-L1 could be blocked by an IFNγ-neutralizing antibody. Inhibition of HER2 intrinsic signaling via HER2 expression knockdown or kinase inhibition had variable and cell-context-specific effects on downregulating the PD-L1 level. Analysis of The Cancer Genome Atlas database showed no direct correlation between HER2 and PD-L1 at the messenger RNA level. Trastuzumab-mediated upregulation of PD-L1 through engagement of immune effector cells may function as a potential mechanism of trastuzumab resistance. Our data justify further investigation of the value of adding anti-PD-1 or anti-PD-L1 therapy to trastuzumab-based treatment.

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.

The adenoviral E1A N-terminal domain represses MYC transcription in human cancer cells by targeting both p300 and TRRAP and inhibiting MYC promoter acetylation of H3K18 and H4K16.

Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells.

α-linolenic acid and docosahexaenoic acid, alone and combined with trastuzumab, reduce HER2-overexpressing breast cancer cell growth but differentially regulate HER2 signaling pathways.

BackgroundDiets rich in the n-3 fatty acid alpha-linolenic acid (ALA) have been shown to reduce breast tumor growth, enhance the effectiveness of the HER2-targeted drug trastuzumab (TRAS) and reduce HER2 signaling in mouse models. It is unclear whether this is due to direct effects of ALA or due to its long-chain n-3 fatty acids metabolites including docosahexaenoic acid (DHA).MethodsThe ability of HER2-overexpressing BT-474 human breast cancer cells to convert ALA to long-chain n-3 fatty acids was determined by measurement of phospholipid fatty acids by gas chromatography following treatment with 100 μM ALA. The effects of 96 h treatment with ALA or DHA, at serum levels seen in mice (50–100 μM), alone and combined with TRAS (10 μg/ml), on BT-474 cell growth measured by trypan blue exclusion, apoptosis measured by flow cytometric analysis of Annexin-V/7-AAD stained cells (ALA and TRAS treatment only) and protein biomarkers HER2 signaling measured by western blot were determined.ResultsALA-treated BT-474 cells had higher phospholipid ALA but no increase in downstream n-3 metabolites including DHA. Both ALA and DHA reduced cell growth with and without TRAS. ALA had no effect on apoptosis. ALA and DHA showed opposite effects on Akt and MAPK phosphorylation; ALA increased and DHA decreased phosphorylation.ConclusionsTogether these data suggest that, while both ALA and its DHA metabolite can reduce HER2-overexpressing breast cancer growth with and without TRAS, they demonstrate for the first time that DHA is responsible for the effects of ALA-rich diets on HER2 signaling pathways.

Abstract 720: Downregulation of ATP6V1B1 in HER2-overexpressing human breast cancer cell line leads resistance against trastuzumab-mediated antibody-dependent cellular cytotoxicity

Abstract Introduction: PIK3CA mutation has been becoming widely recognized as a resistant mechanism or therapeutic biomarker for trastuzumab based anti HER2 targeting therapy, however little is clear on resistant mechanism of trastuzumab mediated antibody dependent cellular cytotoxicity (ADCC) in HER2 overexpressing (HER2+) breast cancer patients. In the current study, we investigated the role of Vacuolar-ATPase subunit ATP6V1B1 on the resistant mechanism of trastuzumab mediated ADCC in HER2+ human breast cancer cell line SK-BR-3. Methods and Results:To establish the trastuzumab mediated ADCC resistant SK-BR-3, isolated healthy PBMCs are co-cultured with 1-10 μg/ml of trastuzumab and SK-BR-3 for 4-9 hours. The cell mixture was incubated with CD45 magnetic beads and CD45 positive PBMCs were removed from the mixture using MACS cell separation system (Miltenyi Biotec, Germany). The survived SK-BR-3 thereafter co-cultured with 1-10 μg/ml of trastuzumab and PBMCs for several times. After 7 passages of the resistant cell making procedure (approximately for 6 months), cytotoxicity was measured by conventional ADCC assay (E/T ratio = 10/1) with various concentration of trastuzumab. The cytotoxicity of ADCC resistant SK-BR-3 was significantly reduced as compared with normal SK-BR-3 cytotoxicity in 10μg/ml of trastuzumab (15.9% and 42.8%, respectively). Then, we analyzed the gene expression profile of ADCC resistant SK-BR-3 by using DNA microarray to sort candidate genes for the resistance. Several genes were up- or down-regulated in ADCC resistant SK-BR-3 and we focused on ATP6V1B1 gene that was significantly reduced on the resistant cells. To clarify the result of comprehensive gene expression analysis, we investigated ATP6V1B1 mRNA level of SK-BR-3 and resistant cell by using real time PCR and found the 50% reduction of ATP6V1B1 mRNA level in ADCC resistant cells. Thus, to evaluate the role of ATP6V1B1 on the trastuzumab mediated ADCC resistant mechanism, ATP6V1B1-knocked down SK-BR-3 (sh-RNA knockdown method) were tested in trastuzumab mediated ADCC assay and found that ATP6V1B1-knocked down SK-BR-3 were significantly less ADCC activity with LDH assay as compared to control SK-BR-3 (19.1% and 40.0%, respectively).Conclusions: ATP6V1B1 might be one factor for trastuzumab mediated ADCC resistance in HER2+ breast cancer cells. Citation Format: Mariko Nishie, Eiji Suzuki, Kosuke Kawaguchi, Keiko Sakamoto, Yuji Fukushima, Masakazu Hattori, Tomoharu Sugie, Masakazu Toi. Downregulation of ATP6V1B1 in HER2-overexpressing human breast cancer cell line leads resistance against trastuzumab-mediated antibody-dependent cellular cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 720. doi:10.1158/1538-7445.AM2015-720

Abstract 1814: Effective auger electron radioimmunotherapy using modified anti-HER2 antibody with nuclear localizing signal

Abstract Objective: Radioimmunotherapy has an advantage in treating tumors scattered in the body. Auger electrons have high cell killing effect because of its high LET, and the short range can prevent damages to normal tissue. However, to achieve higher therapeutic effect, it is desirable to deliver Auger electrons into nucleus and locate close to DNA. The use of nuclear localizing signal (NLS) has been proposed to transport Auger electron emitters into nucleus. To evaluate the cytotoxicity of nuclear localized Auger electron emitter, we performed in-vitro assay using 111In-labeled anti-HER2 antibody attached with NLS peptides to human breast cancer cell lines overexpressing HER2. Material and methods: Trastuzumab, a monoclonal antibody to HER2, was conjugated with CHX-A”-DTPA (DTPA) and derivatized with sulfosuccinimdyl-4-(N-maleimidomethyl) cyclohexane-1carboxylate (sulfo-SMCC) for reaction with synthetic 13-mer NLS peptides (CGYGPKKKRKVGG). 111In was labeled with either trastuzumab-DTPA or NLS-trastuzumab-DTPA, NLS-trastuzumab-DTPA-S carried approximately 4 NLS peptides per antibody and NLS-trastuzumab-DTPA-L carried 10. The radiochemical labeling of 111In-NLS-trastuzumab and 111In-trastuzumab was determined by HPLC. Human mammary epithelial cells HMEpC and two types of human breast cancer cells with low and high HER2 expression, MCF-7 and SKBR3, respectively, were employed for in-vitro study. Nuclear accumulation of 111In was measured in SKBR3 cells by subcellular fraction. Cell viability was evaluated with dye-based cell viability assays in all cell lines. DNA damages were examined with γ-H2AX immunohistochemistry in SKBR3 cells. Results: Nuclear uptake of 111In was 1.3-1.5-fold higher in SKBR3 cells treated with 111In-NLS-trastuzumab than 111In-trastuzumab and increased depending on the number of NLS peptides. Cell viability was significantly reduced with 111In-NLS-trastuzumab comparing 111In-trastuzumab in SKBR3 cells. Both of 111In-NLS-trastuzumab had little or no cytotoxicity on MCF-7 cells and HMEpC cells. SKBR3 cells treated with 111In-NLS-trastuzumab-L showed more γ-H2AX foci than 111In-trastuzumab. Conclusions: The cytotoxicity of 111In-NLS-trastuzumab was higher than that of 111In-trastuzumab by inducing more DNA damages in HER2-overexpressing human breast cancer cells. These data demonstrate that delivery of 111In into cell nucleus by anti-HER2 antibody harboring NLS is an effective strategy for targeted cell killing of the human cancer cells overexpressing HER2. Citation Format: Huizi Li, Sumitaka Hasegawa, Tadashi Kamada, Tsuneo Saga. Effective auger electron radioimmunotherapy using modified anti-HER2 antibody with nuclear localizing signal. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1814. doi:10.1158/1538-7445.AM2015-1814

Expression and characterization of recombinant humanized anti-HER2 single-chain antibody in Pichia pastoris for targeted cancer therapy.

The availability of self-targeting and low immunogenic therapeutic agents is critical to efficient cancer therapy. Therefore, the development of humanized therapeutic antibodies is particularly appealing. A humanized single-chain variable fragment (scFv) antibody that can target human epidermal growth factor receptor-2 (HER2)-overexpressing cancer cells was designed and produced via expression in Pichia pastoris. The expression gave a high yield of 8 mg protein/l (with a purity of 92 %) using shake-flask cultures. Functional studies also revealed that the purified recombinant anti-HER2 scFv exhibited anti-proliferative activity and could bind efficiently to HER2-overexpressing human breast cancer cell line SKBR3. The recombinant scFv offers promising therapeutic and binding efficiencies that are desirable for targeted cancer therapies.

In Vitro Assessment of the Effects of Anti-HER2 Monoclonal Antibodies on Proliferation of HER2-Overexpressing Breast Cancer Cells

HER2 proto-oncogene is critical in the biology of breast cancer and an important therapeutic target of monoclonal antibodies (mAbs). We have recently established a panel of anti-HER2 mAbs recognizing different epitopes within the extracellular domain of HER2. In the present study the antiproliferative effect of these mAbs was investigated on HER2-overexpressing human breast cancer cell line BT474, using radioactive thymidine incorporation assay. Our results demonstrated that while two of the mAbs (1T0 and 2A8) inhibited cell proliferation dose dependently, similar to trastuzumab, six mAbs (1F2, 1B5, 1H9, 4C7, 1H6 and 2A9) augmented cell proliferation. Treatment of BT474 cells with different combinations of two mAbs induced either synergistic inhibitory or stimulatory effects. Our findings indicate that combination of some stimulatory mAbs could completely abolish the inhibitory effect of other mAbs against HER2. Employment of some combinations of mAbs with significant synergistic inhibitory function may improve the therapeutic efficacy of HER2-specific mAbs.

STAT3 activation in HER2-overexpressing breast cancer promotes epithelial-mesenchymal transition and cancer stem cell traits

Clinically, HER2 proto-oncogene amplification is found in about 25–30% of human breast cancers, where it is correlated to a poor prognosis. Constitutive STAT3 activation is found in about 50–60% of the breast tumors and associated with tumorigenesis and drug resistance. In this study, we showed that STAT3 was phosphorylated in HER2-overexpressing, ER-positive human breast tumors and, furthermore, phosphorylated STAT3 promoted the stem-like cell phenotype. We examined the dysregulation of the stem cell markers (Oct-4, Sox-2 and CD44) and the tumorsphere formation in HER2-overexpressing human breast cancer cell lines. We demonstrated that the STAT3 inhibitor, Stattic, treatment abolished the cancer stem cell phenotype in HER2-positive breast cancers. Combined treatment of Herceptin and Stattic showed the synergistic effect on the cancer cell growth in vitro. In addition, when the STAT3 gene was knocked down, the expression of the stem cell markers Oct-4, Sox-2 and CD44 were downregulated and tumorsphere formation was abolished. HER2-elicited STAT3 signaling may provide a potential model for drug resistance induced by stem-like cell characteristics. This mechanism may be responsible for acquiring resistance to Herceptin in the treatment of HER2-overexpressing breast tumors. Based on our findings, targeting pSTAT3 could overcome Herceptin-induced resistance in HER2-overexpressing breast tumors.

Lapatinib induces p27Kip1-dependent G₁ arrest through both transcriptional and post-translational mechanisms

Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor, has been shown to have potent antitumor effects against human breast cancer. Recent studies have shown that lapatinib upregulates p27Kip1 (here after referred to as p27) expression and induces G1 cell cycle arrest in various types of cancer cells. However, the regulation of p27 in lapatinib-induced cell cycle arrest is not well studied. Here we demonstrate that lapatinib-induced cell growth inhibition and G1 cell cycle arrest in HER2-overexpressing human breast cancer cells were dependent on p27. We also show that lapatinib-induced upregulation of p27 expression was mediated through both transcriptional and post-translational mechanisms. On the one hand, lapatinib treatment led to increased FOXO3a expression and enhanced p27 transcription. On the other hand, lapatinib treatment resulted in increased DYRK1B expression, which correlated with increased p27 phosphorylation at Ser10 and decreased p27 degradation. Interestingly, we found that ERβ1 but not ERβ2 expression also upregulated p27 and enhanced lapatinib-induced cell proliferation inhibition and G1 cell cycle arrest in HER2-overexpressing breast cancer cells. Taken together, our results suggest that lapatinib induces p27 expression via both transcriptional and post-translational upregulations, leading to cell cycle arrest and cell proliferation inhibition, and that its effect on breast cancer cells may be modified by ER expression status.

Glycoengineering in cancer therapeutics

Experimentally modified trastuzumab antibodies show increased cytotoxic potency when used with human effector cells against HER2-overexpressing human breast cancer cells in vitro and ex vivo. Furthermore, the superior efficacy of 'glycoengineered' trastuzumab has been confirmed in vivo utilizing a preclinical xenograft model of human HER2-amplified, trastuzumab-resistant human breast cancer. The increased cytotoxic potency coupled with other improvements are achieved by a seemingly modest change in trastuzumab's structure, that is, depletion of two α-L-fucose residues from trastuzumab's heavy chains. Fucose-free trastuzumab binds with much greater affinity to human natural killer cells. This improved binding induces much greater antibody-dependent cellular cytotoxicity against HER2-overexpressing cells. The pharmaceutical industry has recognized the advantages of fucose-free therapeutic antibodies and has developed technologies that aim to mass produce such antibodies for human use. Here, we summarize data from multiple academic and pharmaceutical laboratories highlighting fucose depletion of antibodies as a key strategy of glycoengineering in cancer therapeutics. We use fucose-depleted trastuzumab as a model to show the advantages of this new class of anticancer agents. We predict that these advantages will translate clinically into improved therapeutics for many patients including those with HER2-overexpressing neoplasms.

Abstract P4-02-06: Molecular imaging with trastuzumab and pertuzumab of HER2-positive breast cancer in mice: a step towards personalized medicine

Abstract Introduction: The accurate and reliable assessment of HER2 status is an essential step in the diagnostic workup and selection of targeted treatment strategy in breast cancer patients. However, current ex vivo methods do not evaluate HER2 status possible discordance between primary tumor and distant metastases, nor immediate response to therapeutic intervention. Non-invasive imaging of HER2 expression could be a relevant alternative with additional benefits such as a better selection of patients for HER2-targeted therapies and the possible optimization of treatment strategies. Recent clinical trials suggest a better efficacy of trastuzumab/pertuzumab doublet but a lower activity of pertuzumab monotherapy over trastuzumab alone. Aim: The aim of our study was therefore to evaluate preclinically the use of radiolabeled DOTAGA (2, 2′, 2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) conjugated trastuzumab and pertuzumab for SPECT/CT (Single Photon Emission Computed Tomography/Computed Tomography) HER2 imaging purposes. Methods: Trastuzumab and pertuzumab were conjugated to a new bifunctional chelating agent: DOTAGA anhydride (DOTA analog) and subsequently radiolabeled with the gamma-emitter Indium 111. The functionality of [111In]-DOTAGA-antibodies analogs was evaluated by in vitro saturation assays using HER2-overexpressing human breast cancer cell line (HCC1954). In vivo biodistribution was studied by SPECT/CT imaging at 24h, 48h and 72h after intravenous injection of [111In]-DOTAGA-antibodies in subcutaneous BT474 tumor-bearing rodents. Results: [111In]-DOTAGA-trastuzumab and [111In]-DOTAGA-pertuzumab showed respectively 2.6 and 2.7 DOTAGA/antibody. Both [111In]-DOTAGA-trastuzumab and [111In]-DOTAGA-pertuzumab achieved a radiochemical purity > 95%. Biological activity of [111In]-DOTAGA-trastuzumab and [111In]-DOTAGA-pertuzumab was maintained: the affinity determined on HER2 expressing HCC1954 cell lines, was 5.5 and 5.6 nM. SPECT/CT imaging experiments in tumor-bearing rodents and gamma-counting of organs both showed that at 72h post-injection, more than 60 % of the activity was localized in the tumor (66.9±0.9 %ID/g for [111In]-DOTAGA-trastuzumab and 63.8±6.3 %ID/g for [111In]-DOTAGA-pertuzumab) and that an excess of non-radiolabeled corresponding antibody significantly shifted down tumor-targeting (to 17.6±2.4 %ID/g for [111In]-DOTAGA-trastuzumab and 8.7±0.8 %ID/g for [111In]-DOTAGA-pertuzumab. Conclusions: Our results demonstrate a similar HER2 binding of trastuzumab and pertuzumab. Both radiolabeled [111In]-DOTAGA-trastuzumab and [111In]-DOTAGA-pertuzumab should be considered and might be promising tools for molecular imaging diagnosis of HER2 overexpression in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-02-06.

Modulation of MicroRNA-194 and Cell Migration by HER2-Targeting Trastuzumab in Breast Cancer

Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 3′-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.

Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition.

Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable whereas active PI3K-Akt and MAPK were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFK) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.

Abstract 4519: In vitro cell and in vivo animal studies of the demethylating agent in treating HER2-amplified breast cancer

Abstract PURPOSE: The recent evidence shows that HER2-amplified breast cancers exhibit more frequent CpG island methylation than other types of breast cancers, suggesting epigenetic alterations are involved in HER2-mediated tumorigenesis. In this study, we addressed the possibility of the use of the demethylating agent (5-aza-2’-deoxycytidine, decitabine) for treatment of HER2-overexpressing breast cancer. EXPERIMENTAL DESIGN: Two cell model systems were used in this study: murine primary Her2-overexpressing tumor cell line (A6O5, derived from the tumor naturally developed in HER2/neu transgenic mice) and human HER2-overexpressing luminal breast cancer cell line MCF7-HER2-18. Cells were cultured in regular cell culture dishes with the serum-containing medium (adhesive culture) or in ultra-low attachment dishes with the serum-free sphere culture medium (tumorsphere culture) for drug testing. Tumors developed from A6O5 cells injected into mammary glands of HER2/neu transgenic mice were used as the xenograft model. The early spontaneous mammary tumors naturally developed in HER2/neu transgenic mice (size < 5 mm) were used for drug treatment. RESULTS: The in vitro growth of A6O5 was more significantly inhibited by decitabine than that of non-tumorigenic murine mammary epithelial cells. Consistently, MCF7-HER2-18 cells were more sensitive to decitabine-induced growth suppression than its control vector-transfected cell line MCF7-Neo. These results suggest that HER2 overexpression renders cancer cells more sensitive to the effect of decitabine. Surprisingly, the tumorsphere formation of A6O5 and MCF7-HER2-18 was dramatically abrogated by decitabine, suggesting that self-renewal of cancer stem cells in these two cell lines was annihilated by decitabine. Importantly, decitabine inhibited the in vivo growth of A6O5 xenograft tumors. For proof-in-principle of this study, we treated the HER2/neu transgenic mice bearing early developing mammary tumors with decitabine. Indeed, decitabine effectively suppressed the in vivo tumor growth of the decitabine-treated group compared to that of the vehicle-treated group. CONCLUSIONS: This study for the first time explores the potential of the demethylating agent in the treatment of HER2-amplified breast cancers using in vitro cell and in vivo animal models. These findings shed light on the translational application of epigenetic modifiers with the HER2-signaling blockers to treatment of HER2-amplified breast cancer patients. 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 4519. doi:10.1158/1538-7445.AM2011-4519

Furanodienone induces cell cycle arrest and apoptosis by suppressing EGFR/HER2 signaling in HER2-overexpressing human breast cancer cells

Overexpression of EGFR and HER2 is seen in breast cancers and results in poor prognosis and decreased patient survival. Clinically, EGFR and HER2 are effective therapeutic targets. The objective of this study is to investigate the in vitro effects of furanodienone, an active chemical component isolated from Rhizoma Curcumae, on the activation of EGFR/HER2 signaling, cell cycle, and apoptosis in HER2-overexpressing BT474 and SKBR3 cells. Cell growth was assessed by SRB protein assay. Cell cycle analysis was carried out by flow cytometry, and apoptosis was observed by Annexin V and DAPI staining. Effects of furanodienone on the activation of EGFR/HER2 signaling-related proteins were analyzed by western blotting. Furanodienone inhibited cell growth in BT474 and SKBR3 cells. Furanodienone caused G1 arrest in BT474 cells and induced apoptosis in SKBR3 cells. Furanodienone interfered with EGFR/HER2 signaling in treated cells as shown by decreases in phosphorylated EGFR, HER2, Akt, Gsk3β and an increase in p27(kip1) protein. Accordingly, furanodienone inhibited EGF-induced phosphorylation of EGFR, HER2, Akt, and Gsk3β. EGFR-specific siRNA knockdown did not affect the cell growth inhibitory effect of furanodienone. On the contrary, specific siRNA knockdown of HER2 increased cellular resistance to furanodienone toxicity. In HER-2-deficient MDA-MB-231 cells, the transfection and expression of HER2 increased the sensitivity of cells to furanodienone toxicity. Furanodienone inhibited EGFR/HER2 signaling pathway in BT474 and SKBR3 cells. More importantly, the effect of furanodienone was specifically dependent on HER2, but not EGFR, expression.