ErbB2-expressing Cells Research Articles

Janus Kinase 1 Plays a Critical Role in Mammary Cancer Progression

Janus kinases (JAKs) and their downstream STAT proteins play key roles in cytokine signaling, tissue homeostasis, and cancer development. Using a breast cancer model that conditionally lacks Janus kinase 1, we show here that JAK1 is essential for IL-6-class inflammatory cytokine signaling and plays a critical role in metastatic cancer progression. JAK1 is indispensable for the oncogenic activation of STAT1, STAT3, and STAT6 in ERBB2-expressing cancer cells, suggesting that ERBB2 receptor tyrosine kinase complexes do not directly activate these STAT proteins invivo. A genome-wide gene expression analysis revealed that JAK1 signaling has pleiotropic effects on several pathways associated with cancer progression. We established that FOS and MAP3K8 are targets of JAK1/STAT3 signaling, which promotes tumorsphere formation and cell migration. The results highlight the significance of JAK1 as a rational therapeutic target to block IL-6-class cytokines, which are master regulators of cancer-associated inflammation.

Designing a cancer therapeutic peptide by combining the mitochondrial targeting domain of Noxa and ErbB2-targeting moieties.

Many anticancer drugs target epidermal growth factor receptors to inhibit receptor tyrosine kinases and tumor growth. Here, we show that an ErbB2-targeting pronecrotic peptide (KWSY:MTD) selectively kills tumor cells expressing ErbB2 in vitro. An antibody against ErbB2 inhibits KWSY:MTD-induced cell death. KWSY:MTD causes membrane permeability which allows propidium iodide entry into the cytosol and the release of HMGB1 into the media, indicative of necrosis. Mitochondrial swelling occurs in response to KWSY:MTD. Moreover, in vivo analysis using a mouse model shows that KWSY:MTD partially suppressed growth in tumor tissue bearing ErbB2-expressing cells, but did not have obvious toxicity in mouse liver or kidney tissue. Taken together, KWSY:MTD has potential as an ErbB2-targeting anticancer drug.

Overcoming the Challenge of Transduction of Human T-cells with Chimeric Antigen Receptor (CAR) Specific for ERBB2 Antigen

Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor (CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene. CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabead-conjugated CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells.

Superior Suppression of ErbB2-positive Tumor Cells by a Novel Human Triparatopic Tribody.

Downregulation of the epidermal growth factor receptor family of receptors is improved by combining different antibodies to noncompetitive epitopes. For ErbB2/HER2 this has already been translated into clinical practice by using a combination of trastuzumab and pertuzumab. Moreover, cocktails of 2 or 3 anti-epidermal growth factor receptor antibodies show an enhanced downregulation of the receptor due to the induction of matrix formation. A more efficient method for inducing matrix formation and receptor downregulation might include the use of trispecific reagents. A triparatopic Tribody consisting of 3 noncompeting ErbB2 binders was compared with equivalent trivalent monoparatopic counterparts, as well as to a cocktail of 3 monoclonal antibodies for its effects on downregulation of the ErbB2 receptor's kinase activity and survival of several ErbB2-expressing cancer cell lines. The triparatopic Tribody was significantly more efficient in downregulating ErbB2 and inhibiting tumor cell growth than either the control monoparatope tribodies or the combinatorial treatment with the 3 different parental antibodies on all the tested tumor cells, including trastuzumab-resistant cell lines. The enhancement of effectivity was dependent on all 3 binding moieties. Because the novel Tribody allows reduction of the costs of production (as only 1 construct provides the antitumor effects of 3 antibodies) and has an intermediate molecular size (∼100 kDa) well suited for both tumor penetration and acceptable half-life, it has the potential to become a precious tool for therapeutic use particularly in trastuzumab-resistant cancer patients.

Abstract 716: Targeting Mek-Erk pathway abrogates hypoxia-mediated lapatinib resistance in ErbB2-positive breast cancer cells

Abstract Overexpression/amplification of the tyrosine kinase ErbB2 is often observed in breast cancers. ErbB2 expression activates EGFR signaling pathway promoting tumorigenesis. Breast cancer ErbB2-positive patients are treated with lapatinib, a dual EGFR/ERBB2 inhibitor. Despite lapatinib clinical efficacy, acquired resistance has been reported and its mechanism is poorly understood. Another factor that is associated with mammary tumors is hypoxia. Hypoxia stabilizes hypoxia inducible factor 1 (HIF-1), a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors are often chemoresistant. Here, we show that hypoxia decreases lapatinib-mediated growth inhibition and apoptosis in three-dimensional cultures. In agreement with this data, hypoxia also promotes activation of signaling pathways downstream from ErbB2/EGFR including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 and can be abrogated by HIF-1 RNAi. Moreover stable overexpression HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to rescue cell proliferation and survival and maintain ERK and AKT signaling in the presence of lapatinib. We also show that activation of ERK signaling is required for hypoxia-mediated lapatinib resistance and inhibition of MEK (a kinase upstream of Erk) reverses hypoxia protective effect on ErbB2-expressing cells. One potential mechanism of how hypoxia/HIF-1 may lead to activation of ERK pathway is via regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of ERK pathway. Here we demonstrate that cells exposed to hypoxia have decreased levels of DUSP2 and it correlate with increased activation of Erk signaling. Moreover, reducing DUSP2 expression alone in ErbB2-positive breast cancer cells is sufficient to reduce lapatinib-mediated effects. And DUSP2 overexpression abrogates hypoxia-mediated protective effect on ErbB2-expressing cells treated with lapatinib. All these data suggests that targeting MEK-Erk signaling pathway may reverse hypoxia mediated lapatinib resistance and might be a viable option for lapatinib resistant ErbB2-posive tumors. Indeed, a combination of lapatinib and an FDA approved MEK inhibitor trametinib abrogates hypoxia protective effect on cell growth inhibition and apoptosis in three dimensional (3D) cultures. Thus targeting MEK/ERK pathway in hypoxic breast cancer may improve an anti-ErbB2 therapy in breast cancer. Citation Format: Sergey Karakashev, Mauricio Reginato. Targeting Mek-Erk pathway abrogates hypoxia-mediated lapatinib resistance in ErbB2-positive breast cancer cells. [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 716. doi:10.1158/1538-7445.AM2015-716

EMT Transition Alters Interstitial Fluid Flow-Induced Signaling in ERBB2-Positive Breast Cancer Cells.

A variety of biophysical forces are altered in the tumor microenvironment (TME) and these forces can influence cancer progression. One such force is interstitial fluid flow (IFF)-the movement of fluid through the tissue matrix. IFF was previously shown to induce invasion of cancer cells, but the activated signaling cascades remain poorly understood. Here, it is demonstrated that IFF induces invasion of ERBB2/HER2-expressing breast cancer cells via activation of phosphoinositide-3-kinase (PI3K). In constitutively activate ERBB2-expressing cells that have undergone epithelial-to-mesenchymal transition (EMT), IFF-mediated invasion requires the chemokine receptor CXCR4, a gradient of its ligand CXCL12, and activity of the PI3K catalytic subunits p110α and β. In wild-type ERBB2-expressing cells, IFF-mediated invasion is chemokine receptor-independent and requires only p110α activation. To test whether cells undergoing EMT alter their signaling response to IFF, TGFβ1 was used to induce EMT in wild-type ERBB2-expressing cells, resulting in IFF-induced invasion dependent on CXCR4 and p110β. This study identifies a novel signaling mechanism for interstitial flow-induced invasion of ERBB2-expressing breast cancer cells, one that depends on EMT and acts through a CXCR4-PI3K pathway. These findings suggest that the response of cancer cells to interstitial flow depends on EMT status and malignancy.

The Role of Multicellular Aggregation in the Survival of ErbB2-positive Breast Cancer Cells during Extracellular Matrix Detachment

The metastasis of cancer cells from the site of the primary tumor to distant sites in the body represents the most deadly manifestation of cancer. In order for metastasis to occur, cancer cells need to evade anoikis, which is defined as apoptosis caused by loss of attachment to extracellular matrix (ECM). Signaling from ErbB2 has previously been linked to the evasion of anoikis in breast cancer cells but the precise molecular mechanisms by which ErbB2 blocks anoikis have yet to be unveiled. In this study, we have identified a novel mechanism by which anoikis is inhibited in ErbB2-expressing cells: multicellular aggregation during ECM-detachment. Our data demonstrate that disruption of aggregation in ErbB2-positive cells is sufficient to induce anoikis and that this anoikis inhibition is a result of aggregation-induced stabilization of EGFR and consequent ERK/MAPK survival signaling. Furthermore, these data suggest that ECM-detached ErbB2-expressing cells may be uniquely susceptible to targeted therapy against EGFR and that this sensitivity could be exploited for specific elimination of ECM-detached cancer cells.

Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.

Hypoxia/HIF1α induces lapatinib resistance in ERBB2-positive breast cancer cells via regulation of DUSP2.

ERBB2/HER2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can promote tumor progression. Breast cancer patients with ERBB2 amplifications are currently treated with lapatinib, a small-molecule kinase inhibitor that specifically blocks EGFR/ERBB2 signaling. Here, we show that hypoxia, via HIF-1, induces resistance to lapatinib-mediated effects in ERBB2-expressing mammary epithelial and ERBB2-positive breast cancer cells. Lapatinib-mediated growth inhibition and apoptosis in three-dimensional (3D) cultures are decreased under hypoxic conditions. Hypoxia can maintain activation of signaling pathways downstream from ERBB2 including AKT and ERK in the presence of lapatinib. HIF-1 is both required and sufficient to induce lapatinib resistance as overexpression of stable HIF-1 in ERBB2-expressing cells blocks lapatinib-mediated effects and maintains ERBB2-downstream signaling under normoxic conditions. Under hypoxia, activation of ERK signaling is required for lapatinib resistance as treatment with MEK inhibitor trametinib reverses hypoxia-mediated lapatinib resistance. HIF-1 can bypass the lapatinib-treated inhibition of the ERK pathway via inhibition of the dual-specificity phosphatase 2 (DUSP2). Indeed, overexpression of DUSP2 in ErbB2-positve breast cancer cells reverses hypoxia-mediated lapatinib resistance. Thus, our results provide rationale for therapeutic evaluation of the treatment of hypoxic ERBB2 expressing breast tumors with a combination of lapatinib and MEK inhibitors.

Abstract 1824: Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2

Abstract Breast cancer is one of the most common cancers among women. Many factors are associated with this disease including overexpression/amplification of the tyrosine kinase HER2/ErbB2. ErbB2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can activate multiple signaling pathways that can promote tumor progression via regulation of cell growth, proliferation, and survival. Breast cancer patients with ErbB2 amplifications are currently treated with lapatinib, a small-molecule kinase inhibitor that blocks the active site of ErbB2 to inhibit EGFR/ErbB2 downstream signaling. While lapatinib has displayed clinical efficacy in breast cancer patients, acquired resistance has been reported and the mechanism of this resistance is poorly understood. Hypoxia or low oxygen tension occurs in many breast cancers and is associated with poor patient survival. One of the major players in the cellular response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors often exhibit weak response to chemotherapeutic agents and are associated with poor prognosis. Here, we show that hypoxia, via HIF-1α, blocks lapatinib-mediated effects on ErbB2-expressing mammary epithelial and ErbB2-positive breast cancer cells. Lapatinib-mediated growth inhibition and apoptosis in three dimensional (3D) cultures are decreased under hypoxic conditions (1%O2) Consistent with these results, we find that hypoxia can maintain activation of signaling pathways downstream from ErbB2 including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 expression as cells expressing HIF-1 RNAi no longer are resistant to lapatinib treatment. Moreover, overexpression of stable HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to inhibit lapatinib-mediated effects on proliferation and survival and maintain ERK and AKT signaling. Moreover, hypoxia-mediated activation of ERK signaling, but not AKT, is required for lapatinib resistance as MEK inhibitors (including clinically approved tramatinib) reverses hypoxia-mediated lapatinib resistance in standard and 3D cultures. HIF-1 may bypass lapatinib-treated inhibition of the ERK pathway via its regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of the ERK pathway and we show that cells exposed to hypoxia contain decreased DUSP2 levels and correlate with increased Erk activation. Indeed, reducing DUSP2 expression via RNAi in ErbB2-positive breast cancer cells is sufficient to reduce lapatanib-mediated effects under normal oxygen. Thus, hypoxia, via HIF-1, causes lapatinib resistance in ErbB2-expressing breast cancer cells and suggests that targeting MEK/ERK pathway in hypoxic breast cancer may increase sensitivity to ErbB2-targeted therapy. Citation Format: Sergey Karakashev. Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1824. doi:10.1158/1538-7445.AM2014-1824

O6.05 * ERBB2/HER2-SPECIFIC NATURAL KILLER CELLS FOR ADOPTIVE IMMUNOTHERAPY OF GLIOBLASTOMA

BACKGROUND: While EGFRvIII appears a logical target for immunotherapy, only a subpopulation of tumor cells express EGFRvIII and immune escape has been demonstrated. ErbB2 is overexpressed in a substantial proportion of glioblastomas and has been successfully utilized in immunotherapies. Natural killer (NK) cells are the first line of defense against viral infections and malignant cells. The continuously growing cytotoxic cell line NK-92 holds promise for cancer immunotherapy. Safety of infusion of high doses of NK-92 was established in previous phase I clinical trials utilizing irradiated cells to prevent permanent engraftment. METHODS: To provide NK-92 cells with pre-determined tumor-cell specificity, we generated a lentiviral second generation chimeric antigen receptor (CAR) construct (5.28.z) employing the ErbB2 (HER2)-specific scFv(FRP5) antibody fragment for target cell recognition, and human CD28-CD3 ζ as a composite signaling moiety. An ErbB2-specific single cell clone (NK-92/5.28.z) was isolated, which showed high and selective cytotoxicity towards ErbB2-expressing tumor cells of various origins in vitro. We evaluated the cytotoxicity of NK-92/5.28.z cells against a panel of glioblastoma cell lines and primary glioblastoma cultures with different levels of Erb2 expression in vitro and in vivo. RESULTS: 13 out of a total of 15 established glioblastoma cell lines and 5 out of 5 primary glioblastoma cell lines were specifically lysed by NK-92/5.28.z cells, although to varying degrees. ErbB2 expression was necessary for cytotoxicity of NK-92/5.28.z cells towards glioblastoma cells, and ectopic overexpression of ErbB2 enhanced their cytotoxicity. In subcutaneous glioblastoma xenograft models, injections of NK-92/5.28.z induced prolonged regression in NOD-scid/gamma (NSG) mice with grafts of LN-319 cells and retarded the growth of LNT-229 grafts. In an orthotopic intracranial model utilizing LN-319 cells, tumor size in mice intratumorally injected with NK-92/5.28.z cells weekly was substantially smaller compared to controls as assessed by MR imaging. Injections of NK-92/5.28.z cells also conferred an impressive advantage in symptom free survival in two independent experiments (median 73 days in controls versus 140 days in NK-92/5.28.z treated animals in experiment A, p < 0,05, and median 77 days in controls versus 163 days in NK-92/5.28.z treated animals in experiment B, p < 0,01). In preparation of a phase I trial, GMP-compliant protocols for expansion of the NK-92/5.28.z line were established. CONCLUSIONS: Adoptive immunotherapy with application of ErbB2-specific NK-92/5.28.z cells may be a promising new immunotherapy approach for ErB2 positive glioblastoma. A phase I trial for glioblastoma patients with local injections of NK-92/5.28.z cells is in preparation.

ERBB2/HER2-SPECIFIC NATURAL KILLER CELLS FOR ADOPTIVE IMMUNOTHERAPY OF GLIOBLASTOMA

BACKGROUND: While EGFRvIII appears a logical target for immunotherapy, only a subpopulation of tumor cells express EGFRvIII and immune escape has been demonstrated. ErbB2 is overexpressed in a substantial proportion of glioblastomas and has been successfully utilized in immunotherapies. Natural killer (NK) cells are the first line of defense against viral infections and malignant cells. The continuously growing cytotoxic cell line NK-92 holds promise for cancer immunotherapy. Safety of infusion of high doses of NK-92 was established in previous phase I clinical trials utilizing irradiated cells to prevent permanent engraftment. METHODS: To provide NK-92 cells with pre-determined tumor-cell specificity, we generated a lentiviral second generation chimeric antigen receptor (CAR) construct (5.28.z) employing the ErbB2 (HER2)-specific scFv(FRP5) antibody fragment for target cell recognition, and human CD28-CD3 ζ as a composite signaling moiety. An ErbB2-specific single cell clone (NK-92/5.28.z) was isolated, which showed high and selective cytotoxicity towards ErbB2-expressing tumor cells of various origins in vitro. We evaluated the cytotoxicity of NK-93/5.28.z cells against a panel of glioblastoma cell lines and primary glioblastoma cultures with different levels of Erb2 expression in vitro and in vivo. RESULTS: To provide NK-92 cells with pre-determined tumor-cell specificity, we generated a lentiviral second generation chimeric antigen receptor (CAR) construct (5.28.z) employing the ErbB2 (HER2)-specific scFv(FRP5) antibody fragment for target cell recognition, and human CD28-CD3 ζ as a composite signaling moiety. An ErbB2-specific single cell clone (NK-92/5.28.z) was isolated, which showed high and selective cytotoxicity towards ErbB2-expressing tumor cells of various origins in vitro. We evaluated the cytotoxicity of NK-93/5.28.z cells against a panel of glioblastoma cell lines and primary glioblastoma cultures with different levels of Erb2 expression in vitro and in vivo. CONCLUSIONS: Adoptive immunotherapy with application of ErbB2-specific NK-92/5.28.z cells may be a promising new immunotherapy approach for ErB2 positive glioblastoma. A phase I trial for glioblastoma patients with local injections of NK-92/5.28.z cells is in preparation. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Activated ERBB2/HER2 licenses sensitivity to apoptosis upon endoplasmic reticulum stress through a PERK-dependent pathway.

HER2/Neu/ERBB2 is a receptor tyrosine kinase overexpressed in approximately 20% of human breast tumors. Truncated or mutant isoforms that show increased oncogenicity compared with the wild-type receptor are found in many breast tumors. Here, we report that constitutively active ERBB2 sensitizes human breast epithelial cells to agents that induce endoplasmic reticulum stress, altering the unfolded protein response (UPR) of these cells. Deregulation of the ERK, AKT, and mTOR activities elicited by mutant ERBB2 was involved in mediating this differential UPR response, elevating the response to endoplasmic reticulum stress, and apoptotic cell death. Mechanistic investigations revealed that the increased sensitivity of mutant ERBB2-expressing cells to endoplasmic reticulum stress relied upon a UPR effector signaling involving the PERK-ATF4-CHOP pathway, upregulation of the proapoptotic cell surface receptor TRAIL-R2, and activation of proapoptotic caspase-8. Collectively, our results offer a rationale for the therapeutic exploration of treatments inducing endoplasmic reticulum stress against mutant ERBB2-expressing breast tumor cells.

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1+/− mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1+/+ and Becn1+/− immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.

Abstract A084: TGFβ induces Chordin-like 1 (Chrdl1) expression in ErbB2 expressing breast cancer cells with attenuated ShcA signaling

Abstract Background: In 25 to 30% of human primary breast cancer, the ErbB2 receptor is overexpressed and is associated with poor prognosis. Transgenic mouse models have demonstrated that ErbB2 co-operates with TGFβ signaling pathway to promote the invasive and metastatic behavior of breast cancer cells. By transient knockdown approaches, a role for ShcA in mediating cooperation between the ErbB2 and TGFβ pathways has been established. Furthermore, a stable knockdown of ShcA in NMuMG cells overexpressing oncogenic ErbB2 significantly reduced tumor growth, which was found to be the consequence of reduced proliferation and endothelial cell recruitment coupled with increased apoptosis. Many of the migratory and invasive effects induced by TGFβ are secondary to the induction of a transcriptional program and TGFβ is known to uniquely induce the expression of specific genes in cells overexpressing ErbB2. Given that ShcA is required for TGFβ induced migration and invasion of ErbB2 expressing cells, we hypothesize that the signaling molecules engaged downstream of ShcA will ultimately modulate gene expression induced by TGFβ stimulation. Experimental Approach: To characterize ShcA dependent gene expression changes induced by TGFβ in ErbB2-expressing cells, we generated NMuMG-ErbB2 cells harboring dox-inducible ShcA shRNAs. These cell populations were divided into two groups treated with (ShcAlow) or without (ShcAhigh) Doxycycline, and each group was subjected to three conditions: one was left untreated and the others stimulated for 3 and 24 hours with TGFβ. Using gene expression profiling, we have identified a list of differentially expressed genes that are regulated by TGFβ signaling in the context of acute ShcA loss. Subsequently, we identified genes that have functions associated with migratory and invasive phenotypes. Results: Among the candidates identified by these approaches, Chrdl1, which has been demonstrated to interact with and neutralize several different members of the TGFβ family including BMP4, BMP5, BMP6, TGFβ1 and TGFβ2, displayed elevated transcript levels in ShcAlow cells following TGFβ treatment. ELISA assays reveal that reduced ShcA signaling results in the up-regulation of secreted Chrdl1 protein in ErbB2-expressing cells following TGFβ stimulation. Similar results were also obtained using additional human and mouse breast cancer cell lines. We next determined whether Chrdl1 found in the conditioned media of our NMuMG-ErbB2 ShcAlow cells, is functional. We observed that conditioned media from ShcAlow cells, which was harvested from cells stimulated with TGFβ (high Chrdl1), was able to decrease the activation of Smad1/5/8 following BMP4 stimulation, as measured by immunblot analysis for phospho-Smad1/5/8. Furthermore, immunohistochemical analysis confirmed that Chrdl1 induction, in response to reduced ShcA expression, is also observed in mammary tumors in vivo. Moreover, high levels of Chrdl1 correlated with diminished pSmad2 and pSmad1/5/8 in these tumor lysates. Conclusion: We have identified Chrdl1, a BMP inhibitor, as an interesting candidate that may account for the reduced growth and metastasis that accompany ShcA loss in ErbB2-expressing breast cancer cells. Citation Format: Chanèle Cyr-Depauw, Jason Northey, Zhifeng Dong, Sean Cory, Michael Hallett, Peter M. Siegel. TGFβ induces Chordin-like 1 (Chrdl1) expression in ErbB2 expressing breast cancer cells with attenuated ShcA signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A084.

Abstract PR15: Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2

Abstract Breast cancer is one of the most common cancers among women. Many factors are associated with this disease including overexpression/amplification of the tyrosine kinase HER2/ErbB2. ErbB2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can activate multiple signaling pathways that can promote tumor progression via regulation of cell growth, proliferation, metabolism, and survival. Breast cancer patients with ErbB2 amplifications are currently treated with lapatinib (Tykerb), a small-molecule kinase inhibitor that specifically blocks the active site of ErbB2 to inhibit EGFR/ErbB2 downstream signaling. While lapatinib has displayed clinical efficacy in breast cancer patients, acquired resistance has been reported and the exact mechanism of this resistance is poorly understood. Hypoxia or low oxygen tension occurs in many breast cancers and is associated with poor patient survival. One of the major players in the cellular response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors often exhibit weak response to different chemotherapeutic agents and poor prognosis. Here, we show that hypoxia, via HIF-1α, blocks lapatinib-mediated effects on ErbB2-expressing mammary epithelial and ErbB2-positive breast cancer cells. Lapatinib-mediated growth inhibition is reduced under hypoxic conditions (1%O2). Consistent with these results, we find that hypoxia can maintain activation of signaling pathways downstream from ErbB2 including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 expression as cells expressing HIF-1 RNAi no longer are resistant to lapatinib treatment. Moreover, overexpression of stable HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to inhibit lapatinib-mediated effects on proliferation and survival and maintain ERK and AKT signaling. One possible mechanism of how HIF-1 may bypass lapatinib-treated inhibition of the ERK pathway is via regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of the ERK pathway and we show that cells exposed to hypoxia contain decreased DUSP2 levels and correlate with increased Erk activation. Indeed, reducing DUSP2 expression alone in ErbB2-positive breast cancer cells is sufficient to reduce lapatinib-mediated effects. Thus, hypoxia, via HIF-1, causes lapatinib resistance in ErbB2-expressing breast cancer cells and suggests that targeting ERK/MAPK pathway in hypoxic breast cancer may increase sensitivity to ErbB2-targeted therapy. This abstract is also presented as poster C63. Citation Format: Sergey V. Karakashev, Reginato J. Mauricio. Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR15.

Chimeric Antigen Receptor-Redirected T Cells Display Multifunctional Capacity and Enhanced Tumor-Specific Cytokine Secretion Upon Secondary Ligation of Chimeric Receptor

The aim of the current study was to fully elucidate the functions of T cells genetically modified with an erbB2-specific chimeric antigen receptor (CAR). In this study, key functional parameters of CAR T cells were examined following antigen-specific stimulation of the chimeric anti-erbB2 receptor. Gene-modified T cells produced the cytokines IFN-γ, IL-2, IL-4, IL-10, TNF-α and IL-17, and the chemokine RANTES upon CAR ligation. A multifunctional capacity of these CAR T cells was also demonstrated, where 13.7% of cells were found to simultaneously express IFN-γ and CD107a, indicative of cytolytic granule release. In addition, the CAR T cells were able to respond to a greater degree on the second ligation of CAR, which has not been previously shown. IFN-γ secretion levels were significantly higher on second ligation than those secreted following initial ligation. CAR-expressing T cells were also demonstrated to lyze erbB2-expressing tumor cells in the absence of activity against bystander cells not expressing the erbB2 antigen, thereby demonstrating exquisite specificity. This study demonstrates the specificity of CAR gene-engineered T cells and their capacity to deliver a wide range of functions against tumor cells with an enhanced response capability after initial receptor engagement.

Abstract 3967: ErbB2/HER2-specific natural killer cells for adoptive immunotherapy of glioblastoma.

Abstract In addition to primary natural killer (NK) cells, continuously growing cytotoxic cell lines such as NK-92 are being considered for adoptive cancer immunotherapy. High cytotoxicity of NK-92 has been shown against malignant cells of hematologic origin in preclinical studies, and general safety of infusion of NK-92 cells has been established in phase I clinical trials. To enhance their therapeutic utility, we genetically modified NK-92 cells to express chimeric antigen receptors (CAR) specific for different tumor-associated surface antigens including ErbB2 (HER2). Such CAR were composed of a tumor-specific scFv antibody fragment fused via hinge and transmembrane domains to intracellular signaling proteins such as CD3 zeta chain or a composite CD28-CD3 zeta fusion molecule. Glioblastoma multiforme (GBM) is the most common and severe intracranial malignant tumor in humans. Despite aggressive therapy, recurrence of GBM is very frequent, and the median survival of GBM patients is only 12 to 15 months. Since enhanced ErbB2 expression was found in up to 80% of GBM cases, adoptive ErbB2-targeted immunotherapy may represent a more efficient alternative to standard therapy. For development towards clinical applications, here we generated a lentiviral second generation CAR construct (5.28.z) specific for the ErbB2 antigen, and established GMP-compliant protocols for transduction and expansion of NK-92 cells. An ErbB2-specific single cell clone (NK-92/5.28.z) was isolated, which showed high and selective cytotoxicity towards different established ErbB2-expressing glioblastoma cells and tumor cells of various other origins in vitro, as well as specific tumor homing in murine in vivo models. Treatment with NK-92/5.28.z cells also resulted in marked inhibition of the growth of subcutaneous glioblastoma xenografts in NOD/SCID γc KO mice. Ongoing work now focuses on evaluating the feasibility and efficacy of intracranial application of NK-92/5.28.z cells in orthotopic xenograft models of ErbB2-positive glioblastoma cells as a basis for further development of these cells as an adoptive immunotherapy for glioblastoma patients. Citation Format: Concong Zhang, Kurt Schönfeld, Michael Burger, Sabrina Genßler, Christiane Sahm, Christian Brendel, Sonja Naundorf, Marcus Odendahl, Ulrike Köhl, Torsten Tonn, Manuel Grez, Joachim P. Steinbach, Winfried S. Wels. ErbB2/HER2-specific natural killer cells for adoptive immunotherapy of glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3967. doi:10.1158/1538-7445.AM2013-3967

Abstract A65: ShcA is required for the TGFβ-induced repression of Chordin-like-1 in ErbB2-expressing breast cancer cells

Abstract Clinical data and transgenic mouse models have provided clear evidence of a critical role for the adaptor protein ShcA in mammary tumor formation and metastasis downstream of several oncogenes. Indeed, we have demonstrated that stable silencing of ShcA expression in ErbB2-expressing breast cancer cells impairs tumor outgrowth and metastasis in vivo. In addition, we have identified a requirement for an intact PTB domain and the three tyrosine residues in ShcA for mediating TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. Together, these observations position the ShcA adaptor as an important signaling node that facilitates cooperation between the ErbB2 and TGFβ signaling pathways in breast cancer progression. To understand how ShcA signaling contributes to TGFβ-induced gene expression changes, we generated a doxycycline-inducible system to deplete ShcA levels from ErbB2-expressing breast cancer cells. The inducible silencing of ShcA in pre-established ErbB2-positive tumors resulted in impaired tumor cell proliferation and survival in vivo. Gene expressing profiling was then performed on cells with an inducible loss of ShcA expression to identify ShcA-dependent transcriptional responses downstream of active TGFβ and ErbB2 signaling in breast cancer cells. ShcA was found to mediate the suppression of Chordin-like 1, a BMP antagonist. Conditioned media taken from TGFβ-stimulated ErbB2-expressing cells treated with doxycycline to achieve reduced ShcA levels was confirmed to contain increased levels of Chordin-like-1 compared with unstimulated controls. This same conditioned media was efficient at inhibiting the BMP-stimulated phosphorylation of Smad proteins 1/5/8 in ErbB2-expressing cells. Furthermore, this TGFβ upregulation of Chordin-like 1 production resulting from ShcA loss correlated with decreased activation of Smad1/5/8 in ShcA-deficient tumor cells in vivo, and may represent a transcriptional mechanism through which ShcA coordinates ErbB2 and TGFβ initiated tumor growth and invasion. Citation Format: Jason J. Northey, Zhifeng Dong, Chanele Cyr-Depauw, Sean Cory, Peter M. Siegel. ShcA is required for the TGFβ-induced repression of Chordin-like-1 in ErbB2-expressing breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A65.

IL-15 augments antitumoral activity of an ErbB2/HER2 cancer vaccine targeted to professional antigen-presenting cells.

Targeted delivery of tumor-associated antigens to professional antigen-presenting cells (APC) is being explored as a strategy to enhance the antitumoral activity of cancer vaccines. Here, we generated a cell-based system for continuous in vivo production of a CTLA-4-ErbB2 fusion protein as a therapeutic vaccine. The chimeric CTLA-4-ErbB2 molecule contains the extracellular domain of CTLA-4 for specific targeting to costimulatory B7 molecules on the surface of APC, genetically fused to residues 1-222 of human ErbB2 (HER2) as an antigenic determinant. In wild-type BALB/c mice, inoculation of syngeneic epithelial cells continuously secreting the CTLA-4-ErbB2 fusion vaccine in the vicinity of subcutaneously growing ErbB2-expressing renal cell carcinomas resulted in the rejection of established tumors, accompanied by the induction of ErbB2-specific antibodies and cytotoxic T cells. In contrast, treatment with CTLA-4-ErbB2 vaccine-secreting producer cells alone was insufficient to induce tumor rejection in ErbB2-transgenic WAP-Her-2 F1 mice, which are characterized by pronounced immunological tolerance to the human self-antigen. When CTLA-4-ErbB2 producer cells were modified to additionally secrete interleukin (IL)-15, antigen-specific antitumoral activity of the vaccine in WAP-Her-2 F1 mice was restored, documented by an increase in survival, and marked inhibition of the growth of established ErbB2-expressing, but not antigen-negative tumors. Our results demonstrate that continuous in vivo expression of an APC-targeted ErbB2 fusion protein results in antigen-specific immune responses and antitumoral activity in tumor-bearing hosts, which is augmented by the pleiotropic cytokine IL-15. This provides a rationale for further development of this approach for specific cancer immunotherapy.