Heregulin Research Articles

The HER2- and Heregulin β1 (HRG)–Inducible TNFR Superfamily Member Fn14 Promotes HRG-Driven Breast Cancer Cell Migration, Invasion, and MMP9 Expression

HER2 overexpression occurs in 15% to 20% of all breast cancers and is associated with increased metastatic potential and poor patient survival. Abnormal HER2 activation, either through HER2 overexpression or heregulin (HRG):HER3 binding, elicits the formation of potent HER2-HER3 heterodimers and drives breast cancer cell growth and metastasis. In a previous study, we found that fibroblast growth factor-inducible 14 (Fn14), a member of the TNF receptor superfamily, was frequently overexpressed in human HER2+ breast tumors. We report here that HER2 and Fn14 are also coexpressed in mammary tumors that develop in two different transgenic mouse models of breast cancer. In consideration of these findings, we investigated whether HER2 activation in breast cancer cells could directly induce Fn14 gene expression. We found that transient or stable transfection of MCF7 cells with a HER2 expression plasmid increased Fn14 protein levels. Also, HRG1-β1 treatment of MCF7 cells transiently induced Fn14 mRNA and protein expression. Both the HER2- and HRG1-β1-induced increase in Fn14 expression in MCF7 cells as well as basal Fn14 expression in HER2 gene-amplified AU565 cells could be reduced by HER2 kinase inhibition with lapatinib or combined HER2 and HER3 depletion using siRNA. We also report that Fn14-depleted, HER2-overexpressing MCF7 cells have reduced basal cell migration capacity and reduced HRG1-β1-stimulated cell migration, invasion, and matrix metalloproteinase (MMP)-9 expression. Together, these results indicate that Fn14 may be an important downstream regulator of HER2/HER3-driven breast cancer cell migration and invasion.

Abstract 4633: MM-111, a bispecific HER2 and HER3 antibody, synergistically combines with trastuzumab and paclitaxel in preclinical models of gastric cancer.

Abstract Increasing evidence suggests amplification or overexpression of human epidermal growth factor receptor-2 (HER2), and HER3 levels are correlated to decreased survival in gastric cancers. Our previous studies established that MM-111, a novel bispecific antibody that specifically targets the HER2/HER3 heterodimer, blocks heregulin (HRG) binding to HER3 and corresponding downstream signaling pathways. In this study we used computational and experimental biology to assess the activity of MM-111 combination therapies in treating gastric cancer. First, we utilized a multi-scale computational network model of HER2-positive tumors that relates HER family signal transduction with cell growth to predict that MM-111 plus trastuzumab would synergistically inhibit tumor growth. The mechanism underlying this prediction is that the combinatorial blockade of HER2 and HER3 suppresses signal propagation through both the AKT and ERK cascades and this leads to synergistic cell growth arrest. Cell viability and signaling experiments in a gastric cancer model (NCI-N87) confirmed these predictions. In particular, MM-111 plus trastuzumab caused 25% greater cell growth inhibition than the additive effects of the individual treatments (compared to Bliss independence; p< 0.05). Next, we assessed the effect of exogenous HRG on this combination therapy. HRG reduced the activity of trastuzumab but did not affect MM-111’s activity, and it enhanced the synergy of this combination (50% greater than Bliss independence; p < 10−8). We then assessed the effect of adding paclitaxel to the trastuzumab plus MM-111 combination. Our key finding was that activation of HER3 by HRG greatly reduced paclitaxel's activity. MM-111, but not trastuzumab, restored cell sensitivity to paclitaxel in the presence of HRG. A synergistic effect (35% greater than Bliss independence; p < 10−8) was observed for the combination of MM-111, trastuzumab and paclitaxel. Similar combination effects were also achieved using a three-dimensional NCI-N87 spheroid assay: Inhibition of HER2, HER3, and the AKT and ERK pathways correlated with spheroid growth inhibition. Furthermore, HRG-overexpressing NCI-N87 (NCI-N87-HRG) xenografts were less sensitive to paclitaxel than the wild type xenografts. MM-111 restored the sensitivity to paclitaxel in these xenografts, even when given as a second line treatment regimen after tumor progression on the front-line regimen (trastuzumab plus 5-fluorouracil). Trastuzumab alone did not overcome the HRG-induced paclitaxel resistance, but its combination with MM-111 showed significantly greater activity than either drug alone. Taken together, our data suggest that combination of MM-111, trastuzumab and paclitaxel is worthy of investigation as a potential therapeutic strategy for the treatment of HER2-positive gastric cancer. Citation Format: Bo Zhang, Johanna Lahdenranta, Jinyan Du, Daniel Kirouac, Stephanie Nguyen, Ryan Overland, Violette Paragas, Arthur Kudla, Ulrik Nielsen, Charlotte McDonagh, Matthew Onsum. MM-111, a bispecific HER2 and HER3 antibody, synergistically combines with trastuzumab and paclitaxel in preclinical models of gastric cancer. [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 4633. doi:10.1158/1538-7445.AM2013-4633

Abstract 3832: Cancer tissue-derived spheroid, CTOS, for evaluation of drug response from individual patient tumor samples.

Abstract Primary culture of cancer cells can be an excellent platform for investigating the biology of cancer and predicting chemosensitivity for individual patients, yet has been hampered by technical difficulties. We established a method of primary culture for human colorectal cancer: cancer tissue-originated spheroid (CTOS) method. The principle of the method is to maintain cell-cell contact throughout preparation and culture; never dissociating the samples into single cells. The clusters of cancer cells turn out to spheroids, CTOSs, within a short period. CTOSs of colorectal cancer consisted of highly purified cancer cells, and they were prepared with high efficiency. We applied the CTOS method to non-small cell lung cancer and urothelial cancer. For non-small cell lung cancer, we used 125 surgical specimens and 18 pleural effusions. The success rate of CTOS preparation from surgical specimens was 80.0%. CTOSs were subjected to sensitivity assay and pathway activation assay for the EGFR tyrosine kinase inhibitor (TKI) erlotinib. The sensitivity differed among individual patient CTOSs. The EGFR mutation status and expression levels corresponded with erlotinib sensitivity, confirming previous clinical findings. Next, we examined urothelial cancer. The success rate of CTOS preparation from the non muscle-invasive urothelial cancer was 90.7%, whereas that from the muscle-invasive urothelial cancer was 68.2%. Total development rate was 84.2%. Using these CTOSs, we investigated the effects of growth factors on the growth of CTOS in vitro. Among the growth factors examined, including EGF, IGF-1, basic FGF, and activin A, we found that heregulin (HRG)/neuregulin1, a ligand of HER3, potently induced CTOS growth in both lung and urothelial cancers. HRG promoted the growth of primary CTOSs in 10 out of 13 (71.4%) cases of lung cancer, and in 2 out of 4 (50%) cases of urothelial cancer. Along with the growth, HRG most potently stimulated the phosphorylation of AKT. In lung cancer CTOSs, a neutralizing antibody against HER3 inhibited the growth and the phosphorylation of HER3 and AKT. In all three tumor types examined, CTOSs formed xenograft tumors in immunodeficient mice, and the tumors retained the features of the parental tumors. As CTOS can be cryopreserved, we are accumulating stocks of CTOSs derived from patient samples as well as the CTOSs from xenograft tumors. CTOS experiments can be shuttled between in vitro and in vivo. Thus, the CTOS method enables us to obtain primary colorectal, lung, and urothelial tumor cells of high viability and purity. CTOS could be a new platform for studying lung cancer biology, and be useful to select patients for molecular targeting drugs. Citation Format: Masahiro Inoue, Hiroko Endo, Masayuki Ohue, Kazuo Nishimura, Fumio Imamura, Jiro Okami, Hiroaki Okuyama. Cancer tissue-derived spheroid, CTOS, for evaluation of drug response from individual patient tumor samples. [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 3832. doi:10.1158/1538-7445.AM2013-3832

Bone marrow stromal cell transdifferentiation into oligodendrocyte-like cells using triiodothyronine as a inducer with expression of platelet-derived growth factor α as a maturity marker.

The present study investigated the functional maturity of oligodendrocyte derived from rat bone marrow stromal cells (BMSC). The BMSC were isolated from female Sprague-Dawley rats and evaluated for different markers, such as fibronectin, CD106, CD90, Oct-4 and CD45. Transdifferentiation of OLC from BMSC was obtained by exposing the BMSC to DMSO and 1 µM all-trans-retinoic acid during the pre-induction stage and then induced by heregulin (HRG), platelet-derived growth factor AA (PDGFR-alpha), fibroblast growth factor and T3. The neuroprogenitor cells (NPC) were evaluated for nestin, neurofilament 68, neurofilament 160 and glial fibrillary acidic protein gene expression using immunocytochemistry. The OLC were assessed by immunocytochemistry for O4, oligo2, O1 and MBP marker and gene expression of PDGFR-alpha was examined by RT-PCR. Our results showed that the fibronectin, CD106, CD90, CD45 and Oct-4 were expressed after the fourth passage. Also, the yield of OLC differentiation was about 71% when using the O1, O4 and oligo2 markers. Likewise, the expression of PDGFR-alpha in pre-oligodendrocytes was noticed, while MBP expression was detected in oligodendrocyte after 6 days of the induction. The conclusion of the study showed that BMSC can be induced to transdifferentiate into mature OLC.

Reduction of the ST6 β-Galactosamide α-2,6-Sialyltransferase 1 (ST6GAL1)-catalyzed Sialylation of Nectin-like Molecule 2/Cell Adhesion Molecule 1 and Enhancement of ErbB2/ErbB3 Signaling by MicroRNA-199a

Nectin-like molecule 2 (Necl-2)/cell adhesion molecule 1 (CADM1) is shown to be down-regulated by the promoter hypermethylation and/or loss of heterozygosity at chromosome 11q23.2 in many types of cancers, including lung and breast cancers, and is proposed to serve as a tumor suppressor. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers, and other mechanisms for the suppression of Necl-2 are presumed to be present. We previously showed that Necl-2 interacts in cis with ErbB3 and suppresses the heregulin (HRG)-induced ErbB2/ErbB3 signaling for cell movement and death. We studied here the relationship between Necl-2 and microRNA-199a (miR-199a) that is up-regulated or down-regulated in a variety of cancers. miR-199a did not directly target the Necl-2 mRNA or affect its mRNA level in human lung cancer A549 cells and human embryonic kidney HEK293 cells. Necl-2 was at least sialylated by the sialyltransferase ST6 β-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1). miR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2. In addition, miR-199a enhanced the HRG-induced ErbB2/ErbB3 signaling. These results indicate that the suppressive role of Necl-2 in the HRG-induced ErbB2/ErbB3 signaling is regulated by miR-199a at least through the reduction of the ST6GAL1-catalyzed sialylation of Necl-2 and/or through the reduction of the protein level of Necl-2 presumably by the protein degradation.

Scattering of MCF7 Cells by Heregulin ß-1 Depends on the MEK and p38 MAP Kinase Pathway

Heregulin (HRG) β1 signaling promotes scattering of MCF7 cells by inducing breakdown of adherens and tight junctions. Here, we show that stimulation with HRG-β1 causes the F-actin backbone of junctions to destabilize prior to the loss of adherent proteins and scattering of the cells. The adherent proteins dissociate and translocate from cell–cell junctions to the cytosol. Moreover, using inhibitors we show that the MEK1 pathway is required for the disappearance of F-actin from junctions and p38 MAP kinase activity is essential for scattering of the cells. Upon treatment with a p38 MAP kinase inhibitor, adherens junction complexes immediately reassemble, most likely in the cytoplasm, and move to the plasma membrane in cells dissociated by HRG-β1 stimulation. Subsequently, tight junction complexes form, most likely in the cytoplasm, and move to the plasma membrane. Thus, the p38 MAP kinase inhibitor causes a re-aggregation of scattered cells, even in the presence of HRG-β1. These results suggest that p38 MAP kinase signaling to adherens junction proteins regulates cell aggregation, providing a novel understanding of the regulation of cell–cell adhesion.

Abstract P4-08-03: The impact of the heregulin-HER receptor signaling axis on response to HER tyrosine kinase inhibitors.

Abstract Background: HER receptor tyrosine kinases (EGFR/HER1; HER2; HER3; HER4) and their soluble ligands represent a robust system involved in the regulation of a diverse array of cellular processes. Deregulation of HER receptors, notably HER2, has been linked to the initiation and progression of breast cancer and other solid tumors. Relatively less is known about the role of HER receptor soluble ligands in tumorigenesis and responsiveness to HER targeted therapies. Here we will discuss the impact of the HER3 ligand heregulin (HRG) on the sensitivity of breast cancers to HER tyrosine kinase inhibitors (TKIs), and identify TKI strategies to treat HRG-driven breast cancers. Methods: The effects of exogenous HRG (50ng/ml) on the antitumor activity of a panel of TKIs with different enzymatic properties e.g. a reversible, selective inhibitor (lapatinib) and irreversible, pan-HER inhibitors (neratinib; CI-1033) were assessed in HER2+ breast cancer (BC) cell lines. The concentration of TKIs used was in the 1–2.5 uM range, and cells were treated over a 72 hr course. Vehicle treatment alone served as controls. The impact of the above mentioned treatments on total HER receptor and specific EGFR, HER2, and HER3 phosphotyrosine sites, in addition to the phosphorylation state of components of downstream MAPK and PI3K signaling pathways was analyzed through western blot. In addition to studying the effects of exogenous HRG, the impact of TKIs on a model of triple negative BC (TNBC) known to produce HRG in an autocrine manner was also evaluated. QRT-PCR was used to assess the effects of TKIs on HER receptor mRNA levels. Results: Lapatinib inhibited proliferation and phosphorylation of EGFR, HER2, HER3, and downstream MAPK and PI3K signaling pathways in HER2+ SKBR3 and BT474 cell lines. Pre-treatment with HRG abrogated the antitumor effects of a therapeutic concentration of lapatinib (1 uM), and reversed the inhibitory effects of lapatinib on the phosphorylation of HER receptors and components of their downstream signaling pathways e.g. Erk1/2 and Akt. Neratinib also blocked proliferation and phosphorylation in HER2+ BC cells. In contrast to lapatinib, the antitumor effects of neratinib were not reversed by exogenous HRG. Interestingly, treatment with neratinib and similar pan-HER irreversible TKIs, but not reversible TKIs, resulted in loss of HER2 and EGFR protein expression. QRT-PCR was used to evaluate if this effect was at the level of transcription. Furthermore, neratinib inhibited cell proliferation and blocked EGFR signaling in a TNBC model driven by autocrine produced HRG. Conclusions: Our findings suggest that HRG is a mediator of therapeutic resistance to lapatinib in HER2+ and TNBC. Neratinib demonstrates profound effects on HER signaling by markedly reducing HER2 and EGFR protein expression and blocking the pro-tumorigenic effects of autocrine or paracrine expression of HRG. In contrast to HRG, we previously showed that EGF, an EGFR specific ligand did not reverse the antitumor effects of lapatinib in breast cancer cells. Thus, the selection of HER targeted therapies in a given tumor should take into account not only the HER receptor expression profile, but also the presence of autocrine or paracrine derived ligands activating HER receptors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-03.

ErbB3-binding protein EBP1 decreases ErbB2 levels via a transcriptional mechanism

Ectopic expression of EBP1, an ErbB3-interacting protein, reduces the expression of the ErbB2 protein and mRNA. However, the mechanism of EBP1-induced decrease in ErbB2 mRNA levels has not yet been determined. Since EBP1 affects both transcriptional and post-transcriptional processes, we evaluated the ability of EBP1 to regulate ErbB2 transcription and RNA stability. We discovered that while wild-type EBP1 decreased the activity of a proximal ErbB2 promoter, EBP1 mutants unable to interact with the Sin3A transcriptional repressor inhibited activity to a lesser extent. EBP1 also decreased the activity of distal ErbB2 promoters. Chromatin immunoprecipitation analysis indicated that EBP1 bound both distal and proximal endogenous ErbB2 promoters in serum-starved conditions. The ErbB3 ligand heregulin (HRG) at growth-promoting concentrations reduced EBP1 binding to the ErbB2 promoter. Although endogenous EBP1 bound ErbB2 mRNA, EBP1 overexpression or ablation of EBP1 protein by shRNA failed to alter ErbB2 mRNA stability. These results suggest that the major effect of EBP1 on ErbB2 mRNA levels is at the transcriptional level.

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces of MCF-7 human breast cancer cells. The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al. (2006) EMBO J 25:4215-4222], suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow. The dissociation rate constant from the liganded monomer was intermediate. The dynamic changes in the association and dissociation kinetics in relation to the dimerization of ErbB displayed negative cooperativity, which resulted in apparent low- and high-affinity sites of HRG association on the cell surface.

Cellular Effects of HER3-Specific Affibody Molecules

Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.

Targeting the eIF4A RNA helicase blocks translation of the MUC1-C oncoprotein

The oncogenic MUC1-C subunit is aberrantly overexpressed in most human breast cancers by mechanisms that are not well understood. The present studies demonstrate that stimulation of non-malignant MCF-10A cells with epidermal growth factor (EGF) or heregulin (HRG) results in marked upregulation of MUC1-C translation. Growth factor-induced MUC1-C translation was found to be mediated by PI3K->AKT, and not MEK->ERK1/2, signaling. We also show that activation of the mTORC1->S6K1 pathway decreases PDCD4, an inhibitor of the eIF4A RNA helicase, and contributes to the induction of MUC1-C translation. In concert with these results, treatment of growth factor-stimulated MCF-10A cells with the eIF4A RNA helicase inhibitors, silvestrol and CR-1-31-B, blocked increases in MUC1-C abundance. The functional significance of the increase in MUC1-C translation is supported by the demonstration that MUC1-C, in turn, forms complexes with EGFR and promotes EGFR-mediated activation of the PI3K->AKT pathway and the induction of growth. Compared to MCF-10A cells, constitutive overexpression of MUC1-C in breast cancer cells was unaffected by EGF stimulation, but was blocked by inhibiting PI3K->AKT signaling. The overexpression of MUC1-C in breast cancer cells was also inhibited by blocking eIF4A RNA helicase activity with silvestrol and CR-1-31-B. These findings indicate that EGF-induced MUC1-C expression is mediated by the PI3K->AKT pathway and the eIF4A RNA helicase, and that this response promotes EGFR signaling in an autoinductive loop. The findings also indicate that targeting the eIF4A RNA helicase is a novel approach for blocking MUC1-C overexpression in breast cancer cells.

Downregulation of the tumor-suppressor miR-16 via progestin-mediated oncogenic signaling contributes to breast cancer development

IntroductionExperimental and clinical evidence points to a critical role of progesterone and the nuclear progesterone receptor (PR) in controlling mammary gland tumorigenesis. However, the molecular mechanisms of progesterone action in breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are short ribonucleic acids which have also been found to play a pivotal role in cancer pathogenesis. The role of miRNA in progestin-induced breast cancer is poorly explored. In this study we explored progestin modulation of miRNA expression in mammary tumorigenesis.MethodsWe performed a genome-wide study to explore progestin-mediated regulation of miRNA expression in breast cancer. miR-16 expression was studied by RT-qPCR in cancer cell lines with silenced PR, signal transducer and activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. Breast cancer cells were transfected with the precursor of miR-16 and proliferation assays, Western blots or in vivo experiments were performed. Target genes of miR-16 were searched through a bioinformatical approach, and the study was focused on cyclin E. Reporter gene assays were performed to confirm that cyclin E 3'UTR is a direct target of miR-16.ResultsWe found that nine miRNAs were upregulated and seven were downregulated by progestin in mammary tumor cells. miR-16, whose function as a tumor suppressor in leukemia has already been shown, was identified as one of the downregulated miRNAs in murine and human breast cancer cells. Progestin induced a decrease in miR-16 levels via the classical PR and through a hierarchical interplay between Stat3 and the oncogenic transcription factor c-Myc. A search for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR region. We found that, similar to the molecular mechanism underlying progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the induction of cyclin E expression by progestin. Moreover, overexpression of miR-16 abrogated the ability of progestin to induce cyclin E upregulation, revealing that cyclin E is a novel target of miR-16 in breast cancer. Overexpression of miR-16 also inhibited progestin-induced breast tumor growth in vitro and in vivo, demonstrating for the first time, a role for miR-16 as a tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand heregulin (HRG) downregulated the expression of miR-16, which then participates in the proliferative activity of HRG in breast tumor cells.ConclusionsIn this study, we reveal the first progestin-regulated miRNA expression profile and identify a novel role for miR-16 as a tumor suppressor in progestin- and growth factor-induced growth in breast cancer.

Regulation of HRG-β1-induced proliferation, migration and invasion of MCF-7 cells by upregulation of GPR30 expression

The cooperation and communication between different cell signaling transduction pathways are considered critical in the development of various types of cancer as well as drug resistance. There is evidence of crosstalk between the G protein-coupled receptor 30 (GPR30), the newly discovered estrogen receptor (ER), and the ErbB family. Heregulin (HRG)-β1, the ligand for ErbB3 and ErbB4, upregulates GPR30 expression in MCF-7, T-47D and BT-474 breast cancer cell lines that express ERα. In the present study, recombinant human HRG-β1 was used to investigate the upregulation of GPR30 expression by HRGs in MCF-7 breast cancer cells which were ERα-positive. In MCF-7 cells, the ErbB2 inhibitor, AG825, the MAPK inhibitor, PD98059, and the MEK1/2 inhibitor, U0126, blocked the HRG-β1-induced GPR30 expression. 17-β-estradiol (E2) boosted the HRG-β1-induced proliferation, migration and invasion of MCF-7 cells. Similar to E2, the specific GPR30 agonist, G-1, promoted HRG-β1-induced migration and invasion, but inhibited growth. Using the specific GPR30 antagonist, G-15, or the small interfering RNA for GPR30, the functions of GPR30 after treatment with HRG-β1 were further investegated. The results from our study indicate that the interruption between GPR30 signaling and the ErbB family system may serve as a promising therapeutic strategy for breast cancer.

Abstract 1063: The ErbB3/4 receptor ligand Heregulin negatively regulates ErbB2 and ErbB3 receptor levels

Abstract ErbB2 is overexpressed in 30% of breast cancers and its overexpression is associated with poor prognosis. In addition, the ErbB2/3 heterodimer forms the most potent mitogenic receptor in vitro and is key to the proliferation of breast cancer cells. Despite the importance of this heterodimer in breast cancer progression, the negative regulation of these ErbB tyrosine kinases is still poorly understood. We demonstrate here for the first time that the ErbB3/4 ligand Heregulin (HRG) reduces mRNA and protein levels of both ErbB2 and ErbB3 in human breast cancer cell lines. We have found that after 24h of HRG treatment, ErbB2 expression decreased at both the RNA and protein levels in ErbB2 overexpressing AU565, BT474 and LTLT-Ca breast cancer cell lines. In contrast, EGFR levels were unaffected by HRG treatment. An initial decline in steady state mRNA levels was first noted at 1 hour after HRG treatment. ErbB3 levels also decreased in LTLT-Ca cells after HRG treatment. Lapatinib, which inhibits ErbB2 and EGFR kinase activity and HRG induced-ErbB3 phosphorylation, diminished the HRG- induced decrease in ErbB2 and ErbB3 in AU565 and LTLT-Ca cells. This finding suggests that the kinase activity of EGFR/ErbB2 may be involved in the HRG-induced down-regulation. HRG did not change the activity of ErbB2 promoter reporter constructs spanning 215 kb to 6007 kb upstream of the ErbB2 transcription start site as indicated by luciferase assays. An ongoing study suggests that HRG could be involved in decreasing ErbB2 mRNA stability. Our findings suggest that resistance to Lapatinib may be due in part to the fact that inhibition of ErbB2/ EGFR kinase activity dampens the negative effect of HRG on ErbB2 expression, resulting in elevated levels of ErbB2 in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1063. doi:1538-7445.AM2012-1063

Abstract 2719: MM-141, a novel bispecific antibody co-targeting IGF-1R and ErbB3, blocks ligand-induced signaling and demonstrates antitumor activity

Abstract Monoclonal antibodies have significantly advanced our ability to treat cancer, but most patients do not show durable responses to therapy. This, in part, can be explained by the tumor cells responding to two or more growth factors in a redundant fashion. We show that cancer cell lines frequently rely on Insulin-like growth factor 1 (IGF-1) and Heregulin (HRG) signaling to support their survival and proliferation. Using a Network Biology approach, we have designed, constructed and optimized a novel bispecific antibody, MM-141, for co-targeting of ErbB3 and IGF-1R. MM-141 blocks IGF-1, IGF-2, and HRG binding to IGF1R and ErbB3, and causes downregulation of these receptors. MM-141 inhibits phosphorylation of IGF1R and ErbB3 as well as downstream activation of the PI3K/Akt pathway. In our in vitro and in vivo testing, MM-141 displays potency in the presence of single or multiple ligands, over a broad range of receptor profiles. Inhibition of growth by MM-141 has been observed in vitro as well as in vivo in multiple xenograft models including human pancreatic cancer (BxPC-3) and human prostate cancer (DU145). MM-141 does not bind the insulin receptor, thus reducing the risk of metabolic side effects. Our in vivo and in vitro studies reveal that MM-141 has favorable pharmaceutical properties and pharmacokinetic profiles. We are advancing MM-141 into preclinical development. Taken together, these results suggest that MM-141 has the potential to be an effective therapeutic for treatment of patients with solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2719. doi:1538-7445.AM2012-2719

ErbB receptor tyrosine kinase/NF-κB signaling controls mammosphere formation in human breast cancer

Breast cancer is one of the most common cancers in humans. However, our understanding of the cellular and molecular mechanisms underlying tumorigenesis in breast tissues is limited. Here, we identified a molecular mechanism that controls the ability of breast cancer cells to form multicellular spheroids (mammospheres). We found that heregulin (HRG), a ligand for ErbB3, induced mammosphere formation of a breast cancer stem cell (BCSC)-enriched population as well as in breast cancer cell lines. HRG-induced mammosphere formation was reduced by treatment with inhibitors for phosphatidyl inositol 3-kinase (PI3K) or NF-κB and by expression of IκBα-Super Repressor (IκBαSR), a dominant-negative inhibitor for NF-κB. Moreover, the overexpression of IκBαSR in breast cancer cells inhibited tumorigenesis in NOD/SCID mice. Furthermore, we found that the expression of IL8, a regulator of self-renewal in BCSC-enriched populations, was induced by HRG through the activation of the PI3K/NF-κB pathway. These findings illustrate that HRG/ErbB3 signaling appears to maintain mammosphere formation through a PI3K/NF-κB pathway in human breast cancer.

In Vitro Differentiation of Bone Marrow Stromal Cells into Oligodendrocyte-like Cells Using Triiodothyronine as Inducer

ABSTRACTAn in vitro technique was devised to induced autologous adult stem cells into oligodendrocyte-like cells. In this study, a protocol was developed for the induction of bone marrow stromal cells (BMSCs) into oligodendrocyte-like cells. BMSCs were incubated in one of these three pre-inducers: dimethyl sulfoxide (DMSO), β-mercaptoethanol (βME) or biotylated hydroxyanisol (BHA), each followed by retinoic acid (RA) treatment. The percentage of viable cells in BHA-RA preinduced cells was significantly lower than the others. The results showed that the preinduced cells were immunoreactive for nestin and NF-68; among the mentioned protocols, the immunoreactivity yielded by following the DMSO-RA protocol was significantly higher than the others. Moreover, no significant immunoreactivity was observed for preinduced cells to O4, O1, MBP (myelin basic protein), S100, and GFAP (glial fibrillary acidic protein). The cells were immunoreactive to oligo-2. Two phases of induction were done: the first was a combination of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and heregulin (HRG), followed by either triiodothyronine (T3) or Forskolin (FSK) as the second phase. The conclusion is that the trans-differentiation of BMSCs by DMSO followed by RA (preinduction stage) then bFGF-PDGF-HRG followed by T3 (10 ng/ml) (induction stage) can be a potential source for oligodendrocyte-like cells preparation.

ASSOCIATION BETWEEN GROWTH FACTOR HEREGULIN -1^|^alpha; AND RECEPTORS IN GROWTH OF OVARIAN CANCER CELL LINE WITH HIGH POTENTIALITY OF PERITONEAL DISSEMINATION

Ovarian cancer, one of the poor-prognosis gynecological malignancies, is often associated with extensive peritoneal carcinomatosis when initially treated. The mechanism of the formation of peritoneal carcinomatosis from ovarian cancer is still unknown. It has been reported that overexpression of cancer-related growth factors and/or receptors may worsen the prognosis of diseases. In the previous paper, we had established the human ovarian serous adenocarcinoma cell lines from those with no potentiality of peritoneal dissemination (FOC-3) to high potentiality (MFOC-3), however, the mechanism of its phenotypical change remains unknown. In this paper, we compared these two cell lines for growth potential and the expression of growth factor heregulin (HRG)-1α and HER-2, HER-3, and HER-4 receptors. In addition, the effect of anti-receptor antibodies on cell growth was investigated. RT-PCR and Western blot analysis found the promotion of the expression of HRG precursor and HRG-1α in MFOC-3. Examination of the number of growing cells over time revealed a statistically significant increase in the number of cells in MFOC-3 compared with FOC-3. In a study using the addition of exogenous HRG-1α, no changes were observed in FOC-3 while statistically significant cell growth was noted in MFOC-3. In a growth inhibition study, statistically significant cell growth inhibition was achieved with the addition of anti-HER2 receptor antibody. Taken together, the results of this study suggested that HRG may play an important role in the increased growth potential of peritoneal dissemination of ovarian cancer. In particular, HER-2 receptors that can act as a starting point to trigger intracellular signaling pathways are strongly involved in the progression of cancer. Therefore, molecular target drug therapies blocking the HER-2 receptor are promising candidates for ovarian cancer treatment in the future.

Abstract C145: Compounds that inhibit the activation of Grb2 by EGFR, HER2, and HER3 complexes.

Abstract Purpose: To demonstrate that ORIL003 (Glc4-Rha2-Glc-diosgenin; full structure will be disclosed) inhibits the activation of Grb2 by EGFR, HER2 and HER3 complexes using the Receptor-Heteromer Investigation Technology (Receptor-HIT) on the bioluminescence resonance energy transfer (BRET) platform. Introduction: The unique property of inhibiting a range of receptor tyrosine kinases (RTKs) in the cell plasma membrane has been demonstrated in a family of steroid saponins. As the compounds have a pan-inhibitory effect, they are effective in inhibiting a wide range of cancer cell types, including lung, breast, prostate, colorectal, melanoma, ovarian and leukemia. Methods: Receptor-HIT, previously published in the form of G protein-coupled receptor heteromer identification technology (GPCR-HIT; See et al, 2011 Assay Drug Dev Technol 9:21–30), was used to determine close proximity and activation of ErbB growth factor receptors and Grb2. EGFR, HER2 and HER3 were tagged with Rluc8 (a variant of Renilla luciferase) as donor; Grb2 was tagged with GFP2 (a variant of green fluorescent protein) as acceptor. Human embryonic kidney 293 cells were stimulated with either epidermal growth factor (EGF) or heregulin (HRG) at a concentration of 20–50 nM. ORIL003 (as an example of the steroid saponin family) was applied to the cells at a concentration of 10 μM, which was known from in vitro studies to completely inhibit cancer cell growth. Results: Stimulation by EGFR or HRG resulted in recruitment of Grb2. This recruitment was inhibited by the use of ORIL003 in a dose-dependent manner. Furthermore, constitutive interactions between the ErbB growth factor receptors and Grb2 also appear to be inhibited by ORIL003. Conclusions: ORIL003 is effective in inhibiting the activation of Grb2 by EGFR, HER2 and HER3 complexes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C145.

Abstract B205: Therapeutically targeting redundant, growth factor-induced prosurvival signaling with MM-141, a novel bispecific antibody targeting IGF-1R and ErbB3.

Abstract Monoclonal antibodies have significantly advanced our ability to treat cancer, but most patients do not have durable responses to therapy. We find that the majority of tumor cell lines are responsive to activation by two or more growth factors. In particular cell lines grown in culture and in mice appear to frequently rely on Insulin-like growth factor 1 (IGF-1) and Heregulin (HRG) signaling to redundantly support their proliferation. Using a Network Biology approach, we have designed, constructed and optimized a novel bispecific antibody, MM-141, for simultaneous targeting and inhibition of ErbB3 and IGF-1R. MM-141 blocks IGF-1, IGF-2, and heregulin-induced AKT signaling, inhibits growth of multiple cancer cell lines, and is active in xenograft models of human pancreatic cancer (BxPC-3) and human prostate cancer (DU145). MM-141 does not bind the insulin receptor, thus strongly reducing the risk of metabolic side effects. MM-141 has favorable CMC and pharmacokinetic profiles and is being advanced into preclinical development. Taken together, these results suggest that MM-141 has the potential to be an effective therapeutic for treatment of patients with solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B205.