ErbB2 Overexpression Research Articles

Germline and Somatic NF1 Alterations Are Linked to Increased HER2 Expression in Breast Cancer.

NF1 germline mutation predisposes to breast cancer. NF1 mutations have also been proposed as oncogenic drivers in sporadic breast cancers. To understand the genomic and histologic characteristics of these breast cancers, we analyzed the tumors with NF1 germline mutations and also examined the genomic and proteomic profiles of unselected tumors. Among 14 breast cancer specimens from 13 women affected with neurofibromatosis type 1 (NF1), 9 samples (NF + BrCa) underwent genomic copy number (CN) and targeted sequencing analysis. Mutations of NF1 were identified in two samples and TP53 were in three. No mutation was detected in ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, and STK11 HER2 (ErbB2) overexpression was detected by IHC in 69.2% (9/13) of the tumors. CN gain/amplification of ERBB2 was detected in 4 of 9 with DNA analysis. By evaluating HER2 expression and NF1 alterations in unselected invasive breast cancers in TCGA datasets, we discovered that among samples with ERBB2 CN gain/amplification, the HER2 mRNA and protein expression were much more pronounced in NF1-mutated/deleted samples in comparison with NF1-unaltered samples. This finding suggests a synergistic interplay between these two genes, potentially driving the development of breast cancer harboring NF1 mutation and ERBB2 CN gain/amplification. NF1 gene loss of heterozygosity was observed in 4 of 9 NF + BrCa samples. CDK4 appeared to have more CN gain in NF + BrCa and exhibited increased mRNA expression in TCGA NF1--altered samples. Cancer Prev Res; 11(10); 655-64. ©2018 AACR.

Biomarkers for Predicting Clinical Outcomes of Chemoradiation-Based Bladder Preservation Therapy for Muscle-Invasive Bladder Cancer.

Chemoradiation-based bladder preservation therapy (BPT) is currently a curative option for non-metastatic muscle-invasive bladder cancer (MIBC) patients at favorable risk or an alternative to radical cystectomy (RC) for those who are unfit for RC. In BPT, only patients who achieve complete response (CR) after chemoradiation have a favorable prognosis and quality of life with a preserved functional bladder. Thus, predicting CR and favorable prognosis is important for optimal patient selection for BPT. We reviewed biomarkers for predicting the clinical outcomes of chemoradiation-based BPT. The biomarkers studied were categorized into those related to apoptosis, cell proliferation, receptor tyrosine kinases, DNA damage response genes, hypoxia, molecular subtype, and others. Among these biomarkers, the Ki-67 labeling index (Ki-67 LI) and meiotic recombination 11 may be used for selecting BPT or RC. Ki-67 LI and erythroblastic leukemia viral oncogene homolog 2 (erbB2) may be used for predicting both the chemoradiation response and the prognosis of patients on BPT. Concurrent use of trastuzumab and a combination of carbogen and nicotinamide can overcome chemoradiation resistance conferred by erbB2 overexpression and tumor hypoxia. Further studies are needed to confirm the practical utility of these biomarkers for progress on biomarker-directed personalized management of MIBC patients.

Protein kinase C mediated internalization of ErbB2 is independent of clathrin, ubiquitination and Hsp90 dissociation

Overexpression of ErbB2 is frequent in cancer and understanding the mechanisms which regulate its expression is important. ErbB2 is considered endocytosis resistant. It has no identified ligand, but upon heterodimerization it is a potent mediator of proliferative signaling. A recent study established a role for protein kinase C (PKC) in internalization and recycling of ErbB2. We have now further investigated the molecular mechanisms involved in PKC-mediated downregulation of ErbB2. We confirm that PMA-induced PKC activation causes ErbB2 internalization, but while the Hsp90 inhibitor 17-AAG induced ErbB2 degradation, PMA had no such effect. When combined with 17-AAG, PMA had additive effect on ErbB2 internalization indicating that Hsp90 inhibition and PKC activation induce internalization by alternative mechanisms. We confirm that while 17-AAG-induced internalization was clathrin-mediated, PMA-induced internalization was clathrin independent. This difference may be explained by while both 17-AAG and PMA reduced the constitutive tyrosine phosphorylation of ErbB2, only 17-AAG induced Hsp90 dissociation, Hsp70 recruitment and ubiquitination of ErbB2. Importantly, since PMA induced internalization of ErbB2, but not dissociation of Hsp90, Hsp90 does not per se retain ErbB2 at the plasma membrane. The morphology of the compartment into which receptors are sorted upon PKC activation has not previously been identified. By immuno-electron microscopy, we show that PMA sorts ErbB2 into a complex tubulovesicular or cisternal organelle resembling a previously described endocytic recycling compartment.

Chromoanasynthesis is a common mechanism that leads to ERBB2 amplifications in a cohort of early stage HER2+ breast cancer samples

BackgroundHER2 positive (HER2+) breast cancers involve chromosomal structural alterations that act as oncogenic driver events.MethodsWe interrogated the genomic structure of 18 clinically-defined HER2+ breast tumors through integrated analysis of whole genome and transcriptome sequencing, coupled with clinical information.ResultsERBB2 overexpression in 15 of these tumors was associated with ERBB2 amplification due to chromoanasynthesis with six of them containing single events and the other nine exhibiting multiple events. Two of the more complex cases had adverse clinical outcomes. Chromosomes 8 was commonly involved in the same chromoanasynthesis with 17. In ten cases where chromosome 8 was involved we observed NRG1 fusions (two cases), NRG1 amplification (one case), FGFR1 amplification and ADAM32 or ADAM5 fusions. ERBB3 over-expression was associated with NRG1 fusions and EGFR and ERBB3 expressions were anti-correlated. Of the remaining three cases, one had a small duplication fully encompassing ERBB2 and was accompanied with a pathogenic mutation.ConclusionChromoanasynthesis involving chromosome 17 can lead to ERBB2 amplifications in HER2+ breast cancer. However, additional large genomic alterations contribute to a high level of genomic complexity, generating the hypothesis that worse outcome could be associated with multiple chromoanasynthetic events.

Abstract 4215: Comprehensive investigation of programmed death receptor ligand 1 (PD-L1) expression and associated molecular features in gastric cancer patients

Abstract Purpose We explored PD-L1 expression and analyzed its association with molecular subtypes, clinical features and other molecular markers in Korean gastric cancer (GC) patients. Experimental design As with the Asian Cancer Research Group (ACRG) GC cohort, 300 primary tumor specimens were procured from Samsung Medical Center. Tumor PD-L1 expression was measured by immunohistochemistry (IHC) using the 22C3 PD-L1 antibody pharmDx (Dako) kit. PD-L1 positivity was defined as CPS ≥1%, where CPS is PD-L1+ cells (tumor cells, macrophages, lymphocytes) over the total number of tumor cells, expressed as a percentage. ACRG and The Cancer Genome Atlas (TCGA) molecular subtypes, and other molecular and clinical features including PD-L1 mRNA expression by microarray, tumor mutation burden (TMB) by targeted sequencing, microsatellite instability (MSI) status by IHC, Epstein-Barr virus (EBV) infection by in situ hybridization, Helicobacter pylori (H. pylori) infection by microscope, ERBB2 (HER2) and EGFR overexpression by IHC, signet ring cell and Lauren classification were provided as part of the ACRG collaboration. Spearman correlation test, Wilcoxon rank-sum test, Kruskal-Wallis test were utilized to test the association with PD-L1 expression, and survival was analyzed by Kaplan-Meier method and log-rank test. Results The median age was 64 years (range, 24-86), 101 (34%) were female, and 127 (42%) had stage I/II disease. Prevalence of PD-L1 positivity was 59.3%, with significantly higher expression in stage I samples (P = 0.002). PD-L1 positivity was associated with good prognosis (P = 0.008) especially for early stage (I/II) subjects (N = 127, P = 0.007). PD-L1 mRNA was moderately correlated with protein expression (R = 0.430, P < 0.001). For molecular subtype, PD-L1 expression was associated with ACRG subtype (P < 0.001) with significantly higher expression in MSI subtype (P < 0.001). And for TCGA subtype, the association with PD-L1 expression also existed (N = 238, P < 0.001) with significantly higher expression in MSI and EBV subtype (P < 0.001). H. pylori infection was also associated with higher PD-L1 expression (N = 127, P = 0.001). Signet ring cell carcinoma had significantly lower PD-L1 expression (P < 0.001). PD-L1 expression was also significantly higher in ARID1A or PIK3CA mutant samples (N = 233, FDR adjusted P = 0.038). TMB was found to be weakly correlated with PD-L1 expression (N = 233, R = 0.266, P < 0.001). No significant association was observed between PD-L1 expression and overexpression of ERBB2 or EGFR. Conclusion Our study provides evidence that several molecular features, especially MSI and EBV infection, are significantly associated with higher PD-L1 protein expression, which suggests that PD-L1 IHC may be an appropriate biomarker to screen GC patients for immune checkpoint inhibitors. Citation Format: Xiaoqiao Liu, Jeeyun Lee, Kyoung-Mee Kim, Seung Tae Kim, Se Hoon Park, Won Ki Kang, Razvan Cristescu, Senaka Peter. Comprehensive investigation of programmed death receptor ligand 1 (PD-L1) expression and associated molecular features in gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4215.

Abstract 5849: Valproic acid reverses trastuzumab resistance in erbB2-positive breast cancer cells via downregulation of ErbB2/ErbB3 signaling through induction of ErbB family members-targeting miRNAs

Abstract Amplification and/or overexpression of erbB2 occur in approximately 25% of invasive breast cancer and are significantly associated with a worse prognosis for breast cancer patients. Trastuzumab, a humanized monoclonal antibody against erbB2 receptor, has been successfully used in early-stage and metastatic breast cancer therapy of patients with erbB2-overexpressing tumors as monotherapy and in combination with other agents; however, both primary and acquired resistances to trastuzumab are common and currently represent a significant clinical problem. Thus, seeking of novel therapeutic strategies/agents to overcome trastuzumab resistance is vital to improve the survival of breast cancer patients whose tumors overexpress erbB2. In our current study, we explored whether valproic acid (VPA), a clinically used anticonvulsant drug with reported HDACi activity, reverses trastuzumab resistance in erbB2-positive breast cancer cells. We have found that VPA inhibits proliferation of both trastuzumab-sensitive SKBR3 and BT474 cell lines as well as their corresponding trastuzumab-resistant SKBR3-pool2 and BT474-HR20 cell lines in a dose- and time-dependent manner, which is accompanied by significant reduction of cyclin D1 as well as induction of P21. Meanwhile, VPA also induces caspase-dependent apoptosis in all aforementioned four cell lines. More interestingly, VPA not only significantly enhances the anti-proliferative effects of trastuzumab, but also potentiates trastuzumab-induced apoptosis in trastuzumab-sensitive and resistant cells. Our further mechanistic studies revealed that VPA treatment results in significant inactivation of PI3K/Akt signaling via simultaneous down-regulation of ErbB2 and ErbB3 in erbB2-positive breast cancer cancer cells, an effect which could be mainly arrtibuted to induction of ErbB family members-targeting microRNAs including miR-125a, miR-125b and miR-205 by VPA. Taken together, we presented here that VPA reverses trastuzumab resistance in erbB2-positive breast cance cells via downregulation of ErbB2/ErbB3 signaling in a miRNAs-inducing-dependent manner. Our data suggested that further investigation regarding the in vivo therapeutic effect of VAP against trastuzumab-resistant breast cancer should be of especial interest. Citation Format: Tingting Lin, Qun Ren, Weimin Zuo, Rong Lin, Hu Zhao, Ping Wang, Jianming Tan, Hui Lyu, Bolin Liu, Shuiliang Wang. Valproic acid reverses trastuzumab resistance in erbB2-positive breast cancer cells via downregulation of ErbB2/ErbB3 signaling through induction of ErbB family members-targeting miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5849.

Abstract 92: CHIP/STUB1 ubiquitin ligase targets MZF1 and loss of its expression in breast cancer unleashes a MZF1-cathepsin pro-oncogenic program

Abstract Triple-negative and ErbB2+ breast cancer subtypes carry inherently poorer prognosis. The overall therapeutic response and survival are markedly lower in metastatic disease. This identification of novel pathways that collide with driver oncogenes to promote more aggressive disease and metastasis could help identify biomarkers for selection of patients for more intensive therapy and provide avenues of targeted therapy for new combinatorial therapies. CHIP/STUB1 is a U-box containing ubiquitin ligase that has been established as a negative co-chaperone for HSP90/HSC70. Recent studies have shown its expression to be reduced or lost in several cancers, including breast cancer. However, the relationship of loss of CHIP with breast cancer subtypes and mechanisms by which loss of CHIP promotes oncogenesis in breast cancer are incompletely understood. IHC analysis of an extensive breast cancer tissue collection revealed that loss of nuclear but not cytoplasmic CHIP predicted aggressive tumorigenesis and shorter survival, with loss of nuclear CHIP in two-thirds of TNBC/ErbB2+ and one-third of ER+ breast cancers. Reduced CHIP expression was confirmed in a panel of breast cancer patient-derived xenograft tumors and in ErbB2+ and TNBC cell lines. Ectopic CHIP expression in TNBC and ErbB2+ breast cancer cell lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth in nude mice. These analyses established CHIP as a barrier to tumorigenic progression and loss of CHIP as a key event in aggressive tumorigenesis in breast cancer. To gain mechanistic insights into the linkage of the loss of nuclear CHIP expression with breast cancer aggressiveness, we carried out an unbiased screen for CHIP-regulated nuclear transcription factor-binding to their cognate DNA elements using an array methodology. This analysis identified many candidate transcription factors whose DNA-binding activity was up- or down-regulated upon re-expression of CHIP in CHIP-low ErbB2+ or TNBC cell line models. Western blotting of selected candidates confirmed the CHIP dependence of their expression levels. We focused our further efforts on the Myeloid Zinc Finger 1 (MZF1) protein as a CHIP target since it was recently identified as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion in a model of oncogenic mutant ErbB2 overexpression in MCF-7 cells. We show that CHIP negatively regulates the expression of CTSB/L in TNBC and other breast cancer cell lines. Chemical inhibition of CTSB abrogated breast cancer cell invasion and matrix degradation in vitro and retarded xenograft tumor growth. We suggest that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways that can provide new therapeutic opportunities in breast cancer. Citation Format: Bhopal C. Mohapatra, Haitao Luan, Timothy A. Bielecki, Insha Mushtaq, Sameer Mirza, Tameka A. Bailey, Robert Clubb, Wei An, Dena Ahmed, Rokaya El Ansari, Matthew D. Storck, Chittibabu Guda, Yuri Sheinin, Jane L. Meza, Srikumar Raja, Emad Rakha, Vimla Band, Hamid Band. CHIP/STUB1 ubiquitin ligase targets MZF1 and loss of its expression in breast cancer unleashes a MZF1-cathepsin pro-oncogenic program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 92.

Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

Although ErbB2-targeted therapeutics have significantly improved ErbB2+ breast cancer patient outcomes, therapeutic resistance remains a significant challenge. Therefore, the development of novel ErbB2-targeting strategies is necessary. Importantly, ErbB2 is a sensitive client protein of heat shock protein 90 (HSP90), which regulates client protein folding, maturation, and stabilization. HSP90 inhibition provides an alternative therapeutic strategy for ErbB2-targeted degradation. In particular, ganetespib, a novel HSP90 inhibitor, is a promising agent for ErbB2+ cancers. Nevertheless, the anti-cancer efficacy and clinical application of ganetespib for ErbB2+ breast cancer is largely unknown. In our study, we examined the anti-cancer effects of ganetespib on ErbB2+ BT474 and SKBR3 breast cancer cells, and isogenic paired cancer cell lines with lentivirus-mediated ErbB2 overexpression. Ganetespib potently inhibited cell proliferation, cell cycle progression, survival, and activation/phosphorylation of ErbB2 and key downstream effectors in ErbB2+ breast cancer cells. Moreover, ganetespib decreased the total protein levels of HSP90 client proteins and reduced ErbB2 protein half-life. ErbB2-overexpressing cancer cells were also more sensitive to ganetespib-mediated growth inhibition than parental cells. Ganetespib also strikingly potentiated the inhibitory effects of lapatinib in BT474 and SKBR3 cells. Ultimately, our results support the application of ganetespib-mediated HSP90 inhibition as a promising therapeutic strategy for ErbB2+ breast cancer.

STAR‐RELATED LIPID TRANSFER PROTEIN 10 AS NOVEL KEY PLAYER IN ETHANOL‐INDUCED ERBB2 BREAST CANCER PROGRESSION

ProposeEpidemiological studies demonstrated that ethanol administration promotes breast cancer development and metastasis. Human breast cancer cells with a high expression of Erb‐B2 Receptor Tyrosine Kinase 2 (ErbB2) exhibited an enhanced response to ethanol‐stimulated cell invasion in vitro triggering the phosphorylation status of mitogen‐activated protein kinase (MAPKs). ErbB2 amplification and/or overexpression has been described in 20–30% of human breast cancers and correlates with poor prognosis. StAR‐related lipid transfer protein 10 (StarD10) is a member of the StarD protein family and lipid transfer protein with selective binding site to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), two potential precursors for lipid metabolism and a major constituent of cell membranes. Choline metabolism and its derived metabolites can produce extensive alterations on phospholipid membrane composition that usually occurs before the morphological tumorigenic changes. StarD10 is highly expressed in 35% of ErbB2‐positive breast cancer suggesting a selective growth advantage and cellular transformation for tumor expressing both proteins. The goal of this study is to investigate the role of StarD10 and ErbB2 cross‐talk in breast cancer as consequence of ethanol administration and elucidate the molecular mechanisms.MethodsThe experiments were performed using MCF‐7 and SKBR‐3 human carcinoma cell lines. mRNA and protein levels were analyzed by Real‐Time PCR and Western Blotting, respectively. StarD10 promoter activity and cell proliferation were performed by promoter reporter and MTT assay, respectively. Phosphatidylcholine level was measured using an enzyme‐coupled assay.ResultsCells treated with 100 mM ethanol for 48 hours showed increased expression of StarD10 and ErbB2. Consistently, ErbB2 overexpression caused an increase of StarD10 expression. Using PROMO program to predict transcription factor binding sites in DNA sequences, we found out that p65, c‐MYC, c‐FOS and c‐JUN, well known to be transcription factors (TFs) ErbB2 downstream targets, are highly predicted to bind StardD10 promoter sequence. Overexpression of all predicted TFs induced StarD10 promoter activity mimicking ErbB2 trigger function. Also, we demonstrated that StarD10 and ErbB2 positively regulate each other's expression. Moreover, ethanol increased phosphatidylcholine level supporting the proposed role of StarD10 as player on tumorigenesis via its lipid transporter function. Interesting, we found that both Stard10 overexpression and silencing induced cell growth and migration in MCF‐7 and SKBR‐3 cells. This data validated the hypothesis of StarD10 is a key protein on induced‐ethanol breast cancer development accordingly with its pathophysiological level. High level of secreted PC was found in ethanol‐treated cells media, while StarD10 silencing completely prevented it. In contrast, StarD10 overexpression promoted PC secretion and induced it further in co‐treatment with ethanol. This finding could help us to explain how potentially StarD10 controls the changes in cell membrane properties during malignancy may be modulating the membrane fluidity.ConclusionsThe ability of Stard10 to influence ErbB2 expression and activity may be involve both dependent and independent lipid binding function. This is the first report demonstrating that ethanol can modulate in dynamic manner the ErbB2 role through StarD10 involvement in breast cancer.Support or Funding InformationNIAAA 5 K01 AA022372‐06This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High glucose forces a positive feedback loop connecting ErbB4 expression and mTOR/S6K pathway to aggravate the formation oftau hyperphosphorylation in differentiated SH-SY5Y cells.

High glucose (HG)-induced mammalian target of rapamycin (mTOR) overactivation acts as a signaling hub for the formation of tau hyperphosphorylation, which contributes to the development of diabetes-associated cognitive deficit. How HG induces the sustained activation of mTOR in neurons is not clearly understood. ErbB4, a member of the receptor tyrosine kinase family, plays critical roles in development and function of neural circuitry, relevant to behavioral deficits. Here, we showed HG-induced ErbB4 overexpression in differentiated SH-SY5Y cells and primary hippocampal neurons and hippocampal pyramidal neurons of streptozotocin-induced diabetic rats. Inhibition of ErbB4 signaling prevented the HG-induced activation of mTOR/S6K signaling to suppress tau hyperphosphorylation. In contrast, ErbB4 overexpression increased the activation of mTOR/S6K signaling, resulting in tau hyperphosphorylation similar to HG treatment. We also demonstrated that HG upregulated the expression of ErbB4 at a mTOR-dependent posttranscriptional level. Together, our results provide the first evidence for the presence of a positive feedback loop for the sustained activation of mTOR involving overexpressed ErbB4, leading to the formation of tau hyperphosphorylation under HG condition. Therefore, ErbB4 is a potential therapeutic target for diabetes-associated neurodegeneration.

Abstract P2-02-12: Cell free DNA analysis identifies actionable ERBB2 amplifications in patients with HER2 negative breast cancer

Abstract Identification of ERBB2 (HER2) overexpression in metastatic breast cancer informs utilization of HER2 targeted therapy. The NCCN recommends HER2 expression re-evaluation at the first disease recurrence in patients with negative or equivocal tissue status given results discrepancies due to inadequate tissue biopsy, tumoral heterogeneity, biopsy technique or fixation as well as discordance in ERBB2 (HER2) expression between primary and metastatic lesions. We examined the incidence of ERBB2 (HER2) negative to positive “flips” (e.g. to ERBB2-amplified in plasma) in a cohort of patients who underwent a blood-based cell-free DNA (cfDNA) assay at a CLIA-certified/CAP-accredited/NYSDOH-approved molecular diagnostic laboratory. Laboratory database was queried for samples from patients with a breast cancer diagnosis. The query was filtered to ensure patients with multiple cfDNA timepoints were counted only once. Patients without a pathology report submitted at any cfDNA collection timepoint or the pathology report did not include ERBB2 (HER2) status, results were inconclusive or quantity not sufficient were excluded. Between March 2014 and April 2017, 1,853 unique patients were identified with reported ERBB2 (HER2) status. For patients with more than one cfDNA timepoint collected (N=349; 18.8%), the earliest pathology report was referenced. 1,386 patient tumor samples were negative for HER2 overexpression (74.8%), 325 (17.5%) were positive, and 142 (7.7%) were equivocal. Twenty-nine of the 1,386 patients with reported tumor negative HER2 status had amplification on subsequent cfDNA analysis (2.1%). All 29 patients were female. Most patients (N=21) had a single cfDNA timepoint collected. Median age at cfDNA blood draw was 58 years (range 28–68). Median length of time between reported tissue negative status and cfDNA blood draw was 405 days (range 21–4,060). Median plasma ERBB2 copy number was 2.44 (greater than 50th-centile per laboratory data) (range 2.15–16.5). Clinical follow-up was obtained for 19 patients (65%). Nine patients were lost to follow-up or succumbed to disease prior to initiation of a new therapeutic regimen. One patient was known HER2 positive prior to receipt of the cfDNA results. In the remaining nine patients, six initiated targeted HER2 therapy following receipt of the cfDNA results, with five of six (83%) demonstrating a clinical response. In one patient with known ER/PR positive, HER2 negative disease, progressing through multiple lines of therapy, addition of trastuzumab and pertuzumab to her paclitaxel regimen following identification of the cfDNA ERBB2 amplification resulted in a significant reduction in CEA levels (238 to 37.9 ng/mL) by week five. In a second patient, following identification of the cfDNA ERBB2 amplification, she was treated with trastuzumab and pertuzumab along with docetaxel and had a dramatic response. She continues on trastuzumab and pertuzumab alone. Although a modest sample size, this is the second cfDNA series demonstrating that ERBB2 (HER2) status may flip from negative to positive upon recurrence or metastasis, and that targeting plasma-detected ERBB2 amplification with anti-HER2 has clinical benefit. cfDNA is a viable alternative to tissue rebiopsy in this patient population. Citation Format: Raymond VM, Diaz J, Banks KC, Ahn E, Brufsky A, Ellis M, Lippman M, Lee C, Pluard T, Schreeder M, Schwab R, Lanman RB. Cell free DNA analysis identifies actionable ERBB2 amplifications in patients with HER2 negative 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 P2-02-12.

Nucleolin and ErbB2 inhibition reduces tumorigenicity of ErbB2-positive breast cancer

ErbB2, a member of the ErbB family of receptor tyrosine kinases, is an essential player in the cell’s growth and proliferation signaling pathways. Amplification or overexpression of ErbB2 is observed in ∼30% of breast cancer patients, and often drives cellular transformation and cancer development. Recently, we have shown that ErbB2 interacts with the nuclear-cytoplasmic shuttling protein nucleolin, an interaction which enhances cell transformation in vitro, and increases mortality risk and disease progression rate in human breast cancer patients. Given these results, and since acquired resistance to anti-ErbB2-targeted therapy is a major obstacle in treatment of breast cancer, we have examined the therapeutic potential of targeting the ErbB2–nucleolin complex. The effect of the nucleolin-specific inhibitor GroA (AS1411) on ErbB2-positive breast cancer was tested in vivo, in a mouse xenograft model for breast cancer; as well as in vitro, alone and in combination with the ErbB2 kinase-inhibitor tyrphostin AG-825. Here, we show that in vivo treatment of ErbB2-positive breast tumor xenografts with GroA reduces tumor size and leads to decreased ErbB2-mediated signaling. Moreover, we found that co-treatment of breast cancer cell lines with GroA and the ErbB2 kinase-inhibitor tyrphostin AG-825 enhances the anti-cancer effects exerted by GroA alone in terms of cell viability, mortality, migration, and invasiveness. We, therefore, suggest a novel therapeutic approach, consisting of combined inhibition of ErbB2 and nucleolin, which has the potential to improve breast cancer treatment efficacy.

Cooperative antitumor activities of carnosic acid and Trastuzumab in ERBB2+ breast cancer cells

BackgroundERBB2 is overexpressed in up to 20–30% of human breast cancers (BCs), and it is associated with aggressive disease. Trastuzumab (Tz), a humanized monoclonal antibody, improves the prognosis associated with ERBB2-amplified BCs. However, the development of resistance remains a significant challenge. Carnosic acid (CA) is a diterpene found in rosemary and sage, endowed with anticancer properties. In this in vitro study, we have investigated whether Tz and CA have cooperative effects on cell survival of ERBB2 overexpressing (ERBB2+) cells and whether CA might restore Tz sensitivity in Tz-resistant cells.MethodsWe have studied BC cell migration and survival upon CA and Tz treatment. In particular, migration ability was assessed by transwell assay while cell survival was assessed by MTT assay. In addition, we have performed cell cycle and apoptosis analysis by high-resolution DNA flow cytometry and annexin-V, resazurin and sytox blue staining by flow cytometry, respectively. The expression of proteins involved in cell cycle progression, ERBB2 signaling pathway, and autophagy was evaluated by immunoblot and immunofluorescence analysis. Cellular structures relevant to the endosome/lysosome and autophagy pathways have been studied by immunofluorescence and transmission electron microscopy.ResultsWe report that, in ERBB2+ BC cells, CA reversibly enhances Tz inhibition of cell survival, cooperatively inhibits cell migration and induces cell cycle arrest in G0/G1. These events are accompanied by ERBB2 down-regulation, deregulation of the PI3K/AKT/mTOR signaling pathway and up-regulation of both CDKN1A/p21WAF1 and CDKN1B/p27KIP1. Furthermore, we have demonstrated that CA impairs late autophagy and causes derangement of the lysosomal compartment as shown by up-regulation of SQSTM1/p62 and ultrastructural analysis. Accordingly, we have found that CA restores, at least in part, sensitivity to Tz in SKBR-3 Tz-resistant cell line.ConclusionsOur data demonstrate the cooperation between CA and Tz in inhibiting cell migration and survival of ERBB2+ BC cells that warrant further studies to establish if CA or CA derivatives may be useful in vivo in the treatment of ERBB2+ cancers.

Evaluation of Protein Levels of the Receptor Tyrosine Kinase ErbB3 in Serum.

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTK) consists of four members: EGFR1/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and HER4/ErbB4. Signaling through these receptors regulates many key cellular activities, such as cell division, migration, adhesion, differentiation, and apoptosis. The ErbB family has been shown to be overexpressed in different types of cancers and is a target of several inhibitors already in clinical trials. ErbB3 lacks a functional tyrosine kinase domain and therefore has not been as extensively studied as the other members of this family, but its importance in activating downstream pathways, such as the PI3K/Akt pathway, makes this RTK a worthy investigation target, especially in urothelial carcinoma where the PI3K/Akt pathway is vital for progression. In recent times, ErbB3 overexpression has been linked to drug resistance and progression of various diseases, especially cancer. ErbB3 levels in the serum were shown in many cases to be reflective of its role in disease progression, and therefore detection of serum ErbB3 levels during treatment may be of importance.Here we describe two methods for detecting ErbB3 protein in serum from patients who have undergone a clinical trial, utilizing two well-established methods in molecular biology-western blotting and ELISA, focusing on sample preparation and troubleshooting.

ErbB2 signaling epigenetically suppresses microRNA-205 transcription via Ras/Raf/MEK/ERK pathway in breast cancer.

We previously reported that microRNA‐205 (miR‐205) is downregulated by overexpression of the receptor tyrosine kinase ErbB2 and that ectopic transfection of miR‐205 precursor decreases ErbB2 tumorigenicity in soft agar. In this study, we further analyzed the regulatory mechanisms linking ErbB2 overexpression and miR‐205 downregulation. In ErbB2‐overexpressing breast epithelial cells, miR‐205 expression was significantly increased by treatment with MEK inhibitor U0126 or PD98059, Raf‐1 inhibitor ZM‐336372, and ERK inhibitor SCH772984, but PI3K inhibitor LY294002 and p38 MAPK inhibitor SB203580 had no effect. We established breast epithelial cells overexpressing RafCAAX, a constitutively active form of Raf‐1, and showed that overexpression of RafCAAX dramatically reduced miR‐205 expression. In RafCAAX‐overexpressing cells, miR‐205 expression was also significantly increased by SCH772984. Moreover, miR‐205 expression was significantly increased by treatment with DNA methyltransferase (DNMT) inhibitor 5‐aza‐2′‐deoxycytidine and expression of several DNMT family members was increased in both ErbB2‐ and RafCAAX‐overexpressing cells. DNA methylation analysis by bisulfite sequencing revealed that the putative miR‐205 promoters were predominantly hypermethylated in both ErbB2‐ and RafCAAX‐overexpressing cells. Reporter activity of the putative miR‐205 promoters was reduced in both ErbB2‐overexpressing and RafCAAX‐overexpressing cells. Together, these findings indicate that ErbB2 signaling epigenetically suppresses miR‐205 transcription via the Ras/Raf/MEK/ERK pathway.

Identification and characterization of ErbB4 kinase inhibitors for effective breast cancer therapy

The overexpression of ErbB4 is associated with aggressive disease biology and reduced the survival of breast cancer patients. We have used ErbB4 receptor as a novel drug target to spearhead the rational drug design. The present study is divided into two parts. In the first part, we have exploited the hidden information inside ErbB4 kinase receptor both at sequence and structural level. PSI-BLAST algorithm is used to search similar sequences against ErbB4 kinase sequence. Top 15 sequences with high identity were selected for finding conserved and variable regions among sequences using multiple sequence alignment. In the second part, available 3 D structure of ErbB4 kinase is curated using loop modeling, and anomalies in the modeled structure is improved by energy minimization. The resultant structure is validated by analyzing dihedral angles by Ramachandran plot analysis. Furthermore, the potential binding sites were detected by using DoGSite and CASTp server. The similarity-search criterion is used for the preparation of our in-house database of drugs from DrugBank database. In total, 409 drugs yet to be tested against ErbB4 kinase is used for screening purpose. Virtual screening results in identification of 11 compounds with better binding affinity than lapatinib and canertinib. Study of protein–ligand interactions reveals information about amino acid residues; Lys726, Thr771, Met774, Cys778, Arg822, Thr835, Asp836 and Phe837 at the binding pocket. The physicochemical properties and bioactivity score calculation of selected compounds suggest them as biological active. This study presents a rich array that assist in expediting new drug discovery for breast cancer.

ErbB4 signaling in the prelimbic cortex regulates fear expression

Many psychiatric diseases such as post-traumatic stress disorder (PTSD) are characterized by abnormal processing of emotional stimuli particularly fear. The medial prefrontal cortex (mPFC) is critically involved in fear expression. However, the molecular mechanisms underlying this process are largely unknown. Neuregulin-1 (NRG1) reportedly regulates pyramidal neuronal activity via ErbB4 receptors, which are abundant in parvalbumin (PV)-expressing interneurons in the PFC. In this study, we aimed to determine how NRG1/ErbB4 signaling in the mPFC modulates fear expression and found that tone-cued fear conditioning increased NRG1 expression in the mPFC. Tone-cued fear conditioning was inhibited following neutralization of endogenous NRG1 and specific inhibition or genetic ablation of ErbB4 in the prelimbic (PL) cortex but not in the infralimbic cortex. Furthermore, ErbB4 deletion specifically in PV neurons impaired tone-cued fear conditioning. Notably, overexpression of ErbB4 in the PL cortex is sufficient to reverse impaired fear conditioning in PV-Cre;ErbB4−/− mice. Together, these findings identify a previously unknown signaling pathway in the PL cortex that regulates fear expression. As both NRG1 and ErbB4 are risk genes for schizophrenia, our study may shed new light on the pathophysiology of this disorder and help to improve treatments for psychiatric disorders such as PTSD.

Abstract 24: HMBD001, a novel anti-ErbB3 antibody with a unique mechanism of action, effectively inhibits tumor growth in pre-clinical models of ErbB3+ solid tumors

Abstract Gastric cancer is the second highest cause of cancer mortality in Asia. Over-expression of ErbB3, a cell surface RTK, occurs in ~60% of mGC patients where it is significantly associated with poorer survival. Ligand binding to ErbB3 triggers heterodimerization and downstream signaling through Akt. Neutralizing antibodies that inhibit ligand binding show poor efficacy in clinical trials, likely due to high levels of ligand independent activation in many tumor types. Directly inhibiting heterodimerization could be more effective, however, current methods of antibody isolation offer limited control over the site of antibody binding and, therefore, mechanism of action, requiring extensive screening of antibodies and posing significant challenges to identifying suitable candidates for therapeutic development. Hummingbird Bioscience has used its proprietary Rational Antibody Development Platform for the design and development of novel therapeutic antibodies against ErbB3. Computational sequence and structural analyses predicted highly specific surface epitopes, with strong antigenicity and good safety profiles, where antibody binding would inhibit dimerization. Subsequently, Hummingbird’s directed evolution mouse immunization strategy efficiently isolated monoclonal antibodies that bound with low nM (Kd < 5nM) affinity to these epitopes on native folded ErbB3. Furthermore, these mAbs showed ELISA cross-reactivity to ErbB3 in model organisms (mouse, rat, monkey) but no binding to other EGFR family proteins. Antibody HMBD001 was found to strongly inhibit the phosphorylation of ErbB3 and decrease downstream signaling through the Akt pathway (90% decrease in p-ErbB3 and 60% decrease in p-AKT observed at 24 hrs) resulting in the inhibition of tumor cell proliferation in models of ErbB3+ gastric cancer (over 90% inhibition of NCI-N87 proliferation after 5 days) and breast cancer (up to 85% inhibition of BT474 proliferation after 5 days). In mouse NCI-N87 gastric cancer CDX models, weekly doses of HMBD001 achieved greater than 90% inhibition of tumor growth (at 25 days) with no observable adverse toxicity. The first in-human trial of HMBD001 is planned for 2018. Citation Format: Jerome D. Boyd-Kirkup, Dipti Thakkar, Piers J. Ingram. HMBD001, a novel anti-ErbB3 antibody with a unique mechanism of action, effectively inhibits tumor growth in pre-clinical models of ErbB3+ solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 24. doi:10.1158/1538-7445.AM2017-24

Abstract 4320: The Survivin-targeting miR-542-3p overcomes erbB3 signaling-mediated chemo-resistance and enhances the antitumor activity of paclitaxel against erbB2-positive breast cancer

Abstract Elevated expression of erbB3 interacts with erbB2 in breast cancer cells to confer chemo-resistance via induction of Survivin. However, the underlying mechanism of erbB3 signaling-induced upregulation of Survivin remains elusive. Ectopic expression or downregulation of erbB3 in erbB2-positive breast cancer cells did not alter Survivin mRNA levels and had no significant effect on Survivin protein stability. We thus hypothesize that the erbB3 signaling modulates certain miRNAs that target Survivin to influence its protein translation. Here we showed that overexpression of erbB3 decreased and specific knockdown of erbB3 enhanced expression of two Survivin-targeting miRNAs, miR-203 and miR-542-3p in breast cancer cells. While the specific inhibitor of either miR-203 or miR-542-3p clearly attenuated an anti-erbB3 antibody (Ab)-mediated reduction of Survivin, a more profound effect on Survivin expression was observed with inhibition of miR-542-3p than miR-203 inhibition. Consistently, miR-542-3p mimic was much more effective than miR-203 mimic not only to downregulate Survivin, but also to enhance paclitaxel-induced apoptosis in the otherwise paclitaxel-resistant breast cancer cells. Moreover, using a murine tumor xenografts model established from a human breast cancer cell line expressing both erbB2 and erbB3, we discovered that the combinations of miR-542-3p mimic and paclitaxel, as compared to either agent alone, significantly inhibited tumor growth in vivo. Collectively, these data demonstrate that the erbB3 signaling upregulates Survivin via inhibition of miR-203 and miR-542-3p in erbB2-positive breast cancer cells. Since miR-542-3p has three binding sites on the 3’UTR of erbB3 mRNA, the miR-542-3p mimic serves as an excellent replacement therapeutic agent to downregulate Survivin, and thereby significantly enhances the antitumor activity of paclitaxel against erbB2-positive breast cancer. Citation Format: Hui Lyu, Shuiliang Wang, Jingcao Huang, Bolun Wang, Bolin Liu. The Survivin-targeting miR-542-3p overcomes erbB3 signaling-mediated chemo-resistance and enhances the antitumor activity of paclitaxel against erbB2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4320. doi:10.1158/1538-7445.AM2017-4320

Involvement of Chromatin Remodeling Genes and the Rho GTPases RhoB and CDC42 in Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinomas (OCCCs) constitute a rare ovarian cancer subtype with distinct clinical features, but may nonetheless be difficult to distinguish morphologically from other subtypes. There is limited knowledge of genetic events driving OCCC tumorigenesis beyond ARID1A, which is reportedly mutated in 30-50% of OCCCs. We aimed to further characterize OCCCs by combined global transcriptional profiling and targeted deep sequencing of a panel of well-established cancer genes. Increased knowledge of OCCC-specific genetic aberrations may help in guiding development of targeted treatments and ultimately improve patient outcome. Gene expression profiling of formalin-fixed, paraffin-embedded (FFPE) tissue from a cohort of the major ovarian cancer subtypes (cohort 1; n = 67) was performed using whole-genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) bead arrays, followed by pathway, gene module score, and gene ontology analyses, respectively. A second FFPE cohort of 10 primary OCCCs was analyzed by targeted DNA sequencing of a panel of 60 cancer-related genes (cohort 2). Non-synonymous and non-sense variants affecting single-nucleotide variations and insertions or deletions were further analyzed. A tissue microarray of 43 OCCCs (cohort 3) was used for validation by immunohistochemistry and chromogenic in situ hybridization. Gene expression analyses revealed a distinct OCCC profile compared to other histological subtypes, with, e.g., ERBB2, TFAP2A, and genes related to cytoskeletal actin regulation being overexpressed in OCCC. ERBB2 was, however, not overexpressed on the protein level and ERBB2 amplification was rare in the validation cohort. Targeted deep sequencing revealed non-synonymous variants or insertions/deletions in 11/60 cancer-related genes. Genes involved in chromatin remodeling, including ARID1A, SPOP, and KMT2D were frequently mutated across OCCC tumors. OCCCs appear genetically heterogeneous, but harbor frequent alterations in chromatin remodeling genes. Overexpression of TFAP2A and ERBB2 was observed on the mRNA level in relation to other ovarian cancer subtypes. However, overexpression of ERBB2 was not reflected by HER2 amplification or protein overexpression in the OCCC validation cohort. In addition, Rho GTPase-dependent actin organization may also play a role in OCCC pathogenesis and warrants further investigation. The distinct biological features of OCCC discovered here may provide a basis for novel targeted treatment strategies.