ERBB2-amplified Breast Cancers Research Articles

Dissecting the Shared and Context-Dependent Pathways Mediated by the p140Cap Adaptor Protein in Cancer and in Neurons.

The p140Cap adaptor protein is a scaffold molecule physiologically expressed in few epithelial tissues, such as the mammary gland, and in differentiated neurons. While the role of p140Cap in mammary gland epithelia is not still understood, we already know that a significant subset of breast cancers express p140Cap. In the subgroup of ERBB2-amplified breast cancers, a high p140Cap status predicts a significantly lower probability of developing a distant event and a clear difference in survival. p140Cap is causal in dampening ERBB2-positive tumor cell progression, impairing tumor onset and growth, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. Since only a few p140Cap interacting proteins have been identified in breast cancer and the molecular complexes and pathways underlying the cancer function of p140Cap are largely unknown, we generated a p140Cap interactome from ERBB2-positive breast cancer cells, identifying cancer specific components and those shared with the synaptic interactome. We identified 373 interacting proteins in cancer cells, including those with functions relevant to cell adhesion, protein homeostasis, regulation of cell cycle and apoptosis, which are frequently deregulated in cancer. Within the interactome, we identified 15 communities (clusters) with topology-functional relationships. In neurons, where p140Cap is key in regulating synaptogenesis, synaptic transmission and synaptic plasticity, it establishes an extensive interactome with proteins that cluster to sub complexes located in the postsynaptic density. p140Cap interactors converge on key synaptic processes, including synaptic transmission, actin cytoskeleton remodeling and cell-cell junction organization. Comparing the breast cancer to the synaptic interactome, we found 39 overlapping proteins, a relatively small overlap. However, cell adhesion and remodeling of actin cytoskeleton clearly emerge as common terms in the shared subset. Thus, the functional signature of the two interactomes is primarily determined by organ/tissue and functional specificity, while the overlap provides a list of shared functional terms, which might be linked to both cancer and neurological functions.

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.

The anti-ErbB2 antibody H2-18 and the pan-PI3K inhibitor GDC-0941 effectively inhibit trastuzumab-resistant ErbB2-overexpressing breast cancer.

Trastuzumab, an anti-ErbB2 humanized antibody, brings benefit to patients with ErbB2-amplified metastatic breast cancers. However, the resistance to trastuzumab is common. Our previously reported H2-18, an anti-ErbB2 antibody, potently induced programmed cell death in trastuzumab-resistant breast cancer cells. Here, we aim to investigate the antitumor efficacy of H2-18 in combination with the pan-PI3K inhibitor GDC-0941 in trastuzumab-resistant breast cancer cell lines. The results showed that H2-18 and GDC-0941 synergistically inhibited the in vitro proliferation of BT-474, SKBR-3, HCC-1954 and HCC-1419 breast cancer cells. H2-18 plus GDC-0941 showed significantly enhanced programmed cell death-inducing activity compared with each drug used alone. The combination of H2-18 and GDC-0941 did not increase the effect of single agent on ROS production, cell cycle and ErbB2 signaling. Importantly, the in vivo antitumor efficacy of H2-18 plus GDC-0941 was superior to that of single agent. Thus, the enhanced in vivo antitumor efficacy of H2-18 plus GDC-0941 may mainly be attributable to its increased programmed cell death-inducing activity. Collectively, H2-18 plus GDC-0941 could effectively inhibit tumor growth, suggesting the potential to be translated into clinic as an efficient strategy for ErbB2-overexpressing breast cancers.

Combating acquired resistance to trastuzumab by an anti-ErbB2 fully human antibody.

Trastuzumab resistance is a common problem that impedes the effectiveness of trastuzumab in ErbB2-amplified cancers. About 70% of ErbB2-amplified breast cancers do not respond to trastuzumab (de novo resistance), and the majority of the trastuzumab-responsive cancers progress within 1 year (acquired resistance). Different mechanisms exist between de novo and acquired resistance. Innate resistance mechanisms are mainly independent of ErbB2 receptor activity, and acquired resistance involves with alterations depending on ErbB2 activity. We previously reported H2-18, an ErbB2 domain I-specific antibody, which could circumvent de novo resistance to trastuzumab. Here, we modeled the development of acquired resistance by treating human gastric cancer cell line NCI-N87 with trastuzumab to obtain the trastuzumab-resistant subline, NCI-N87-TraRT. Next, we investigated the antitumor efficacy of H2-18 in NCI-N87-TraRT cell line. H2-18 exhibited a significantly greater antitumor activity in NCI-N87-TraRT tumor-bearing nude mice than pertuzumab and trastuzumab, either alone or in combination. The unique ability of H2-18 to overcome acquired resistance may be attributable to its potent programmed cell death-inducing activity, which was probably mediated by RIP1-ROS-JNK-c-Jun pathway. In conclusion, H2-18 may have the potential as an effective agent to circumvent acquired resistance to trastuzumab in ErbB2-overexpressing cancers.

The scaffold protein p140Cap limits ERBB2-mediated breast cancer progression interfering with Rac GTPase-controlled circuitries

The docking protein p140Cap negatively regulates tumour cell features. Its relevance on breast cancer patient survival, as well as its ability to counteract relevant cancer signalling pathways, are not fully understood. Here we report that in patients with ERBB2-amplified breast cancer, a p140Cap-positive status associates with a significantly lower probability of developing a distant event, and a clear difference in survival. p140Cap dampens ERBB2-positive tumour cell progression, impairing tumour onset and growth in the NeuT mouse model, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. One major mechanism is the ability of p140Cap to interfere with ERBB2-dependent activation of Rac GTPase-controlled circuitries. Our findings point to a specific role of p140Cap in curbing the aggressiveness of ERBB2-amplified breast cancers and suggest that, due to its ability to impinge on specific molecular pathways, p140Cap may represent a predictive biomarker of response to targeted anti-ERBB2 therapies.

Abstract 3136: Sample specific outlier kinase expression identified by RNA-Seq provide targets for precision therapy.

Abstract Protein kinases represent a most effective class of therapeutic targets in cancer cells, and potent inhibitors of oncogenic kinases including ABL, AKT, ALK, AURKs, BRAF, CDKs, EGFR, ERBB2, FGFRs, KIT, MAPKs, MET, PIK3CA, PLKs, RET, SRC, S6Ks, and VEGFR are in clinical use, trials, or development. Presence of activating mutations, gene fusions or copy number amplification of many of these kinases predict responsiveness to specific inhibitors, for example, imatinib for BCR-ABL-positive chronic myeloid leukemia, trastuzumab and lapatinib for ERBB2-amplified breast cancers, gefitinib for lung cancers with kinase domain mutations in EGFR, and crizotinib for lung cancers with ALK gene fusions. However, tumors with defined kinase aberrations represent a very small proportion of all cancers, and we hypothesize that potentially most, if not all, cancers are dependent on specific kinases, and that “Outlier Expression” of specific kinases in individual samples may be indicative of selection during clonal evolution, and thus may represent a therapeutic avenue. To explore this hypothesis, we analyzed RNA-sequencing data from a compendium of 482 cancer and benign samples belonging to 25 different tissue types to define sample-specific ‘kinome’ expression profiles. Comparing the expression of kinases within a sample and across sample sets, we identified distinct ‘outlier kinases’ in individual samples, defined as genes showing the highest statistically significant levels of absolute and differential expression. Frequently observed outlier kinases included known therapeutic targets like ERBB2 and FGFR4 in breast cancer, distinct from MET, AKT2, and PLK2 in pancreatic cancer. Outlier kinases imparted sample-specific dependencies in various cell lines as assessed by siRNA knockdown or pharmacologic inhibition in vitro and in vivo. Outlier expression of polo-like kinases (PLKs) observed in a subset of KRAS-dependent pancreatic cancer cell lines conferred increased sensitivity to the PLK inhibitor BI 6727. Together, our results suggest that outlier kinases represent effective personalized therapeutic targets that are readily identifiable through RNA-sequencing of tumors. Next, to help translate these observations into treatment options, we are attempting to establish 3D tumoroid cultures from tumor samples that can be used to optimize combinations of therapeutics including outlier kinase inhibitors. Citation Format: Chandan Kumar-Sinha, Vishal Kothari, Iris Wei, Sunita Shankar, Shanker Kalyana-Sundaram, Lidong Wang, Linda W. Ma, Pankaj Vats, Catherine Grasso, Dan Robinson, Yi-Mi Wu, Xuhong Cao, Diane M. Simeone, Arul M. Chinnaiyan. Sample specific outlier kinase expression identified by RNA-Seq provide targets for precision therapy. [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 3136. doi:10.1158/1538-7445.AM2013-3136

Genome profiling of ERBB2-amplified breast cancers

BackgroundAround 20% of breast cancers (BC) show ERBB2 gene amplification and overexpression of the ERBB2 tyrosine kinase receptor. They are associated with a poor prognosis but can benefit from targeted therapy. A better knowledge of these BCs, genomically and biologically heterogeneous, may help understand their behavior and design new therapeutic strategies.MethodsWe defined the high resolution genome and gene expression profiles of 54 ERBB2-amplified BCs using 244K oligonucleotide array-comparative genomic hybridization and whole-genome DNA microarrays. Expression of ERBB2, phosphorylated ERBB2, EGFR, IGF1R and FOXA1 proteins was assessed by immunohistochemistry to evaluate the functional ERBB2 status and identify co-expressions.ResultsFirst, we identified the ERBB2-C17orf37-GRB7 genomic segment as the minimal common 17q12-q21 amplicon, and CRKRS and IKZF3 as the most frequent centromeric and telomeric amplicon borders, respectively. Second, GISTIC analysis identified 17 other genome regions affected by copy number aberration (CNA) (amplifications, gains, losses). The expression of 37 genes of these regions was deregulated. Third, two types of heterogeneity were observed in ERBB2-amplified BCs. The genomic profiles of estrogen receptor-postive (ER+) and negative (ER-) ERBB2-amplified BCs were different. The WNT/β-catenin signaling pathway was involved in ER- ERBB2-amplified BCs, and PVT1 and TRPS1 were candidate oncogenes associated with ER+ ERBB2-amplified BCs. The size of the ERBB2 amplicon was different in inflammatory (IBC) and non-inflammatory BCs. ERBB2-amplified IBCs were characterized by the downregulated and upregulated mRNA expression of ten and two genes in proportion to CNA, respectively. IHC results showed (i) a linear relationship between ERBB2 gene amplification and its gene and protein expressions with a good correlation between ERBB2 expression and phosphorylation status; (ii) a potential signaling cross-talk between EGFR or IGF1R and ERBB2, which could influence response of ERBB2-positive BCs to inhibitors. FOXA1 was frequently coexpressed with ERBB2 but its expression did not impact on the outcome of patients with ERBB2-amplified tumors.ConclusionWe have shown that ER+ and ER- ERBB2-amplified BCs are different, distinguished ERBB2 amplicons in IBC and non-IBC, and identified genomic features that may be useful in the design of alternative therapeutical strategies.

Both t-Darpp and DARPP-32 can cause resistance to trastuzumab in breast cancer cells and are frequently expressed in primary breast cancers

The clinical use of trastuzumab (Herceptin), a humanized antibody against the HER2 growth factor receptor, has improved survival in patients with breast tumors with ERBB2 amplification and/or over-expression. However, most patients with advanced ERBB2 amplified breast cancers whose tumors initially respond to trastuzumab develop resistance to the drug, leading to tumor progression. To identify factors responsible for acquired resistance to trastuzumab, gene expression profiling was performed on subclones of an ERBB2 amplified breast cancer cell line, BT474, which had acquired resistance to trastuzumab. The most overexpressed gene in these subclones was PPP1R1B, encoding the DARPP-32 phosphatase inhibitor. Western analysis revealed that only the truncated isoform of the DARPP-32 protein, t-Darpp, was overexpressed in the trastuzumab resistant cells. Using gene silencing experiments, we confirmed that t-Darpp over-expression was required for trastuzumab resistance in these cells. Furthermore, transfecting t-Darpp in parental BT-474 cells conferred resistance to trastuzumab, suggesting that t-Darpp expression was sufficient for trastuzumab resistance. We also found that t-Darpp over-expression was associated with Akt activation and that the T75 residue in t-Darpp was required for both Akt activation and trastuzumab resistance. Finally, we found that full-length DARPP-32 and t-Darpp are expressed in a majority of primary breast tumors. Over-expression of full-length DARPP-32 can also confer resistance to trastuzumab and, moreover, is associated with a poor prognostic value in breast cancers. Thus, t-Darpp and DARPP-32 expression are novel prognostic and predictive biomarkers in breast cancer.

C- myc gene amplification in selected node-negative breast cancer patients correlates with high rate of early relapse

In breast cancers with histologically negative axillary nodes selected for high frequency of recurrence, the amplification of c- myc, erbB-2 and int-2 genes was found to concern, respectively 25% ( 16 65 ), 31% ( 25 81 ) and 14% ( 10 70 ) of tumours. Their relation with tumour progression expressed by relapse-free survival is reported. Using univariate analyses, c- myc amplified tumours showed significant association with early (30-month period after diagnosis) ( P = 0.0013) and intermediate (50-month period after diagnosis) ( P = 0.0398) risks of recurrence. In contrast, only a trend towards higher relapse was observed in erbB-2 amplified breast cancers with respect to later events (occurring over the first 30-month period). Multivariate analyses indicated that c- myc amplification is an independent prognostic factor stronger than oestrogen receptor status and tumour size to define a high risk subset in node-negative patients selected for high frequency of recurrence.