Kai-Keen Shiu

Functional viability profiles of breast cancer.

UNLABELLED The design of targeted therapeutic strategies for cancer has largely been driven by the identification of tumor-specific genetic changes. However, the large number of genetic alterations present in tumor cells means that it is difficult to discriminate between genes that are critical for maintaining the disease state and those that are merely coincidental. Even when critical genes can be identified, directly targeting these is often challenging, meaning that alternative strategies such as exploiting synthetic lethality may be beneficial. To address these issues, we have carried out a functional genetic screen in >30 commonly used models of breast cancer to identify genes critical to the growth of specific breast cancer subtypes. In particular, we describe potential new therapeutic targets for PTEN-mutated cancers and for estrogen receptor-positive breast cancers. We also show that large-scale functional profiling allows the classification of breast cancers into subgroups distinct from established subtypes. SIGNIFICANCE Despite the wealth of molecular profiling data that describe breast tumors and breast tumor cell models, our understanding of the fundamental genetic dependencies in this disease is relatively poor. Using high-throughput RNA interference screening of a series of pharmacologically tractable genes, we have generated comprehensive functional viability profiles for a wide panel of commonly used breast tumor cell models. Analysis of these profiles identifies a series of novel genetic dependencies, including that of PTEN-null breast tumor cells upon mitotic checkpoint kinases, and provides a framework upon which additional dependencies and candidate therapeutic targets may be identified.

Adenoid cystic carcinomas constitute a genomically distinct subgroup of triple-negative and basal-like breast cancers

Adenoid cystic carcinoma (AdCC) is a rare form of triple‐negative and basal‐like breast cancer that has an indolent clinical behaviour. Four breast AdCCs were recently shown to harbour the recurrent chromosomal translocation t(6;9)(q22–23;p23–24), which leads to the formation of the MYB–NFIB fusion gene. Our aims were (i) to determine the prevalence of the MYB–NFIB fusion gene in AdCCs of the breast; (ii) to characterize the gene copy number aberrations found in AdCCs; and (iii) to determine whether AdCCs are genomically distinct from histological grade‐matched or triple‐negative and basal‐like invasive ductal carcinomas of no special type (IDC‐NSTs). The presence of the MYB–NFIB fusion gene was investigated in 13 AdCCs of the breast by fluorescence in situ hybridization (FISH) and reverse transcriptase‐PCR (RT‐PCR), and MYB and BRCA1 RNA expression was determined by quantitative RT‐PCR. Fourteen AdCCs, 14 histological grade‐matched IDC‐NSTs, and 14 IDC‐NSTs of triple‐negative and basal‐like phenotype were microdissected and subjected to high‐resolution microarray‐based comparative genomic hybridization (aCGH). The MYB–NFIB fusion gene was detected in all but one AdCC. aCGH analysis demonstrated a relatively low number of copy number aberrations and a lack of recurrent amplifications in breast AdCCs. Contrary to grade‐matched IDC‐NSTs, AdCCs lacked 1q gains and 16q losses, and in contrast with basal‐like IDC‐NSTs, AdCCs displayed fewer gene copy number aberrations and expressed MYB and BRCA1 at significantly higher levels. Breast AdCCs constitute an entity distinct from grade‐matched and triple‐negative and basal‐like IDC‐NSTs, emphasizing the importance of histological subtyping of triple‐negative and basal‐like breast carcinomas. Copyright

The genomic profile of HER2‐amplified breast cancers: the influence of ER status

Expression profiling studies have suggested that HER2‐amplified breast cancers constitute a heterogeneous group that may be subdivided according to their ER status: HER2‐amplified ER‐positive breast carcinomas that fall into the luminal B cluster; and HER2‐amplified ER‐negative cancers which form a distinct molecular subgroup, known as the erbB2 or HER2 subgroup. ER‐negative breast cancer differs significantly from ER‐positive disease in the pattern, type, and complexity of genetic aberrations. Here we have compared the genomic profiles of ER‐positive and ER‐negative HER2‐amplified cancers using tiling path microarray‐based comparative genomic hybridization (aCGH). Validation of the differentially amplified regions was performed in an independent series of 70 HER2‐amplified breast cancers. Although HER2‐amplified cancers had remarkably complex patterns of molecular genetic aberrations, ER‐positive and ER‐negative HER2‐amplified breast carcinomas shared most molecular genetic features as defined by aCGH. Genome‐wide Fishers exact test analysis revealed that less than 1.5% of the genome was significantly differentially gained or lost in ER‐positive versus ER‐negative HER2‐amplified cancers. However, two regions of amplification were significantly associated with ER‐positive carcinomas, one of which mapped to 17q21.2 and encompassed GJC1, IGFBP4, TNS4, and TOP2A. Chromogenic in situ hybridization analysis of an independent validation series confirmed the association between ER status and TOP2A amplification. In conclusion, although hormone receptor status does not determine the overall genetic profile of HER2‐amplified breast cancers, specific genetic aberrations may be characteristic of subgroups of HER2 breast cancers. Copyright

Loss of 16q in high grade breast cancer is associated with estrogen receptor status: Evidence for progression in tumors with a luminal phenotype?

Loss of the long arm of chromosome 16 (16q) is observed in the vast majority of low grade/grade I (GI) invasive ductal carcinomas of no special type (IDC‐NSTs), whereas this event is uncommonly seen in high grade/grade III (GIII) IDC‐NSTs. Together with data on the pathology and genetics of breast cancer recurrences, this has led to the proposal that GI and GIII breast cancers evolve through distinct genetic pathways and that progression from GI to GIII is an unlikely biological phenomenon. We compared the genomic profiles of GIII‐IDC‐NSTs with 16q whole arm loss (16qWL) according to estrogen receptor (ER) status. 16qWL was found in 36.5% of cases and was significantly associated with ER expression and luminal phenotype. ER+ GIII‐IDC‐NSTs with 16qWL displayed significantly higher levels of genomic instability than ER+ IDC‐NSTs without 16qWL. Furthermore, ER+ and ER− IDC‐NSTs stratified according to the presence of 16qWL harbored distinct patterns of genetic aberrations. Interestingly, ER+/16qWL tumors displayed genetic features usually found in tumors with homologous DNA repair defects and significantly more frequently harbored heterozygous loss of BRCA2 than the remaining ER+ cancers. Our results demonstrate that approximately one third of GIII tumors harbor 16qWL, confirming that progression from low to high grade breast cancer is not found in the majority of breast cancers. 16qWL was significantly more prevalent in ER+/luminal GIII‐IDC‐NSTs. Given that GI breast cancers harbor a luminal phenotype, our results suggest that if progression from GI to GIII breast cancer does happen, it may preferentially occur in breast cancers of luminal phenotype.

Identification of novel determinants of resistance to lapatinib in ERBB2-amplified cancers

The gene encoding the receptor tyrosine kinase ERBB2, also known as HER2, is amplified and/or overexpressed in up to 15% of breast cancers. These tumours are characterised by an aggressive phenotype and poor clinical outcome. Although therapies targeted at ERBB2 have proven effective, many patients fail to respond to treatment or become resistant and the reasons for this are still largely unknown. Using a high-throughput functional screen we assessed whether genes found to be recurrently amplified and overexpressed in ERBB2+ve breast cancers mediate resistance to the ERBB2-targeted agent lapatinib. Lapatinib-resistant ERBB2-amplified breast cancer cell lines were screened, in the presence or absence of lapatinib, with an RNA interference library targeting 369 genes recurrently amplified and overexpressed in both ERBB2-amplified breast cancer tumours and cell lines. Small interfering RNAs targeting a number of genes caused sensitivity to lapatinib in this context. The mechanisms of resistance conferred by the identified genes were further investigated and in the case of NIBP (TRAPPC9), lapatinib resistance was found to be mediated through NF-κB signalling. Our results indicate that specific amplified and/ or overexpressed genes found in ERBB2-amplified breast cancer may mediate response to ERBB2-targeting agents.

DNA amplifications in breast cancer: genotypic–phenotypic correlations

DNA copy number changes in cancer cells, in particular, amplifications, occur frequently, have prognostic impact and are associated with subtypes of breast cancer. Some amplicons contain well-characterized oncogenes, including 11q13 (CCND1) and 17q12 (HER2). HER2 amplification and overexpression defines the HER2+ subgroup of breast cancer patients and is both a prognostic marker for poor outcome and a predictive marker for response to anti-HER2 targeted therapies. Therefore, there is considerable interest in documenting the locations of other recurring amplifications in breast cancers as they may also provide a rich source of new biomarkers and novel therapeutic targets for these subgroups. This article focuses on the genomic profiling of breast cancer, with an emphasis on the characteristics of the amplifications found in subtypes of breast cancer, including luminal (ER+)/HER2(-)), HER2+ and basal-like (ER(-)/HER2(-)), and discusses their known or potential roles in cancer biology and their clinical implications.

Development of therapeutic approaches to ‘triple negative’ phenotype breast cancer

Background: Triple negative phenotype (TNP) breast cancers are characterised by the lack of expression of oestrogen and progesterone receptors and of human EGF receptor 2 (HER2) overexpression/amplification. This subgroup of cancers has an aggressive clinical behaviour and is associated with poorer overall survival compared with other subtypes. Given the lack of targets for current tailored therapies in TNP tumours, chemotherapy is the only systemic treatment available; however, overall outcomes remain poor. Therefore, optimal treatment regimens and targeted therapies are urgently needed. Objective: We discuss characteristics of TNP cancers that underpin the rationale of current and novel therapeutic strategies, and an approach for finding and validating new therapeutic targets. Results/conclusion: The results of large prospective randomised controlled trials are currently awaited. Efforts to unravel the heterogeneity and complexity of TNP cancers using the latest high-throughput molecular techniques and integrating these findings with biology-driven therapeutic strategies in clinical trials will be of paramount importance for the development of treatment approaches for this breast cancer subtype.

Integrative molecular and functional profiling of ERBB2- amplified breast cancers identifies new genetic dependencies

Overexpression of the receptor tyrosine kinase ERBB2 (also known as HER2) occurs in around 15% of breast cancers and is driven by amplification of the ERBB2 gene. ERBB2 amplification is a marker of poor prognosis, and although anti-ERBB2-targeted therapies have shown significant clinical benefit, de novo and acquired resistance remains an important problem. Genomic profiling has demonstrated that ERBB2+ve breast cancers are distinguished from ER+ve and ‘triple-negative’ breast cancers by harbouring not only the ERBB2 amplification on 17q12, but also a number of co-amplified genes on 17q12 and amplification events on other chromosomes. Some of these genes may have important roles in influencing clinical outcome, and could represent genetic dependencies in ERBB2+ve cancers and therefore potential therapeutic targets. Here, we describe an integrated genomic, gene expression and functional analysis to determine whether the genes present within amplicons are critical for the survival of ERBB2+ve breast tumour cells. We show that only a fraction of the ERBB2-amplified breast tumour lines are truly addicted to the ERBB2 oncogene at the mRNA level and display a heterogeneous set of additional genetic dependencies. These include an addiction to the transcription factor gene TFAP2C when it is amplified and overexpressed, suggesting that TFAP2C represents a genetic dependency in some ERBB2+ve breast cancer cells.

Abstract A184: Integrative molecular and functional profiling of HER2 amplified breast cancers to identify new gene targets.

Up to 15% of all breast cancers are defined as HER2+ve where overexpression is due to amplification of the HER2 oncogene. Treatment of patients with these clinically aggressive tumors has been dramatically improved by the use of trastuzumab and lapatinib. However a significant proportion of patients9 tumors remain resistant to these therapies. Therefore new biomarkers and additional therapeutic targets are urgently required for what remains a clinically challenging and biologically heterogeneous disease. We describe an integrated genetic, transcriptomic and functional analysis of a wide panel of genes amplified and overexpressed in HER2 amplified breast tumors to identify new gene targets. Molecular profiling of 45 sporadic primary HER2 amplified breast cancers, 14 commercially available HER2 amplified and 9 non HER2 amplified breast cancer cell lines was performed. Genome wide copy number and transcriptomic array data were integrated and identified 511 genes that were significantly overexpressed when amplified in the tumors. The functional significance of 369 of these genes, which were recurrently amplified in the cell lines, was examined using parallel high throughput siRNA screens. Multiple genetic dependencies were observed. We demonstrate that loss of cell viability following silencing of HER2 by multiple siRNAs significantly correlated with sensitivity to lapatinib and other HER2 targeted agents. We also show that silencing of TFAP2C caused significant loss of cell viability when amplified and overexpressed in multiple HER2 amplified breast cancer cell lines. Down regulation of HER2 expression and cell apoptosis was also observed. The potential clinical value of TFAP2C overexpression for patients with HER2+ve breast cancer is currently being explored in adjuvant HER2+ breast cancer trials of chemotherapy +/− HER2 targeted agents. We propose that TFAP2C amplification and overexpression represents a genetic dependency in HER2+ breast cancer and is a clinically relevant candidate biomarker. 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 A184.

Abstract 4987: Identification of novel genes and pathways involved in resistance to HER2-targeting agents in breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The HER2 gene ERBB2 is amplified and/ or overexpressed in up to 15% of all invasive breast cancers. These cancers are characterised by an aggressive phenotype and poor clinical outcome. HER2 positive tumours often display multiple high level amplifications of genes in addition to HER2. Although targeted therapies to HER2 have proven clinical benefit, a substantial number of patients have tumours that are either de novo resistant or acquire resistance over time to these agents. Reasons for this are still largely unknown. By using a high throughput siRNA screen we sought to determine whether genes recurrently amplified and overexpressed in HER2-amplified breast cancer mediate resistance to HER2-targeting agents. We profiled 45 HER2-amplified primary breast cancers and 13 HER2-amplified cell lines with high resolution microarray-based comparative genomic hybridisation (aCGH) and Illumina WG6 v2 arrays. Overlaying of aCGH and gene expression data led to the identification of 369 genes recurrently amplified and overexpressed when amplified. Lapatinib-resistant HER2-amplified breast cancer cell lines were treated with lapatinib, a HER2 small molecule inhibitor, and screened with an RNA interference (RNAi) library targeting these 369 genes and controls. This RNAi screen and subsequent validation screens identified eight genes that when silenced lead to a significant sensitization to lapatinib. The mechanisms of resistance conferred by the identified genes were further investigated and pointed to the involvement of NF-kappaB-signalling, Wnt-signalling and reactive oxygen species scavenging. Our results indicate that specific amplified and overexpressed genes found in HER2 positive breast cancers mediate resistance to anti-HER2 agents. These findings might be used to identify patients likely to be de novo resistant to lapatinib and provide new targets for combined targeted therapies to overcome resistance to anti-HER2 therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4987. doi:10.1158/1538-7445.AM2011-4987