Comprehensive Molecular Portraits of Invasive Lobular Breast Cancer.

Invasive lobular carcinoma (ILC) is the second most prevalent histologic subtype of invasive breast cancer. Here, we comprehensively profiled 817 breast tumors, including 127 ILC, 490 ductal (IDC), and 88 mixed IDC/ILC. Besides E-cadherin loss, the best known ILC genetic hallmark, we identified mutations targeting PTEN, TBX3, and FOXA1 as ILC enriched features. PTEN loss associated with increased AKT phosphorylation, which was highest in ILC among all breast cancer subtypes. Spatially clustered FOXA1 mutations correlated with increased FOXA1 expression and activity. Conversely, GATA3 mutations and high expression characterized luminal A IDC, suggesting differential modulation of ER activity in ILC and IDC. Proliferation and immune-related signatures determined three ILC transcriptional subtypes associated with survival differences. Mixed IDC/ILC cases were molecularly classified as ILC-like and IDC-like revealing no true hybrid features. This multidimensional molecular atlas sheds new light on the genetic bases of ILC and provides potential clinical options.

Proteogenomics connects somatic mutations to signalling in breast cancer.

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

Emerging Role of SOX Proteins in Breast Cancer Development and Maintenance.

The SOX genes encode a family of more than 20 transcription factors that are critical regulators of embryogenesis and developmental processes and, when aberrantly expressed, have been shown to contribute to tumor development and progression in both an oncogenic and tumor suppressive role. Increasing evidence demonstrates that the SOX proteins play essential roles in multiple cellular processes that mediate or contribute to oncogenic transformation and tumor progression. In the context of breast cancer, SOX proteins function both as oncogenes and tumor suppressors and have been shown to be associated with tumor stage and grade and poor prognosis. Experimental evidence demonstrates that a subset of SOX proteins regulate critical aspects of breast cancer biology including cancer stemness and multiple signaling pathways leading to altered cell proliferation, survival, and tumor development; EMT, cell migration and metastasis; as well as other tumor associated characteristics. This review will summarize the role of SOX family members as important mediators of tumorigenesis in breast cancer, with an emphasis on the triple negative or basal-like subtype of breast cancer, as well as examine the therapeutic potential of these genes and their downstream targets.

Amplification of SOX4 promotes PI3K/Akt signaling in human breast cancer.

PURPOSE: The PI3K/Akt signaling axis contributes to the dysregulation of many dominant features in breast cancer including cell proliferation, survival, metabolism, motility, and genomic instability. While multiple studies have demonstrated that basal-like or triple-negative breast tumors have uniformly high PI3K/Akt activity, genomic alterations that mediate dysregulation of this pathway in this subset of highly aggressive breast tumors remain to be determined. METHODS: In this study, we present an integrated genomic analysis based on the use of a PI3K gene expression signature as a framework to analyze orthogonal genomic data from human breast tumors, including RNA expression, DNA copy number alterations, and protein expression. In combination with data from a genome-wide RNA-mediated interference screen in human breast cancer cell lines, we identified essential genetic drivers of PI3K/Akt signaling. RESULTS: Our in silico analyses identified SOX4 amplification as a novel modulator of PI3K/Akt signaling in breast cancers and in vitro studies confirmed its role in regulating Akt phosphorylation. CONCLUSIONS: Taken together, these data establish a role for SOX4-mediated PI3K/Akt signaling in breast cancer and suggest that SOX4 may represent a novel therapeutic target and/or biomarker for current PI3K family therapies.

A genomic strategy to elucidate modules of oncogenic pathway signaling networks.

Recent studies have emphasized the importance of pathway-specific interpretations for understanding the functional relevance of gene alterations in human cancers. Although signaling activities are often conceptualized as linear events, in reality, they reflect the activity of complex functional networks assembled from modules that each respond to input signals. To acquire a deeper understanding of this network structure, we developed an approach to deconstruct pathways into modules represented by gene expression signatures. Our studies confirm that they represent units of underlying biological activity linked to known biochemical pathway structures. Importantly, we show that these signaling modules provide tools to dissect the complexity of oncogenic states that define disease outcomes as well as response to pathway-specific therapeutics. We propose that this model of pathway structure constitutes a framework to study the processes by which information propogates through cellular networks and to elucidate the relationships of fundamental modules to cellular and clinical phenotypes.

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

Cross-species DNA copy number analyses identifies multiple 1q21-q23 subtype-specific driver genes for breast cancer.

A large number of DNA copy number alterations (CNAs) exist in human breast cancers, and thus characterizing the most frequent CNAs is key to advancing therapeutics because it is likely that these regions contain breast tumor 'drivers' (i.e., cancer causal genes). This study aims to characterize the genomic landscape of breast cancer CNAs and identify potential subtype-specific drivers using a large set of human breast tumors and genetically engineered mouse (GEM) mammary tumors. Using a novel method called SWITCHplus, we identified subtype-specific DNA CNAs occurring at a 15% or greater frequency, which excluded many well-known breast cancer-related drivers such as amplification of ERBB2, and deletions of TP53 and RB1. A comparison of CNAs between mouse and human breast tumors identified regions with shared subtype-specific CNAs. Additional criteria that included gene expression-to-copy number correlation, a DawnRank network analysis, and RNA interference functional studies highlighted candidate driver genes that fulfilled these multiple criteria. Numerous regions of shared CNAs were observed between human breast tumors and GEM mammary tumor models that shared similar gene expression features. Specifically, we identified chromosome 1q21-23 as a Basal-like subtype-enriched region with multiple potential driver genes including PI4KB, SHC1, and NCSTN. This step-wise computational approach based on a cross-species comparison is applicable to any tumor type for which sufficient human and model system DNA copy number data exist, and in this instance, highlights that a single region of amplification may in fact harbor multiple driver genes.

Interaction of E2F7 transcription factor with E2F1 and C-terminal-binding protein (CtBP) provides a mechanism for E2F7-dependent transcription repression.

Previous work has identified distinct functions for E2F proteins during a cellular proliferative response including a role for E2F1-3 in the activation of transcription at G1/S and a role for E2F4-8 in repressing the same group of E2F1-3 target genes as cells progress through S phase. We now find that E2F7 and E2F8, which are induced by E2F1-3 at G1/S, can form a heterodimer with E2F1 through interactions involving the DNA-binding domains of the two proteins. In vitro DNA interaction assays demonstrate the formation of an E2F1-E2F7 complex, as well as an E2F7-E2F7 complex on adjacent E2F-binding sites. We also show that E2F7 recruits the co-repressor C-terminal-binding protein (CtBP) and that CtBP2 is essential for E2F7 to repress E2F1 transcription. Taken together, these findings suggest a mechanism for the repression of transcription by E2F7.

A pathway-based classification of human breast cancer.

The hallmark of human cancer is heterogeneity, reflecting the complexity and variability of the vast array of somatic mutations acquired during oncogenesis. An ability to dissect this heterogeneity, to identify subgroups that represent common mechanisms of disease, will be critical to understanding the complexities of genetic alterations and to provide a framework to develop rational therapeutic strategies. Here, we describe a classification scheme for human breast cancer making use of patterns of pathway activity to build on previous subtype characterizations using intrinsic gene expression signatures, to provide a functional interpretation of the gene expression data that can be linked to therapeutic options. We show that the identified subgroups provide a robust mechanism for classifying independent samples, identifying tumors that share patterns of pathway activity and exhibit similar clinical and biological properties, including distinct patterns of chromosomal alterations that were not evident in the heterogeneous total population of tumors. We propose that this classification scheme provides a basis for understanding the complex mechanisms of oncogenesis that give rise to these tumors and to identify rational opportunities for combination therapies.

HCK Promotes High-Grade Serous Ovarian Cancer Tumorigenesis through CD44 and NOTCH3 Signaling.

High-grade serous ovarian cancer (HGSOC) is a highly aggressive and lethal subtype of ovarian cancer. While most patients initially respond to standard-of-care treatment, the majority will eventually relapse and succumb to their disease. Despite significant advances in our understanding of this disease, the mechanisms that govern the distinctions between HGSOC with good and poor prognosis remain unclear. In this study, we implemented a proteogenomic approach to analyze gene expression, proteomic and phosphoproteomic profiles of HGSOC tumor samples to identify molecular pathways that distinguish HGSOC tumors relative to clinical outcome. Our analyses identify significant upregulation of hematopoietic cell kinase (HCK) expression and signaling in poor prognostic HGSOC patient samples. Analyses of independent gene expression datasets and IHC of patient samples confirmed increased HCK signaling in tumors relative to normal fallopian or ovarian samples and demonstrated aberrant expression in tumor epithelial cells. Consistent with the association between HCK expression and tumor aggressiveness in patient samples, in vitro phenotypic studies showed that HCK can, in part, promote cell proliferation, colony formation, and invasive capacity of cell lines. Mechanistically, HCK mediates these phenotypes, partly through CD44 and NOTCH3-dependent signaling, and inhibiting CD44 or NOTCH3 activity, either genetically or through gamma-secretase inhibitors, can revert HCK-driven phenotypes. IMPLICATIONS: Collectively, these studies establish that HCK acts as an oncogenic driver of HGSOC through aberrant activation of CD44 and NOTCH3 signaling and identifies this network as a potential therapeutic opportunity in a subset of patients with aggressive and recurrent HGSOC.

SOX4-SMARCA4 complex promotes glycolysis-dependent TNBC cell growth through transcriptional regulation of Hexokinase 2.

Tumor cells rely on increased glycolytic capacity to promote cell growth and progression. While glycolysis is known to be upregulated in the majority of triple negative (TNBC) or basal-like subtype breast cancers, the mechanism remains unclear. Here, we used integrative genomic analyses to identify a subset of basal-like tumors characterized by increased expression of the oncogenic transcription factor SOX4 and its co-factor the SWI/SNF ATPase SMARCA4. These tumors are defined by unique gene expression programs that correspond with increased tumor proliferation and activation of key metabolic pathways, including glycolysis. Mechanistically, we demonstrate that the SOX4-SMARCA4 complex mediates glycolysis through direct transcriptional regulation of Hexokinase 2 (HK2) and that aberrant HK2 expression and altered glycolytic capacity are required to mediate SOX4-SMARCA4-dependent cell growth. Collectively, we have defined the SOX4-SMARCA4-HK2 signaling axis in basal-like breast tumors and established that this axis promotes metabolic reprogramming which is required to maintain tumor cell growth.

Genetic heterogeneity of Myc-induced mammary tumors reflecting diverse phenotypes including metastatic potential.

Human cancers result from a complex series of genetic alterations, resulting in heterogeneous disease states. Dissecting this heterogeneity is critical for understanding underlying mechanisms and providing opportunities for therapeutics matching the complexity. Mouse models of cancer have generally been used to reduce this complexity and focus on the role of single genes. Nevertheless, our analysis of tumors arising in the MMTV-Myc model of mammary carcinogenesis reveals substantial heterogeneity, seen in both histological and expression phenotypes. One contribution to this heterogeneity is the substantial frequency of activating Ras mutations. Additionally, we show that these Myc-induced mammary tumors exhibit even greater heterogeneity, revealed by distinct histological subtypes as well as distinct patterns of gene expression, than many other mouse models of tumorigenesis. Two of the major histological subtypes are characterized by differential patterns of cellular signaling pathways, including beta-catenin and Stat3 activities. We also demonstrate that one of the MMTV-Myc mammary tumor subgroups exhibits metastatic capacity and that the signature derived from the subgroup can predict metastatic potential of human breast cancer. Together, these data reveal that a combination of histological and genomic analyses can uncover substantial heterogeneity in mammary tumor formation and therefore highlight aspects of tumor phenotype not evident in the population as a whole.

SIGNATURE: a workbench for gene expression signature analysis.

BACKGROUND: The biological phenotype of a cell, such as a characteristic visual image or behavior, reflects activities derived from the expression of collections of genes. As such, an ability to measure the expression of these genes provides an opportunity to develop more precise and varied sets of phenotypes. However, to use this approach requires computational methods that are difficult to implement and apply, and thus there is a critical need for intelligent software tools that can reduce the technical burden of the analysis. Tools for gene expression analyses are unusually difficult to implement in a user-friendly way because their application requires a combination of biological data curation, statistical computational methods, and database expertise. RESULTS: We have developed SIGNATURE, a web-based resource that simplifies gene expression signature analysis by providing software, data, and protocols to perform the analysis successfully. This resource uses bayesian methods for processing gene expression data coupled with a curated database of gene expression signatures, all carried out within a GenePattern web interface for easy use and access. CONCLUSIONS: SIGNATURE is available for public use at http://genepattern.genome.duke.edu/signature/.

Analysis of tumor environmental response and oncogenic pathway activation identifies distinct basal and luminal features in HER2-related breast tumor subtypes.

INTRODUCTION: Breast cancer heterogeneity occurs as a consequence of the dysregulation of numerous oncogenic pathways as well as many non-genetic factors, including tumor microenvironmental stresses such as hypoxia, lactic acidosis, and glucose deprivation. Although the importance of these non-genetic factors is well recognized, it is not clear how to integrate these factors within the genetic framework of cancer as the next logical step in understanding tumor heterogeneity. METHODS: We report here the development of a series of gene expression signatures to measure the influences of microenvironmental stresses. The pathway activities of hypoxia, lactic acidosis, acidosis and glucose deprivation were investigated in a collection of 1,143 breast tumors, which have been separated into 17 breast tumor subgroups defined by their distinct patterns of oncogenic pathways. A validation dataset comprised of 547 breast tumors was also used to confirm the major findings, and representative breast cancer cell lines were utilized to validate in silico results and mechanistic studies. RESULTS: Through the integrative pathway analysis of microenvironmental stresses and oncogenic events in breast tumors, we identified many known and novel correlations between these two sources of tumor heterogeneity. Focusing on differences between two human epidermal growth factor receptor 2 (HER2)-related subgroups, previously identified based on patterns of oncogenic pathway activity, we determined that these subgroups differ with regards to tumor microenvironmental signatures, including hypoxia. We further demonstrate that each of these subgroups have features consistent with basal and luminal breast tumors including patterns of oncogenic signaling pathways, expression of subtype specific genes, and cellular mechanisms that regulate the hypoxia response. Importantly, we also demonstrate that the correlated pattern of hypoxia-related gene expression and basal-associated gene expression are consistent across HER2-related tumors whether we analyze the tumors as a function of our pathway-based classification scheme, using the intrinsic gene list (ERBB2+), or based on HER2 IHC status. Our results demonstrate a cell lineage-specific phenomenon in which basal-like tumors, HER2-related tumors with high hypoxia, as well as normal basal epithelial cells express increased mRNA levels of HIF-1α compared to luminal types and silencing of HIF-1α results in decreased expression of hypoxia-induced genes. CONCLUSIONS: This study demonstrates differences in microenvironmental conditions in HER2-related subgroups defined by distinct oncogenic pathway activities, and provides a mechanistic explanation for differences in the observed hypoxia response between these subgroups. Collectively, these data demonstrate the potential of a pathway-based classification strategy as a framework to integrate genetic and non-genetic factors to investigate the basis of tumor heterogeneity.

Nuclear Receptor-Mediated Metabolic Reprogramming and the Impact on HR+ Breast Cancer.

Metabolic reprogramming enables cancer cells to adapt to the changing microenvironment in order to maintain metabolic energy and to provide the necessary biological macromolecules required for cell growth and tumor progression. While changes in tumor metabolism have been long recognized as a hallmark of cancer, recent advances have begun to delineate the mechanisms that modulate metabolic pathways and the consequence of altered signaling on tumorigenesis. This is particularly evident in hormone receptor positive (HR+) breast cancers which account for approximately 70% of breast cancer cases. Emerging evidence indicates that HR+ breast tumors are dependent on multiple metabolic processes for tumor progression, metastasis, and therapeutic resistance and that changes in metabolic programs are driven, in part, by a number of key nuclear receptors including hormone-dependent signaling. In this review, we discuss the mechanisms and impact of hormone receptor mediated metabolic reprogramming on HR+ breast cancer genesis and progression as well as the therapeutic implications of these metabolic processes in this disease.

Recent Advances in Integrative Multi-Omics Research in Breast and Ovarian Cancer.

The underlying molecular heterogeneity of cancer is responsible for the dynamic clinical landscape of this disease. The combination of genomic and proteomic alterations, including both inherited and acquired mutations, promotes tumor diversity and accounts for variable disease progression, therapeutic response, and clinical outcome. Recent advances in high-throughput proteogenomic profiling of tumor samples have resulted in the identification of novel oncogenic drivers, tumor suppressors, and signaling networks; biomarkers for the prediction of drug sensitivity and disease progression; and have contributed to the development of novel and more effective treatment strategies. In this review, we will focus on the impact of historical and recent advances in single platform and integrative proteogenomic studies in breast and ovarian cancer, which constitute two of the most lethal forms of cancer for women, and discuss the molecular similarities of these diseases, the impact of these findings on our understanding of tumor biology as well as the clinical applicability of these discoveries.

SOX4 and SMARCA4 cooperatively regulate PI3k signaling through transcriptional activation of TGFBR2.

Dysregulation of PI3K/Akt signaling is a dominant feature in basal-like or triple-negative breast cancers (TNBC). However, the mechanisms regulating this pathway are largely unknown in this subset of aggressive tumors. Here we demonstrate that the transcription factor SOX4 is a key regulator of PI3K signaling in TNBC. Genomic and proteomic analyses coupled with mechanistic studies identified TGFBR2 as a direct transcriptional target of SOX4 and demonstrated that TGFBR2 is required to mediate SOX4-dependent PI3K signaling. We further report that SOX4 and the SWI/SNF ATPase SMARCA4, which are uniformly overexpressed in basal-like tumors, form a previously unreported complex that is required to maintain an open chromatin conformation at the TGFBR2 regulatory regions in order to mediate TGFBR2 expression and PI3K signaling. Collectively, our findings delineate the mechanism by which SOX4 and SMARCA4 cooperatively regulate PI3K/Akt signaling and suggest that this complex may play an essential role in TNBC genesis and/or progression.

SMAD4 is critical in suppression of BRAF-V600E serrated tumorigenesis.

BRAF-driven colorectal cancer is among the poorest prognosis subtypes of colon cancer. Previous studies suggest that BRAF-mutant serrated cancers frequently exhibit Microsatellite Instability (MSI) and elevated levels of WNT signaling. The loss of tumor-suppressor Smad4 in oncogenic BRAF-V600E mouse models promotes rapid serrated tumor development and progression, and SMAD4 mutations co-occur in human patient tumors with BRAF-V600E mutations. This study assesses the role of SMAD4 in early-stage serrated tumorigenesis. SMAD4 loss promotes microsatellite stable (MSS) serrated tumors in an oncogenic BRAF-V600E context, providing a model for MSS serrated cancers. Inactivation of Msh2 in these mice accelerated tumor formation, and whole-exome sequencing of both MSS and MSI serrated tumors derived from these mouse models revealed that all serrated tumors developed oncogenic WNT mutations, predominantly in the WNT-effector gene Ctnnb1 (ß-catenin). Mouse models mimicking the oncogenic ß-catenin mutation show that the combination of three oncogenic mutations (Ctnnb1, Braf, and Smad4) are critical to drive rapid serrated dysplasia formation. Re-analysis of human tumor data reveals BRAF-V600E mutations co-occur with oncogenic mutations in both WNT and SMAD4/TGFß pathways. These findings identify SMAD4 as a critical factor in early-stage serrated cancers and helps broaden the knowledge of this rare but aggressive subset of colorectal cancer.

CPT1A and fatty acid ß-oxidation are essential for tumor cell growth and survival in hormone receptor-positive breast cancer.

Chromosome 11q13-14 amplification is a defining feature of high-risk hormone receptor-positive (HR+) breast cancer; however, the mechanism(s) by which this amplicon contributes to breast tumorigenesis remains unclear. In the current study, proteogenomic analyses of >3000 breast tumors from the TCGA, METABRIC and CPTAC studies demonstrated that carnitine palmitoyltransferase 1A (CPT1A), which is localized to this amplicon, is overexpressed at the mRNA and protein level in aggressive luminal tumors, strongly associated with indicators of tumor proliferation and a predictor of poor prognosis. In vitro genetic studies demonstrated that CPT1A is required for and can promote luminal breast cancer proliferation, survival, as well as colony and mammosphere formation. Since CPT1A is the rate-limiting enzyme during fatty acid oxidation (FAO), our data indicate that FAO may be essential for these tumors. Pharmacologic inhibition of FAO prevented in vitro and in vivo tumor growth and cell proliferation as well as promoted apoptosis in luminal breast cancer cells and orthotopic xenograft tumor models. Collectively, our data establish an oncogenic role for CPT1A and FAO in HR+ luminal tumors and provide preclinical evidence and rationale supporting further investigation of FAO as a potential therapeutic opportunity for the treatment of HR+ breast cancer.

FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036.

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.