Pierfrancesco Marra

Integrated genomic analysis of triple-negative breast cancers reveals novel microRNAs associated with clinical and molecular phenotypes and sheds light on the pathways they control

BackgroundThis study focuses on the analysis of miRNAs expression data in a cohort of 181 well characterised breast cancer samples composed primarily of triple-negative (ER/PR/HER2-negative) tumours with associated genome-wide DNA and mRNA data, extensive patient follow-up and pathological information.ResultsWe identified 7 miRNAs associated with prognosis in the triple-negative tumours and an additional 7 when the analysis was extended to the set of all ER-negative cases. miRNAs linked to an unfavourable prognosis were associated with a broad spectrum of motility mechanisms involved in the invasion of stromal tissues, such as cell-adhesion, growth factor-mediated signalling pathways, interaction with the extracellular matrix and cytoskeleton remodelling. When we compared different intrinsic molecular subtypes we found 46 miRNAs that were specifically expressed in one or more intrinsic subtypes. Integrated genomic analyses indicated these miRNAs to be influenced by DNA genomic aberrations and to have an overall influence on the expression levels of their predicted targets. Among others, our analyses highlighted the role of miR-17-92 and miR-106b-25, two polycistronic miRNA clusters with known oncogenic functions. We showed that their basal-like subtype specific up-regulation is influenced by increased DNA copy number and contributes to the transcriptional phenotype as well as the activation of oncogenic pathways in basal-like tumours.ConclusionsThis study analyses previously unreported miRNA, mRNA and DNA data and integrates these with pathological and clinical information, from a well-annotated cohort of breast cancers enriched for triple-negative subtypes. It provides a conceptual framework, as well as integrative methods and system-level results and contributes to elucidate the role of miRNAs as biomarkers and modulators of oncogenic processes in these types of tumours.

The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility

Dimerization of EGFR with an ErbB4 receptor variant increases growth factor–induced migration of breast cancer cells. Drug Resistance Through Dimerization The epidermal growth factor receptor (EGFR) is often targeted in various cancers, including breast cancer. The EGFR can dimerize with related receptors in the ErbB family, and formation of these heterodimers is associated with the development of resistance to EGFR inhibitors. Kiuchi et al. found that binding of EGFR to a naturally occurring variant of the receptor ErbB4 prevented a ubiquitin E3 ligase from associating with EGFR and triggering its breakdown. The migration of breast cancer cells to EGFR ligands was increased when EGFR was overexpressed with the ErbB4 variant, but not with a mutant that could not dimerize with EGFR. Furthermore, the transcript for this ErbB4 variant was increased in a subset of breast cancer patients. The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor–stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2– breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.

IL15RA Drives Antagonistic Mechanisms of Cancer Development and Immune Control in Lymphocyte-Enriched Triple-Negative Breast Cancers

Despite its aggressive nature, triple-negative breast cancer (TNBC) often exhibits leucocyte infiltrations that correlate with favorable prognosis. In this study, we offer an explanation for this apparent conundrum by defining TNBC cell subsets that overexpress the IL15 immune receptor IL15RA. This receptor usually forms a heterotrimer with the IL2 receptors IL2RB and IL2RG, which regulates the proliferation and differentiation of cytotoxic T cells and NK cells. However, unlike IL15RA, the IL2RB and IL2RG receptors are not upregulated in basal-like TNBC breast cancer cells that express IL15RA. Mechanistic investigations indicated that IL15RA signaling activated JAK1, STAT1, STAT2, AKT, PRAS40, and ERK1/2 in the absence of IL2RB and IL2RG, whereas neither STAT5 nor JAK2 were activated. RNAi-mediated attenuation of IL15RA established its role in cell growth, apoptosis, and migration, whereas expression of the IL15 cytokine in IL15RA-expressing cells stimulated an autocrine signaling cascade that promoted cell proliferation and migration and blocked apoptosis. Notably, coexpression of IL15RA and IL15 was also sufficient to activate peripheral blood mononuclear cells upon coculture in a paracrine signaling manner. Overall, our findings offer a mechanistic explanation for the paradoxical association of some high-grade breast tumors with better survival outcomes, due to engagement of the immune stroma.

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple‐negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three‐dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6‐depleted, triple‐negative MDA‐MB‐231 cells rearranged the actin cytoskeleton and restored epidermal growth factor‐mediated invasion. In patients with localized, lymph node‐negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse‐free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6–fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC.

Splicing imbalances in basal-like breast cancer underpin perturbation of cell surface and oncogenic pathways and are associated with patients' survival

Despite advancements in the use of transcriptional information to understand and classify breast cancers, the contribution of splicing to the establishment and progression of these tumours has only recently starting to emerge. Our work explores this lesser known landscape, with special focus on the basal-like breast cancer subtype where limited therapeutic opportunities and no prognostic biomarkers are currently available. Using ExonArray analysis of 176 breast cancers and 9 normal breast tissues we demonstrate that splicing levels significantly contribute to the diversity of breast cancer molecular subtypes and explain much of the differences compared with normal tissues. We identified pathways specifically affected by splicing imbalances whose perturbation would be hidden from a conventional gene-centric analysis of gene expression. We found that a large fraction of them involve cell-to-cell communication, extracellular matrix and transport, as well as oncogenic and immune-related pathways transduced by plasma membrane receptors. We identified 247 genes in which splicing imbalances are associated with clinical patients’ outcome, whilst no association was detectable at the gene expression level. These include the signaling gene TGFBR1, the proto-oncogene MYB as well as many immune-related genes such as CCR7 and FCRL3, reinforcing evidence for a role of immune components in influencing breast cancer patients’ prognosis.

Abstract 178: PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer

Triple-Negative Breast Cancers (TNBCs) are aggressive, associated with poor prognosis and lack targeted therapies, mainly due to the absence of suitable molecular targets. The genomic region 6p21-p25 is recurrently amplified in TNBCs and encompasses the PIM1 oncogene. This study interrogated genomic breast cancer datasets and identified gene copy number-driven increase of PIM1 expression in TNBC. To understand the role of PIM1 in malignant phenotypes of TNBCs, functional studies were carried out in breast cancer and non-malignant mammary epithelial cell line models. RNA interference and rescue-of-function experiments revealed addiction to PIM1 kinase for cell population growth and apoptosis protection in TNBC cells, absent in non-malignant cells. In cells sensitive to PIM1 knockdown, we observed a subsequent reduction of the expression of the anti-apoptotic proteins BCL2 and MCL1. Moreover, exogenous expression of BCL2 prevented apoptosis caused by PIM1 knockdown. BH3-profiling analysis further confirmed that PIM1 blocks apoptosis elicited through the mitochondrial pathway in TNBC cell lines. The activity of PIM1 in other cancers has been closely linked to the regulation of c-MYC, an “un-targetable” oncogene that drives malignancy and that is frequently amplified and highly expressed in primary TNBCs and post-chemotherapy residual disease. Importantly, we found that PIM1 expression associates with several MYC-transcriptional signatures in TNBCs and mediates phosphorylation of c-MYC Ser62 and Histone-H3 Ser10, key targets for MYC-driven transcription. Exogenous expression of MYC rescued the phenotypes caused by PIM1 knockdown, providing mechanistic insights for the observed addiction. Finally, the therapeutic potential of targeting PIM1 was assessed using AZD1208, a small molecule inhibitor of the PIM kinase family. AZD1208 inhibited growth and sensitized TNBC derived cell lines, cell line xenografts and PDXs to chemotherapy by increasing apoptosis. We therefore identify PIM1 as a driver of malignancy in TNBC, illustrating relationships with MYC-activation and regulation of anti-apoptotic BCL2 and MCL1 proteins. PIM1 inhibitors could provide a potential therapeutic to abrogate chemotherapy resistance in TNBCs. Citation Format: Fara Braso-Maristany, Simone Filosto, Steven Catchpole, Rebecca Marlow, Jelmar Quist, Erika Francesch Domenech, Gianmaria Liccardi, Leila Zakka, Violeta Serra, Albert Gris, Maggie Cheang, Nirmesh Patel, Anna Perdrix Rosell, Patrycja Gazinska, Elodie Noel, Jonathan Watkins, Pierfrancesco Marra, Anita Grigoriadis, Andrew Tutt. PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 178.

Abstract P3-04-05: Amplification driven expression of KIAA0020, a PARP1 interacting gene, facilitates repair of replication associated DNA damage in triple-negative breast cancers

Approximately 25% of triple-negative breast cancers display increased copy number in the region of 9p24. Since several genes mapping to this genomic locus have been shown to be involved in the biology of triple-negative breast cancers, a copy number-dependent transcriptional influence of genes at 9p24 on the malignant phenotypes has been suggested. Given the limited options of targeted therapy for triple-negative breast cancer patients, further studies of genes located at this locus could therefore provide potential novel targets or companion biomarkers for this disease. By integrating microarray-based DNA copy number and gene expression of genes located on 9p24 in 111 triple-negative breast cancers, we identified among a wider set of genes three hypothetical genes, KIAA0020, KIAA1432 and KIAA2026 whose expression was highly correlated with their copy number status. External validation through analysis of several comprehensive breast cancer cohorts, confirmed KIAA0020 as being highly abundant in basal-like/ triple-negative breast cancers. Involvement of KIAA0020 in PARP1 activity and the DNA damage response has previously been suggested. To elucidate KIAA00209s functional involvement in cell growth, cell cycle progression, apoptosis and DNA damage response in this subtype of breast cancer, breast cancer cell lines with and without increased 9p24 copy number levels were used as in vitro models. In cell lines with increased DNA copy number at 9p24, depletion of KIAA0020 expression selectively impaired growth causing an accumulation in S-phase and a decrease in cell proliferation. Furthermore, KIAA0020-silencing in such cell lines resulted in decreased repair of hydroxyurea induced replication associated DNA damage, accumulation of DNA double strand breaks and decreased occurrence of RAD51 and PAR foci, all pointing to a decreased repair of inactivated replication forks. Taken together, our data supports the notion that genes of unknown function, residing in the 9p24 copy number aberrant region are involved in DNA repair and may thereby also contribute to the tumourigenesis of triple-negative breast cancers. A subset of such triple-negative tumours seemed to have developed a dependency on the expression of KIAA0020, a PARP1 interacting gene, for replication fork associated repair. Thus, greater understanding of KIAA00209s molecular function may provide additional information for patient selection with regards to DNA damaging chemotherapeutics or PARP inhibitors within triple-negative breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-05.

Abstract B3: Genomic characterization of triple-negative breast cancers with high-resolution SNP 6.0 Affymetrix arrays

Introduction: The histopathological absence of ER, PR, and HER2 defines a subgroup of breast cancers named triple negative, characterized by aggressive behavior and poor prognosis. These tumors represent a significant clinical challenge as little is known about the genetic alterations underlying their development and, as a consequence, no specific targeted therapies are yet available. Using high-resolution SNP6.0 Affymetrix technology, we evaluated genetic copy number alteration events in a collection of 107 fine-needle microdissected breast cancers, including 82 triple-negative and 25 non-triple negative (17 ER+/ HER2- and 8 ER-/HER2+) samples. 18 blood samples from matching patients were also profiled and their pool was used as a baseline. Results: Upon application of circular binary segmentation algorithm, 36 and 190 chromosomal regions could be identified for being respectively frequently amplified and deleted in triple-negative tumors (frequency > 30%, p-value 30% of samples) in the two tumor groups. Among others, the “immunological response” pathway emerged as more frequently affected by events of amplification in triple-negative breast tumors (p-value 20%). A number of chromosome regions were identified for being frequently co-amplified. These include regions on chromosome 1q (with each other), 10p (with each other), 6q11 with 7p12 and 19q13,19q and 5p. Similarly, regions of co-deletions could be identified, such as: 8p21 (with each other), 4p15 (with each other), 17p13 and 5q11,6q26, and 17p12. Conclusions: We have performed a comprehensive survey of copy number variations in triple-negative breast tumors, pinpointing genomic regions and related genes and pathways specifically altered in this class of tumors. Dependencies between individual events of genomic amplification or deletion have been identified, suggesting they are resulting from selective pressure and their coordinated genomic alteration is required for the survival of the cancer cells involved. Our analyses shed further light on the genomically complex and heterogeneous nature of triple-negative tumors in relation to other subgroups of breast cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B3.