Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.

Kinome profiling reveals breast cancer heterogeneity and identifies targeted therapeutic opportunities for triple negative breast cancer.

Our understanding of breast cancer heterogeneity at the protein level is limited despite proteins being the ultimate effectors of cellular functions. We investigated the heterogeneity of breast cancer (41 primary tumors and 15 breast cancer cell lines) at the protein and phosphoprotein levels to identify activated oncogenic pathways and developing targeted therapeutic strategies. Heterogeneity was observed not only across histological subtypes, but also within subtypes. Tumors of the Triple negative breast cancer (TNBC) subtype distributed across four different clusters where one cluster (cluster ii) showed high deregulation of many proteins and phosphoproteins. The majority of TNBC cell lines, particularly mesenchymal lines, resembled the cluster ii TNBC tumors. Indeed, TNBC cell lines were more sensitive than non-TNBC cell lines when treated with targeted inhibitors selected based on upregulated pathways in cluster ii. In line with the enrichment of the upregulated pathways with onco-clients of Hsp90, we found synergy in combining Hsp90 inhibitors with several kinase inhibitors, particularly Erk5 inhibitors. The combination of Erk5 and Hsp90 inhibitors was effective in vitro and in vivo against TNBC leading to upregulation of pro-apoptotic effectors. Our studies contribute to proteomic profiling and improve our understanding of TNBC heterogeneity to provide therapeutic opportunities for this disease.

miR-139-5p is a regulator of metastatic pathways in breast cancer.

Metastasis is a complex, multistep process involved in the progression of cancer from a localized primary tissue to distant sites, often characteristic of the more aggressive forms of this disease. Despite being studied in great detail in recent years, the mechanisms that govern this process remain poorly understood. In this study, we identify a novel role for miR-139-5p in the inhibition of breast cancer progression. We highlight its clinical relevance by reviewing miR-139-5p expression across a wide variety of breast cancer subtypes using in-house generated and online data sets to show that it is most frequently lost in invasive tumors. A biotin pull-down approach was then used to identify the mRNA targets of miR-139-5p in the breast cancer cell line MCF7. Functional enrichment analysis of the pulled-down targets showed significant enrichment of genes in pathways previously implicated in breast cancer metastasis (P < 0.05). Further bioinformatic analysis revealed a predicted disruption to the TGFß, Wnt, Rho, and MAPK/PI3K signaling cascades, implying a potential role for miR-139-5p in regulating the ability of cells to invade and migrate. To corroborate this finding, using the MDA-MB-231 breast cancer cell line, we show that overexpression of miR-139-5p results in suppression of these cellular phenotypes. Furthermore, we validate the interaction between miR-139-5p and predicted targets involved in these pathways. Collectively, these results suggest a significant functional role for miR-139-5p in breast cancer cell motility and invasion and its potential to be used as a prognostic marker for the aggressive forms of breast cancer.

MicroRNA-182-5p targets a network of genes involved in DNA repair.

MicroRNAs are noncoding regulators of gene expression, which act by repressing protein translation and/or degrading mRNA. Many have been shown to drive tumorigenesis in cancer, but functional studies to understand their mode of action are typically limited to single-target genes. In this study, we use synthetic biotinylated miRNA to pull down endogenous targets of miR-182-5p. We identified more than 1000 genes as potential targets of miR-182-5p, most of which have a known function in pathways underlying tumor biology. Specifically, functional enrichment analysis identified components of both the DNA damage response pathway and cell cycle to be highly represented in this target cohort. Experimental validation confirmed that miR-182-5p-mediated disruption of the homologous recombination (HR) pathway is a consequence of its ability to target multiple components in that pathway. Although there is a strong enrichment for the cell cycle ontology, we do not see primary proliferative defects as a consequence of miR-182-5p overexpression. We highlight targets that could be responsible for miR-182-5p-mediated disruption of other biological processes attributed in the literature so far. Finally, we show that miR-182-5p is highly expressed in a panel of human breast cancer samples, highlighting its role as a potential oncomir in breast cancer.

The plasminogen activator inhibitor 2 transcript is destabilized via a multi-component 3' UTR localized adenylate and uridylate-rich instability element in an analogous manner to cytokines and oncogenes.

Plasminogen activator inhibitor type 2 (PAI-2; SERPINB2) is a highly-regulated gene that is subject to both transcriptional and post-transcriptional control. For the latter case, inherent PAI-2 mRNA instability was previously shown to require a nonameric adenylate-uridylate element in the 3' UTR. However, mutation of this site was only partially effective at restoring complete mRNA stabilization. In the present study, we have identified additional regulatory motifs within the 3' UTR that cooperate with the nonameric adenylate-uridylate element to promote mRNA destabilization. These elements are located within a 74 nucleotide U-rich stretch (58%) of the 3' UTR that flanks the nonameric motif; deletion or substitution of this entire region results in complete mRNA stabilization. These new elements are conserved between species and optimize the destabilizing capacity with the nonameric element to ensure complete mRNA instability in a manner analogous to some class I and II adenylate-uridylate elements present in transcripts encoding oncogenes and cytokines. Hence, post-transcriptional regulation of the PAI-2 mRNA transcript involves an interaction between closely spaced adenylate-uridylate elements in a manner analogous to the post-transcriptional regulation of oncogenes and cytokines.

Lefty antagonises TGF-beta1 induced epithelial-mesenchymal transition in tubular epithelial cells.

Lefty is a novel member of the transforming growth factor (TGF) supergene family which has the potential to antagonise actions of TGF-beta1 - the main factor driving fibrotic disease in the kidney and in other organs. TGF-beta1 can induce fibrosis through several mechanisms, including epithelial-mesenchymal transition (EMT) which contributes to myofibroblast accumulation in the renal interstitium. This study examined whether Lefty can antagonise TGF-beta1 mediated EMT. A rat tubular epithelial cell line (NRK52E) was stably transfected with a Lefty expression plasmid (52E-Lefty) or control plasmid (52E-Control). 52E-Control cells underwent TGF-beta1 induced EMT with up-regulation of alpha-smooth muscle actin (alpha-SMA), down-regulation of E-cadherin, and transition to an elongated fibroblast-like morphology. In contrast, 52E-Lefty cells were substantially protected from TGF-beta1 induced EMT. Analysis of signalling pathways showed that 52E-Lefty cells had a marked reduction in TGF-beta1 induced Smad activity and suppression of the secondary phase of JNK (but not p38) signalling. Treatment of NRK52E cells with a JNK inhibitor was shown to suppress TGF-beta1 induced EMT. In conclusion, Lefty can antagonise TGF-beta1 mediated EMT in renal tubular epithelial cells. Lefty may have potential as an anti-fibrotic molecule in the treatment of renal fibrosis.