Biology subjects

Swarbrick, A.

Publications and source records attributed to Swarbrick, A..

25 records · Page 2Linked to original sources

bioRxiv2019

Targeting the Id1-Kif11-Aurka axis in triple negative breast cancer using combination therapy

Evidence points to breast cancer following a hierarchical model, with Cancer Stem Cells (CSCs) driving critical phenotypes of the bulk tumor. Chemoresistant CSCs are not an abstract concept but have clinical consequences as they drive relapse and ultimately lead to mortality in patients, making it imperative to understand how these subpopulations of cells survive. Our previous work (1-2) has demonstrated that the bHLH transcription factor, Inhibitor of Differentiation 1 (Id1) and its closely related family member Id3, have an important role in maintaining the CSC phenotype in the Triple Negative breast cancer (TNBC) subtype. A genetic screen conducted to further elucidate the molecular mechanism underlying the Id (Id1/3) mediated CSC phenotypes in TNBC revealed critical cell cycle genes such as Kif11 and Aurka as putative Id targets. We take this work forward by investigating how alteration in Kif11 and Aurka via Id proteins promotes the CSC phenotype in TNBC. Cells lacking Id are poised in a state of G0/G1 arrest from which they can re-enter the cell cycle. Intriguingly, depletion of Kif11 and Aurka independently did not phenocopy the G0/G1 arrest observed in Id knockdown (Id KD) cells. We have further explored the hypothesis that we can deplete the chemo resistant Id expressing CSC population by combining chemotherapy with targeted therapy using existing small molecule inhibitors (against Id target Kif11) to more effectively debulk the entire tumor. This work opens up exciting new possibilities of targeting Id targets like Kif11, in the TNBC subtype which is currently refractory to chemotherapy.

cancer biology↗

bioRxiv2018

Id proteins promote a cancer stem cell phenotype in triple negative breast cancer via Robo1-dependent c-Myc activation

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed two independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

cancer biology↗

bioRxiv2018

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

High-throughput single-cell RNA-Sequencing is a powerful technique for gene expression profiling of complex and heterogeneous cellular populations such as the immune system. However, these methods only provide short-read sequence from one end of a cDNA template, making them poorly suited to the investigation of gene-regulatory events such as mRNA splicing, adaptive immune responses or somatic genome evolution. To address this challenge, we have developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We use Repertoire And Gene Expression sequencing (RAGE-seq) to accurately characterize full-length T cell (TCR) and B cell (BCR) receptor sequences and transcriptional profiles of more than 7,138 lymphocytes sampled from the primary tumour and draining lymph node of a breast cancer patient. With this method we show that somatic mutation, alternate splicing and clonal evolution of T and B lymphocytes can be tracked across these tissue compartments. Our results demonstrate that RAGE-Seq is an accessible and cost-effective method for high-throughput deep single cell profiling, applicable to a wide range of biological challenges.

genomics↗

bioRxiv2018

A novel role for the HLH protein Inhibitor of Differentiation 4 (ID4) in the DNA damage response in basal-like breast cancer.

Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of Differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC, through unknown mechanisms. Here, we have defined a molecular mechanism of action for ID4 in BLBC and the related disease highgrade serous ovarian cancer (HGSOV), by combining RIME proteomic analysis and ChIP-Seq mapping of genomic binding sites. Remarkably, these studies have revealed novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins ({gamma}H2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage and regulating DNA damage signalling. Clinical analysis demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair pathways. These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOV.

cancer biology↗

bioRxiv2018

Single cell transcriptomics reveals molecular subtype and functional heterogeneity in models of breast cancer

Breast cancer has long been classified into a number of molecular subtypes that predict prognosis and therefore influence clinical treatment decisions. Cellular heterogeneity is also evident in breast cancers and plays a key role in the development, evolution and metastatic progression of many cancers. How clinical heterogeneity relates to cellular heterogeneity is poorly understood, so we approached this question using single cell gene expression analysis of well established in vitro and in vivo models of disease.\n\nTo explore the cellular heterogeneity in breast cancer we first examined a panel of genes that define the PAM50 classifier of molecular subtype. Five breast cancer cell line models (MCF7, BT474, SKBR3, MDA-MB-231, and MDA-MB-468) were selected as representatives of the intrinsic molecular subtypes (luminal A and B, basal-like, and Her2-enriched). Single cell multiplex RT-PCR was used to isolate and quantify the gene expression of single cells from each of these models, and the PAM50 classifier applied. Using this approach, we identified heterogeneity of intrinsic subtypes at single-cell level, indicating that cells with different subtypes exist within a cell line. Using the Chromium 10X system, this study was extended into thousands of cells from the MCF7 cell-line and an ER+ patient derived xenograft (PDX) model and again identified significant intra-tumour heterogeneity of molecular subtype.\n\nEstrogen Receptor (ER) is an important driver and therapeutic target in many breast cancers. It is heterogeneously expressed in a proportion of clinical cases but the significance of this to ER activity is unknown. Significant heterogeneity in the transcriptional activation of ER regulated genes was observed within tumours. This differential activation of the ER cistrome aligned with expression of two known transcriptional co-regulatory factors of ER (FOXA1 and PGR).\n\nTo examine the degree of heterogeneity for other important phenotypic traits, we used an unsupervised clustering approach to identify cellular sub-populations with diverse cancer associated transcriptional properties, such as: proliferation; hypoxia; and treatment resistance. In particular, we show that we can identify two distinct sub-populations of cells that may have denovo resistance to endocrine therapies in a treatment naive PDX model of ER+ breast cancer. One of these consists of cells with a non-proliferative transcriptional phenotype that is enriched for transcriptional properties of ERBB2 tumours. The other is heavily enriched for components of the primary cilia. Gene regulatory networks were used to identify transcription factor regulons that are active in each cell, leading us to identify potential transcriptional drivers (such as E2F7, MYB and RFX3) of the cilia associated endocrine resistant cells. This rare subpopulation of cells also has a highly heterogenous mix of intrinsic subtypes highlighting a potential role of intra-tumour subtype heterogeneity in endocrine resistance and metastatic potential.\n\nOverall, These results suggest a high degree of cellular heterogeneity within breast cancer models, even cell lines, that can be functionally dissected into sub-populations of cells with transcriptional phenotypes of potential clinical relevance.

cancer biology↗

bioRxiv2018

MicroRNAs as potential therapeutics to enhance chemosensitivity in advancedprostate cancer

Docetaxel and cabazitaxel are taxane chemotherapy treatments for metastatic castration-resistant prostate cancer (CRPC). However, therapeutic resistance remains a major issue. MicroRNAs are short non-coding RNAs that can silence multiple genes, regulating several signalling pathways simultaneously. Therefore, synthetic microRNAs may have therapeutic potential in CRPC by regulating genes involved in taxane response and minimise compensatory mechanisms that cause taxane resistance. To identify microRNAs that can improve the efficacy of taxanes in CRPC, we performed a genome-wide screen of 1280 microRNAs in the CRPC cell lines PC3 and DU145 in combination with docetaxel or cabazitaxel treatment. Mimics of miR-217 and miR-181b-5p enhanced apoptosis significantly in PC3 cells in the presence of these taxanes. These mimics downregulated at least a thousand different transcripts, which were enriched for genes with cell proliferation and focal adhesion functions. Individual knockdown of a selection of 46 genes representing these transcripts resulted in toxic or taxane sensitisation effects, indicating that these genes may be mediating the effects of the microRNA mimics. A range of these genes are expressed in CRPC metastases, suggesting that these microRNA mimics may be functional in CRPC. With further development, these microRNA mimics may have therapeutic potential to improve taxane response in CRPC patients.

cancer biology↗

bioRxiv2017

Targeting stromal remodeling and cancer stem cell plasticity to overcome chemoresistance in triple negative breast cancer

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hh ligand produced by neoplastic cells reprogrammed cancer-associated fibroblast (CAF) gene expression, driving tumor growth and metastasis. Hh-activated CAFs upregulated expression of FGF5 and production of fibrillar collagen, leading to FGFR and FAK activation in adjacent neoplastic cells, which then acquired a stem-like, drug-resistant phenotype. Treatment with smoothened inhibitors (SMOi) reversed these phenotypes. Stromal treatment of TNBC patient-derived xenograft (PDX) models with SMOi downregulated the expression of cancer stem cell markers and sensitized tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. Markers of pathway activity correlated with response. These studies identify Hh signaling to CAFs as a novel mediator of cancer stem cell plasticity and an exciting new therapeutic target in TNBC.\n\nSIGNIFICANCECompared to other breast cancer subtypes, TNBCs are associated with significantly worse patient outcomes. Standard of care systemic treatment for patients with non-BRCA1/2 positive TNBC is cytotoxic chemotherapy. However, the failure of 70% of treated TNBCs to attain complete pathological response reflects the relative chemoresistance of these tumors. New therapeutic strategies are needed to improve patient survival and quality of life. Here, we provide new insights into the dynamic interactions between heterotypic cells within a tumor. Specifically, we establish the mechanisms by which CAFs define cancer cell phenotype and demonstrate that the bidirectional CAF-cancer cell crosstalk can be successfully targeted in mice and humans using anti-stromal therapy.

cancer biology↗