Identification, roles and mechanisms of long non-coding RNAs in stem cells and metastasis in breast cancer.

In the fight against breast cancer, there is good and bad news. Since 1990, there has been a decline in the death rate from breast cancer. However, more than 10,000 people die from breast cancer every year in the UK alone. It is still the second leading cause of deaths from cancer in women. We believe that conventional therapies for advanced breast cancer are limited because they target the wrong cells. These therapies were designed to shrink cancers by killing all the cells in a tumour. We believe that treatment could be improved, both interns of effective and also side-effects, if specific cells called cancer stem cells (CSCs) were targeted. (CSCs) are cancer cells found within tumours that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a cancer sample. Hence, CSCs are tumourigenic (tumour-forming), therefore targeting therapy at CSCs holds hope for survival improvement.
To do this, we need to understand what turns genes on and off in breast CSCs. Intermediate molecules produced from DNA called RNA regulate the genome. Recent recognition that long non-coding RNAs (lncRNAs) function in various aspects of cell biology has focused attention on their potential to contribute towards disease aetiology. We wish to study these in breast CSCs, to evaluate exactly how they cause the genes of these cells to remain dormant and then spread to other parts of the body.

As yet, we do not fully understand the role of the lncRNA in breast cancer. Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. Multiple studies indicate that lncRNAs act not only as the intermediary between DNA and protein but also as important protagonists of cellular functions. Long ncRNAs can regulate gene expression in many ways, including chromosome remodeling, transcription and post-transcriptional processing. Moreover, the dysregulation of lncRNAs has increasingly been linked to many human diseases, especially in cancers.

The aim of this project is quite simple; we want to understand how these new molecules (lncRNAs) turn on and off genes in breast cancer stem cells and in doing so, open up an exciting new frontier of research and potential treatment.

This work will be conducted by Mr Paul Cathcart, a senior registrar in general surgery with a special interest in breast oncoplastic surgery, is dedicated towards pursuing an academic surgical career to form a bridge between laboratory and theoretical work with the clinical setting. The project involves the development of a variety of laboratory techniques, stem cell research and processing of tumour samples. By utilising these newly acquired skills, it will be possible to correlate laboratory findings with factors seen in the hospital setting such as response to treatment. If the results of the project can develop a biomarker, this may alter the surgical approach taken to ensure that the affected tissue is excised.

Aims: Dysregulation of lncRNAs have been implicated in many human cancers, however no significant findings have thus far emerged in the cancer stem cell field. Breast cancer stem cell populations capable of self-renewal and tumorigenesis are linked to the most malignant features of the disease. We propose to disentangle the roles of lncRNAs in the genetic circuitry of different breast cancers to facilitate targeted therapeutics of the future.

Objectives:
i) To identify lncRNAs differentially expressed in BCSCs,
ii) To investigate the binding and transcriptional effects of known BCSC transcription factors on the lncRNA transcriptional landscape they produce,
iii) To use loss- and gain-of-function experiments to determine the contribution of selected lncRNAs to the BCSC phenotype,
iv) To investigate mechanisms of lncRNA regulation, focusing on subcellular localisation, association with specific genomic sites, and presence of binding motifs with predictable functions. Further, using Ago2 immunoprecipitation followed by RNA-seq, we will evaluate the binding of Ago2-miRNA complexes to the lncRNAs indicative of miRNA sponge function,
v) To understand their clinical relevance and establish their role as biomarkers.

Methodology:
This project uses a range of cell and molecular biology techniques, bioinformatics and tissue handling techniques, which are well established in the host laboratory.

Scientific and Medical Opportunities: Over the past five years, research in the host laboratory has focused primarily on the role of non-coding RNAs in cancer and their potential as biomarkers and therapeutic targets. The lab has published extensively on the relationship(s) between miRNAs and the estrogen receptor-alpha following their discovery of a novel regulatory feedback loop involving miRNAs and the ER-alpha. Ongoing work aims to demonstrate that transcriptional regulation is a general mechanism of miRNA biogenesis with relevance to breast cancer.

This proposal aims to advance our understanding of the molecular biology of cancer cells, focusing on the interactions between long non-coding RNA and breast cancer stem cells. As such, at this point we are not aiming to commercially exploit any discoveries. Previous data has shown that dysregulation of lncRNAs occurs in many human cancers, but no significant findings have emerged in the cancer stem cell field. Understanding the roles of lncRNAs in the genetic circuitry of different breast cancers will facilitate the targeted therapeutics of the future capable of combating the cancer stem cells within these malignancies.
This project would fit perfectly with previous and ongoing work in the lab. Over the past five years research, the host laboratory has focused primarily on the role of non-coding RNAs in cancer, and their potential as biomarkers and therapeutic targets. The laboratory has published on the relationship between miRNAs and the estrogen receptor-alpha following their discovery of a novel regulatory feedback loop involving miRNAs and the ER-alpha in cancer. Ongoing work aims to demonstrate that transcriptional regulation is a general mechanism of miRNA biogenesis with relevance to breast cancer, and that an estrogen-inducible mechanism is responsible for the processing inhibition of mature miRNAs and that miRNA binding factors are responsible. Furthermore, the laboratory has published on the relationship between non-coding RNAs and p53. Much of the laboratory's focus is now on the role of long non-coding RNAs in breast cancer and to identify the regulatory relationships that exist between them and the ER-alpha. The advent of remarkable sequencing technologies that we regularly now use has made this possible.

The specific beneficiaries are likely to be:

1. Basic Science: We aim to elucidate the role of lncRNA in the regulation of gene expression and therefore the phenotypic outcome within breast cancer stem cells. This will benefit not only ongoing research within the host laboratory, but will benefit other research groups across several disciplines. We would aim to continually present and publish our work throughout the grant period and thereafter.

2. Patients: In years to come, the outcome of this research may lead to prognostic and predictive biomarkers of breast cancer. Powerful effects on cancer phenotypes have already been shown by inhibiting specific lncRNAs in vitro and in vivo make them targets for emerging nucleic acid-based therapies. If we can understand how and why breast cancer metastasises, we can attempt to develop biomarkers and ultimately targeted therapy towards the breast cancer stem cells to reduce the potential of disease recurrence. Most relevant to this proposal, the lncRNAs may help us to understand the resistance to hormonal therapies, and why some women who take medications such as tamoxifen relapse.

3. Training: Working primarily in the Department of Surgery and Cancer, numerous cell and molecular biology techniques that we currently use to study long non-coding RNAs in numerous types of cancer will be learnt. The applicant will learn experimental design, data handling, use of software appropriate to research and statistical analysis, and have Health and Safety training relevant to laboratory research and attend a course about the Human Tissue Act. This project fosters the transfer of knowledge across scientific disciplines.

In summary, the impact of research may not be always immediately evident, and may take several years to develop an understanding of this complex field before further progress can be made. However, excellent research makes significant advances across various disciplines which may not be imminently apparent. A decade ago, genome sequencing provided glimpses into our cellular transcriptome - now regulation is taking over, and understanding this will have enormous impacts.