Recent genome-wide studies revealed that the majority of the human transcriptome consists of non-coding transcripts, but their function and mode of action remains elusive (2-8). Long non-coding RNAs (lncRNAs) provide an exciting class of therapeutic targets to pursue in terms of breast cancer progression and metastasis. To identify lncRNAs that could function as drivers of tumor progression and metastasis in breast cancer, I performed an RNA-sequencing screen on transgenic mouse models representing both luminal B and HER2- amplified subtypes of human breast cancer. I identified hundreds of lncRNAs that are overexpressed in tumors compared to normal mammary glands. As metastasis is the main cause of breast cancer mortality, I will focus specifically on lncRNAs that can impact tumor progression and prevent metastasis. In the proposed project, I aim to assess a previously uncharacterized lncRNA, termed Mammary Tumor Associated RNA 20 (MaTAR20). I performed antisense knockdown assays of MaTAR20 in vitro and in vivo, leading to reduction of tumor cell proliferation and invasion as well as drastically reduced metastasis. I propose to further evaluate MaTAR20 as a driver of metastasis by generating genetic loss-of-function and gain-of-function models using the CRISPR/Cas9 system (Aim 1). These will be used to further investigate which step of the metastatic cascade is impacted by MaTAR20. In addition, I will apply single-cell sequencing to elucidate the expression of MaTAR20 in the leading edge of the tumor, in circulating tumor cells and metastatic nodules (Aim 2). Analysis of the data will reveal differences of MaTAR20 expression in clonal populations throughout the course of tumor progression. Further, functional integration of single-cell transcriptomes, genomes and methylomes will reveal genetic and epigenetic factors regulating MaTAR20 expression. Finally, I will decipher signaling pathways and cellular interaction partners of MaTAR20 to identify the molecular function of the lncRNA (Aim 3). This project will investigate the great potential of MaTAR20 as a new therapeutic and prognostic target. During the mentored K99 phase, I will work closely with my mentor Dr. David Spector, a leader in the field of non-coding RNA biology, and collaborators Drs. Mikala Egeblad, John Wilkinson and Michael Wigler, recognized experts in mouse models of breast cancer, mammary tumor histology and single cell analysis respectively. Training in innovative new technologies like genome editing using CRISPR/Cas9 and single-cell sequencing will enable me to successfully complete the proposed project and to establish myself as an independent investigator. My long- term goal is to become a tenured professor and develop a cutting edge research program studying lncRNAs in breast cancer progression and tumor evolution. Cold Spring Harbor Laboratory provides an outstanding scientific environment for my research and training, being a conference hub for world-renowned meetings and courses. In addition, several programs are available here to assist transition into independence such as grant writing workshops and seminars on leadership in biosciences.
Breast cancer is the most frequent malignancy in women world wide and metastasis is the main cause of breast cancer mortality. To improve the existing treatment regimens, it is critical to identify new molecular targets that act as key drivers of breast cancer progression and metastasis. This proposal investigates the therapeutic and prognostic value of an exciting new class of molecules, long non-coding RNAs (lncRNAs), in the progression of metastatic disease.
|Arun, Gayatri; Diermeier, Sarah D; Spector, David L (2018) Therapeutic Targeting of Long Non-Coding RNAs in Cancer. Trends Mol Med 24:257-277|