Post-transcriptional Regulation of Cancer Traits and Gene Expression in a Genetically Defined, Primary Cell-derived Model of Breast Tumorigenesis

Post-transcriptional events are crucial determinants of gene expression, and aberrant expression patterns resulting from misregulation are evident in many pathological states. Cancer has traditionally been viewed as being driven by aberrant transcriptional regulation and signaling events, though, over the past several years, many RNA binding proteins and non-coding RNAs have emerged as critical players in tumor development. It is now recognized that regulation of post-transcriptional processes, such as mRNA stability and translation, robustly influence cancer-related gene expression patterns of proto-oncogenes, growth factors, cytokines, and cell cycle regulators. Despite its recognized importance, mechanisms of post-transcriptional regulation that influence molecular pathways at the mRNA level are understudied in the context of tumorigenesis. Additionally, cancer cells are derived from normal cells that often evolve step-wise and progressively to a neoplastic state, and the involvement of post-transcriptional regulation has not been looked at in the context of tumor initiation and step-wise progression. Thus, more studies are needed in order to fully understand the post-transcriptional mechanisms activated by cancer driver mutations that coordinate tumor initiation and progression.

In this dissertation, we aimed to elucidate mechanisms of post-transcriptional regulation coordinating tumorigenesis. We first established a genetically defined, primary cell-derived model of breast cancer initiation and progression. In this model, normal human mammary epithelial cells were immortalized through the expression of hTERT, p53DD, cyclin D1, CDK4R24C and c-MYCT58A, and subsequently converted to a tumorigenic state through expression of oncogenic H-RASG12V. Using RNA-sequencing and real-time PCR arrays, we comprehensively quantified changes in mRNA abundance, miRNA expression and alternative splicing in this system, and revealed thousands of changes during immortalization and relatively few changes during RAS transformation. Moreover, pre-malignant, immortalized cells had expression signatures consistent with an epithelial-to-mesenchymal transition (EMT), but they expressed low levels of mesenchymal protein markers and were non-invasive. Activation of RAS in these pre-malignant cells induced an invasive phenotype without major changes in global mRNA expression. Consistent with post-transcriptional mechanisms, RAS increased protein levels of Vimentin and N-cadherin without changing mRNA levels.

We then sought to investigate a mechanism of this RAS-induced post-transcriptional regulation. We used a method developed in our lab called Digestion-Optimized Ribonucleoprotein Immunoprecipitation coupled with RNA-sequencing (DO-RIP-seq) to identify and quantify transcriptome-wide binding sites for the RNA binding protein HuR. Our study is the first to identify and quantify transcriptome wide binding sites for any RBP during tumorigenesis, and we report that HuR quantitatively, but not qualitatively, changed association at individual mRNA binding sites during RAS transformation. We identified a GU-rich secondary motif associated with a decrease in HuR binding during transformation. Furthermore, our data suggest that HuR may cooperate with the CELF1 protein to positively regulate the translation of a subset of mRNAs and promote the EMT phenotype. We generated HuR CRISPR knockout cell lines and demonstrated that HuR expression was necessary for the maintenance of cancer traits, including proliferation, anchorage independent growth, migration and invasion, but it does not regulate mRNA stability in this context. Lastly, we identified a binding site position dependent mechanism by which HuR regulates alternative polyadenylation of mRNAs encoding proteins involved in cancer-related processes.

In conclusion, our findings indicate that EMT-associated invasion can be initiated through two sequential stages: transcriptional priming followed by oncogenic RAS-triggered post-transcriptional regulation. The HuR RNA binding protein is important for maintaining the cancer phenotypes induced by oncogenic RAS, and regulation by HuR may be, at least in part, determined by a GU-rich secondary motif as well as cooperation with the CELF1 RNA binding protein.