Title: Transcriptional control of tumor suppressor genes in cancer
Description: An important hallmark of cancer is the inactivation of tumor suppressor genes. The most common genetic alteration in cancer is the mutation of the TP53 gene occurring in about half of all cancers, but very little progress has been made on how to therapeutically target the signaling defects in these cancers. Additionally, the PTEN tumor suppressor is mutated in a wide variety of cancer types, and its expression is often lost in the absence of mutation. PTEN is a haploinsufficient tumor suppressor that exhibits dose-dependent effects in cells. In the context where PTEN is lost or downregulated, PI3K signaling and downstream signaling through AKT is overactive, leading to an increase in cell growth and proliferation, among other effects. Acting as both a protein and lipid phosphatase, loss of PTEN also affects the PI3K-independent signaling of PTEN, and results in an increase of migration and invasion phenotypes. Importantly, PTEN transcript level is the key determinant for PTEN protein expression, and downregulation of PTEN is part of a poor-prognosis gene expression signature in breast cancer.

Downregulation of tumor suppressor gene expression represents a reversible change that is often sufficient to drive tumorigenesis. However, our understanding of the broad molecular mechanisms by which the expression of these tumor suppressors is lost remains limited, but is required to develop effective therapeutic strategies to target malignancies driven by tumor suppressor loss. In Chapter 2, we characterize the problem of transcriptional downregulation of PTEN in breast cancer. We investigate the expression of PTEN in various normal and tumor cells at both the transcript and protein level. We identify various model systems that we believe are suitable to model normal PTEN expression and the PTEN downregulation that mimics what is observed in tumors. We employ a sophisticated approach that couples RNA-sequencing with Nanostring nCounter analysis in order to obtain a detailed and thorough transcriptional profile of the PTEN and pseudogene PTENP1 genomic loci, as well as expression of the poor-prognosis gene signature associated with PTEN downregulation.

In this study, we obtained an understanding of the changes in the PTEN transcriptional profile that occur in the progression from normal to cancer, and we believe this approach could be applied to other key tumor suppressor genes. In Chapter 3, we discovered that basally expressed p53 maintains expression of thirteen well-validated tumor suppressors. p53 is expressed at low levels under normal, low-stress conditions, and is expressed at much higher levels under enhanced stress, leading to the activation of stress-response genes. We begin the study by highlighting an association between TP53 mutation and downregulation of PTEN expression. Upon performing chromatin immunoprecipitation coupled with next generation sequencing for p53 under normal, low-stress conditions, we found that p53 binds in the vicinity of thirteen tumor suppressor genes, including PTEN. Basally expressed p53 binds to classic consensus binding sites in enhancers and promoters of target tumor suppressors to maintain their expression at baseline.

CRISPR/Cas9-mediated knockout of the endogenous basal p53 binding site upstream of PTEN led to a decrease in PTEN expression and an increase in tumorigenic phenotypes. Given that mutation of TP53 leads to tumorigenesis in mice, but loss of p53 stress-response targets or loss of the ability of p53 to activate these stress-response targets does not lead to spontaneous tumorigenesis, it is likely that these tumor suppressor targets of basal p53 contribute to p53-mediated tumor suppression. In Chapter 4, we identified yet another mechanism by which transcriptional repression of PTEN occurs in triple-negative breast cancer (TNBC) through polycomb repressive complex 2 (PRC2)-mediated repression of the PTEN promoter and upstream regulatory region. Previous research has shown that mutated NOTCH1 represses PTEN through the HES-1 transcription factor in acute myeloid leukemia (AML), and that NOTCH translocations are frequent in TNBC and are sufficient for transformation in vitro. We discovered that NOTCH1 and NOTCH2 mutations and translocations correlate with PTEN downregulation by immunohistochemistry in a cohort of TNBC cases.

The TNBC cell line exhibiting PRC2-mediated repression of PTEN also harbors a SEC22B-NOTCH2 translocation that creates a gene product resembling the NOTCH2 intracellular domain. The NOTCH target HES-1 co-localizes on the PTEN promoter with EZH2 (the lysine methyltransferase involved in PRC2-mediated transcriptional repression), and knockdown of NOTCH2 in this cell line led to decreased expression of EZH2, and restoration of PTEN expression at the transcript and protein level. We also demonstrated that EZH2 inhibitors, HDAC inhibitors, and DNA hypomethylating agents robustly restore PTEN transcript levels. Taken together, these results elucidate another mechanism by which PTEN is transcriptionally repressed in the highly aggressive and poor-prognosis TNBC subtype of breast cancer that may be applicable to other cancer types. The results also suggest that this repression is reversible by pharmacological approaches, highlighting a promising therapeutic avenue. Taken together, the studies presented in this thesis begin to unravel the complex mechanisms of transcriptional repression of tumor suppressor genes in cancer.

As is the case with PTEN and p53, multiple regulatory mechanisms can influence expression in combination or in a context-dependent manner. The loss of expression of tumor suppressor genes is one of the key hallmarks of cancer, yet very few of the therapeutic approaches used in the clinic today aim to restore tumor suppressor expression. Our results demonstrate proof of concept that restoration of tumor suppressor expression is a plausible and promising therapeutic approach for many different types of cancer, but requires a detailed understanding of the underlying molecular mechanisms of transcriptional regulation.