Mechanisms of large-scale gene expression programs in cancer and aging

Over the past decade, the DNA microarray has evolved into one of the most commonly utilized genomic tools in biology labs around the world. While applications for microarrays have significantly grown over the years, they were originally designed for analyzing global gene expression profiles of tissues or cells. Through genetic profiling of thousands of cell lines and tissue samples, an exorbitant amount of data has been obtained, revealing extensive large-scale gene expression programs that occur during essentially all normal and disease processes. However, understanding the basis and consequences of these large-scale programs remains a challenge. To address this problem, we have developed two computational tools to begin to decipher these genetic programs, which further direct hypothesis-driven experiments to study physiological processes in detail. Specifically, we have focused on large-scale gene expression programs observed in mammalian cancer and aging.;Global gene expression profiles of thousands of cancer samples have been completed, giving rise to hundreds of gene expression signatures. Although many expression signatures show promise in predicting patient prognosis or response to therapies, the usefulness of the signatures in understanding the underlying mechanisms of cancer has not been fully exploited. We describe a novel method that can prospectively identify genetic regulators of gene expression signatures in cancer. We used our method to study the wound response signature, a previously defined poor-prognosis gene expression signature in human breast cancer. MYC (encoding an oncogenic transcription factor) and CSN5 (encoding the catalytic component of the COPS signalosome) were identified as candidate regulators of the wound signature in breast cancer. Co-expression of MYC and CSN5 in non-transformed breast cells is sufficient to ectopically activate the wound signature, and biochemical analyses identified CSN5-mediated ubiquitination of MYC as a novel mechanism to activate a biological program favoring metastasis. Subsequent studies in both human and mouse models of breast cancer revealed a critical role for the enzymatic activity of CSN5 in regulating breast cancer progression. These results connect CSN5 and MYC in a new oncogenic pathway that controls the wound signature in breast cancer. Further, we have shown for the first time that CSN5 regulates breast cancer progression in vivo, highlighting CSN5 as a novel therapeutic target.;In addition to analyzing gene expression programs in disease states such as cancer, we also focused on large-scale gene expression changes in normal physiology, particularly chronological aging. Genetic studies in model organisms such as yeast, worms, flies, and mice leading to lifespan extension suggest that longevity is subject to regulation. In addition, various system-wide interventions in old animals can reverse features of aging. To better understand these processes on a molecular level, researchers have performed microarray analysis of differently aged organisms or tissues and have uncovered diverse global expression programs that occur during normal aging. However the underlying mechanisms and functional consequences of these expression differences are not well understood. Before addressing the issue of age-associated gene expression, we first developed a computational approach to discover candidate transcription factors that drive large-scale gene expression changes. We analyzed all human gene promoters for the presence of specific transcription factor binding motifs. Our predictions were then validated by systematically characterizing these motif targets through integrating our results with diverse sources of biological data. We successfully identified transcriptional regulators for numerous well-known and novel biological processes and experimentally validate four novel predictions in controlling cell cycle progression.;We next employed this approach to identify candidate transcription factors responsible for driving gene expression programs with age across multiple tissues and organisms. The transcription factor NF-kappaB was identified as a candidate activator of aging-related transcription in human and mouse. While previous studies have suggested a role for NF-kappaB in aging, a direct role has not been shown. Genetic blockade of NF-kappaB in aged murine skin reversed the global gene expression program and tissue characteristics to those of young mice. This demonstrates for the first time that disruption of a single gene is sufficient to reverse features of aging, and these results reveal a direct role for continual NF-kappaB activity in maintaining features of aging in the skin.;These findings left us with the important question of how NF-kappaB is regulated with age. Candidate regulators of NF-kappaB include the Sir2 family of proteins, which promote longevity in multiple organisms. There are seven mammalian Sir2 homologues (SIRT1-7), however only deficiency of SIRT6 in mice leads to phenotypes resembling premature aging. We found that SIRT6 is a novel negative regulator of NF-kappaB signaling. Specifically, SIRT6 deacetylates the active chromatin mark histone H3 lysine 9 at NF-kappaB target promoters leading to NF-kappaB target gene repression, and loss of SIRT6 increased NF-kappaB activity in vitro and in vivo. Strikingly, haploinsufficiency of the Rela subunit of NF-kappaB in Sirt6-deficient mice rescues the early lethality and many aging-like phenotypes of Sirt6-/- mice. Thus, SIRT6 functions to inhibit NF-kappaB signaling, and defective NF-kappaB termination may contribute to premature or physiologic aging.