Our Research

Regulation of cell proliferation

Regulation of the mammalian cell division cycle is critical to development as cell cycle exit and differentiation are intimately linked with patterning and tissue morphogenesis. Loss of proliferative control during development is catastrophic, as it leads to apoptosis and tissue degeneration. Loss of cell cycle control is also important in disease pathology, as deregulated proliferation is a defining feature of cancer.

The G1 phase of the cell cycle is the stage at which a cell can decide to replicate its DNA and divide, or exit the cell cycle. As such, understanding the molecular mechanisms that control this decision making process is central to understanding development and cancer. The master regulator of progression through the G1 phase of the cell cycle is the Retinoblastoma protein (pRB). Its function is disabled in nearly all forms of human cancer.

Despite the central role of pRB in cell cycle control, it is unclear how it interprets regulatory signals in G1 and controls cyclin dependent kinases and transcription. This deficiency stems from the fact that pRB uses a single growth suppressive domain called the ‘pocket’ to mediate most of its regulatory functions. Dissection of function within this domain has been challenging because cancer derived mutations destroy all interactions with this domain causing a myriad of functional consequences. In sum, without a concise mechanistic understanding of the retinoblastoma protein’s function, much of the regulation of cell proliferation in development and disease will remain a mystery.

Our Research

A structure-function approach to studying cell cycle control

Our goal is to understand the fundamental biochemical function(s) that pRB performs as a negative regulator of the cell cycle. This structure-function type work has involved creating interaction defective mutants of pRB and studying them using in vitro biochemical and cell culture approaches. Mutants with interesting properties in these experiments are then studied further by using gene-targeted mice. By introducing these mutations into the endogenous gene in mice we can relate defects in specific biochemical functions to more complex biological functions like tumor suppression. In this way enhanced cancer susceptibility in our mutant mice will definitively implicate specific biochemical functions in pRB’s tumor suppressor activity. This general approach is being applied to the areas of research described below.

Model of pRB pocket domain, shading based on conservation

1) Differential control of E2F transcription factors by pRB in normal and cancer cells.
During our investigation of pRB regulation of the E2F family of transcription factors we discovered that pRB was capable of interacting with E2F1 using a different interaction domain. Thus, E2F1 can interact with pRB in two distinct configurations, one that is analogous to the other E2F family proteins and one that is unique to E2F1. Our analysis suggests that these different configurations of interaction between E2F1 and pRB can regulate different functional outcomes like cell cycle arrest or apoptosis. We have generated pRB mutant mouse lines that are disrupted for each E2F interaction in isolation. We are using these mice to better understand how pRB-E2F interactions are regulated, and how they contribution to pRB’s and E2F1’s anti-cancer functions.
This work is supported by a grant from the CIHR.
Selected Publications:
Carnevale et al. (2012) DNA damage signals through differentially modified E2F1 molecules to induce apoptosis. Mol. Cell. Biol. 32, 900-912.

Hirschi, A. et al. (2010) An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein. Nat. Struct. Mol. Biol. 17, 1053-1058.

Dick, FA, and Dyson, N. (2003) pRB contains an E2F1 specific binding domain that allows E2F1 induced apoptosis to be regulated separately from other E2F activities. Mol. Cell 12, 639-49.

3) The role of negative growth regulation in tumor suppression.

Loss of responsiveness to negative growth signals is considered necessary for pre-malignant cells to become cancerous. G1 arrest is dependent on the activity of pRB and its function is abrogated in nearly all forms of cancer, emphasizing the importance of its role in negative growth regulation. Negative growth signals can originate from environmental insults that cause DNA damage or extracellular sources, such as the growth inhibitory cytokine, transforming growth factor beta (TGF-β). TGF-β is activated by proteolysis in the extracellular matrix, allowing the loss of tissue integrity to be coupled with growth regulation. Interestingly, deficiency for TGF-β signaling in mammary epithelium, as a result of expressing a dominant negative receptor alone, is insufficient to induce cancer formation. Similarly, we have discovered that loss of negative growth control caused by defective pRB function does not universally enhance cancer susceptibility. This underscores the importance of the initiating oncogenic event, the strength of negative growth signals, and other aspects of the tissue microenvironment as critical factors that determine when negative growth signaling is tumor suppressive. We are generating new genetically modified mouse strains to study how and when negative growth control is most critical in blocking cancer formation. In addition we are studying why TGF-β signalling requires aspects of pRB function that are dispensable in other growth arrest paradigms.

This work is supported by a grant from the CCSRI.

Selected Publications:

Francis, SM, et al. (2009) A functional connection between pRB and transforming growth factor beta in growth inhibition and mammary gland development. Mol. Cell. Biol. 29, 4455-66