mcmahon@cc.ucsf.edu
(415) 502-1317 (lab); (415) 502-3179 (fax)

Box 0128, UCSF; San Francisco, CA 94143-0128

deliveries: 2340 Sutter Street, S-371; San Francisco, CA 94115

Full Biosketch

Robert Cartlidge, PhD; Eric Collisson, MD; David Dankort, PhD; Stephan Gysin, PhD; Takashi Hirano; Megan Salt; Jun Zhang, PhD

Oncogenes, Signal Transduction, and Cancer
Work in my laboratory focuses on the role of oncogenes and tumor suppressors in the aberrant proliferation of cancer cells. In the past 25 years enormous progress has been made in the elucidation of the fundamental mechanisms by which normal cells are converted to a tumorigenic phenotype. The general consensus is that in order for cancer cells to proliferate they must subvert both the machinery that controls the cell division cycle and the process of programmed cell death (apoptosis). This is frequently achieved by mutation of specific proto-oncogenes such as Ras or tumor suppressors such as p53. The Ras-family of membrane associated GTPases transmits signals into the interior of the cell by the activation of a number of cytosolic signal transduction pathways (Figure 1). Prominent among these is the Raf--->MEK--->ERK MAP kinase signaling pathway. Binding of Raf to activated Ras leads to activation of Raf protein kinase activity. Activated Raf phosphorylates to activate a second protein kinase MEK, which in turn phosphorylates to activate the MAP kinases ERK1 and 2. Activated ERKs are pleiotropic modulators of cell physiology that elicit their effects by phosphorylating numerous proteins including several transcription factors. Using conditionally active forms of Raf (deltaRaf:ER) that permit selective activation of the ERK MAP kinase pathway in cells we have explored the regulation of gene expression by this pathway (Figure 2)(references 1,3). It is clear that the Raf--->MEK--->ERK pathway can contribute to many of the phenotypes of the cancer cell by regulating genes involved in the cell division cycle (cyclin D1, p21Cip1), apoptosis (Mdm2, HB-EGF), cell invasion (alphavbeta3-integrin), epithelial cell multilayering (Rnd3, Figure 3) and angiogenesis (VEGF) (references 2-6). Recently we have uncovered a direct link between the ERK MAP kinase pathway and members of the Bcl-2 family of proteins that play a central role in the control of apoptosis. Preliminary indications suggest that direct phosphorylation of a subset of Bcl-2 family proteins influences the predisposition of cells to commit to an apoptotic cell fate.

In 1997 we and others uncovered an interesting connection between oncogenes and tumor suppressors (reference 7). Although Ras and Raf came to view as agents of neoplastic transformation, these genes can have effects that run counter to oncogenic transformation, such as the arrest of the cell division cycle. It appears that sustained activation of Ras and Raf can elicit cell cycle arrest and premature cell senescence (references 2,7). Ras and Raf-induced senescence is mediated by genes such as p53 and p16INK4A, which are tumor suppressors that are frequently mutated in human cancer cells that express activated Ras proteins (reference 9) (Figure 4). It seems likely therefore that the observed induction of cell cycle arrest/senescence may provide a defense mechanism against neoplastic transformation when the Raf--->MEK--->ERK signaling cascade is inappropriately active. Hence, in order for cancer cells that express an activated form of Ras to progress, they must silence the expression of tumor suppressors such as p53 and p16INK4A. The best example of this is in human pancreatic cancer where the extremely high frequency of Ras mutation (~95%) is accompanied by an equally high frequency of mutation/silencing of p16INK4A (~99%) and p53 (~75%).

In 1997 we and others uncovered an interesting connection between oncogenes and tumor suppressors7. Although Ras and Raf came to view as agents of neoplastic transformation, these genes can have effects that run counter to oncogenic transformation, such as the arrest of the cell division cycle. It appears that sustained activation of Ras and Raf can elicit cell cycle arrest and premature cell senescence 2,7. Ras and Raf-induced senescence is mediated by genes such as p53 and p16INK4A, which are tumor suppressors that are frequently mutated in human cancer cells that express activated Ras proteins9 (Figure 4). It seems likely therefore that the observed induction of cell cycle arrest/senescence may provide a defense mechanism against neoplastic transformation when the Raf--->MEK--->ERK signaling cascade is inappropriately active. Hence, in order for cancer cells that express an activated form of Ras to progress, they must silence the expression of tumor suppressors such as p53 and p16INK4A. The best example of this is in human pancreatic cancer where the extremely high frequency of Ras mutation (~95%) is accompanied by an equally high frequency of mutation/silencing of p16INK4A (~99%) and p53 (~75%).

Although the molecular genetics of pancreatic cancer have been explored in some detail there is a large gulf in our understanding of how mutations in oncogenes and tumor suppressors influence the aberrant behavior of pancreatic cancer cells. In addition pancreatic cancer is a disease for which there is an urgent need for new diagnostic and therapeutic tools. Consequently we have initiated a series of new projects to explore the role of oncogenes and tumor suppressors in human pancreatic cancer in more detail. We are taking three main approaches to explore fundamental aspects of the cell and molecular biology of this disease:

1. In order to understand the initiation of pancreatic cancer we need to know more about pancreatic ductal epithelial cells (PDEC), the cells from which pancreatic cancer is derived (Figure 5, Figure 6). Starting with whole pancreas we have established conditions for the isolation and propagation of primary cultures of human and mouse PDECs and we are attempting to isolate immortalized, long term cultures of such cells (reference 8). These cells will then be subjected to an in-depth analysis of the regulation of gene expression with particular emphasis on the control of the cell division cycle, apoptosis and senescence. In addition we hope to use these cell lines as recipients in gene transfer experiments to explore the effects of Ras and Raf on primary epithelial cells (reference 4).

2. Using pancreatic cancer cell lines and patient derived primary pancreatic cancer specimens we are using high throughput microarray techniques (array CGH and cDNA expression arrays) to profile the genetic alterations that occur in pancreatic cancer and the effect of these alterations on patterns of mRNA expression in the cancer cell. As new techniques become available to scan the proteome of the pancreatic cancer cell we will apply the full spectrum of high throughput profiling techniques to understand how alterations in the patterns of mRNA and protein expression contribute to the aberrant properties of pancreatic cancer cells. Although this research has a goal to understand the biology of the cancer cell, we anticipate that this type of analysis may lead to the identification of candidate diagnostic and therapeutic targets to aid in the management of pancreatic cancer.

3. To explore the initiation and progression of pancreatic cancer (Figure 6) in an animal model system we are deriving a set of transgenic mice with an inherited pre-disposition to pancreatic cancer. These mice will then be selectively bred to other transgenic/knock-out mice to explore the role of specific genes in the genesis and progression of pancreatic cancer. These studies will focus in particular on genes that regulate the cell division cycle, apoptosis and senescence. Although these experiments seek to explore the initiation and progression of pancreatic cancer, a transgenic mouse model that accurately recapitulates the features of the human disease may be a useful platform for the design and evaluation of novel diagnostic and therapeutic tools to target this dread disease.

1. Fowles, LF., Martin, ML., Nelsen, L., Stacey, KJ., Douglas D., Clark, YM., Nagamine, Y., McMahon, M., Hume, DA. and Ostrowski, MC. (1998) Persistent Activation of Mitogen-Activated Protein Kinases p42 and p44 and ets-2 Phosphorylation in Response to Colony-Stimulating Factor 1/c-fms Signaling. Mol. Cell. Biol. 18: 5148-5156.

2. Woods, D. et al. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol Cell Biol 17, 5598-611 (1997).

3. Schulze, A., Lehmann, K., Jefferies, H. B., McMahon, M. & Downward, J. Analysis of the transcriptional program induced by Raf in epithelial cells. Genes Dev 15, 981-94. (2001).

4. Hansen, S. H. et al. Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf--->MEK--->ERK pathway. Mol Cell Biol 20, 9364-75. (2000).

5. Ries, S. et al. Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF. Cell 103, 321-30. (2000).

6. Woods, D. et al. Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal-regulated kinase signaling pathway. Mol Cell Biol 21, 3192-205. (2001).

7. Zhu, J., Woods, D., McMahon, M. & Bishop, J. M. Senescence of human fibroblasts induced by oncogenic Raf. Genes Dev 12, 2997-3007 (1998).

8. Venetsanakos, E. et al. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells. Exp Cell Res 273, 21-33. (2002).

9. McMahon, M and Woods, D. Regulation of the p53 pathway by Ras, the plot thickens. BBA Reviews on Cancer Online, 1461: M63-M71 (2001)