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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 transmit signals into the
interior of the cell by the activation of a number of cytosolic signal
transduction pathways (Fig. 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 (DRaf:ER) that permit selective activation of the
ERK MAP kinase pathway in cells we have explored the regulation of gene
expression by this pathway (Fig. 2) 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 (avb3-integrin), epithelial cell
multilayering (Rnd3, Fig. 3) and angiogenesis (VEGF) 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 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 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 9 (Fig. 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 (Figs. 5 & 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 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 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 (Fig.
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 regulated
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.
Selected Publications:
McMahon, M. Steroid receptor fusion proteins for conditional activation
of Raf-MEK- ERK signaling pathway. Methods Enzymol 332, 401-17 (2001).
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).
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).
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).
Ries, S. et al. Opposing effects of Ras on p53: transcriptional activation
of mdm2 and induction of p19ARF. Cell 103, 321-30. (2000).
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).
Zhu, J., Woods, D., McMahon, M. & Bishop, J. M. Senescence of human fibroblasts
induced by oncogenic Raf. Genes Dev 12, 2997-3007 (1998).
Venetsanakos, E. et al. Induction of tubulogenesis in telomerase-immortalized
human microvascular endothelial cells by glioblastoma cells. Exp Cell
Res 273, 21-33. (2002).
McMahon, M and Woods, D. Regulation of the p53 pathway by Ras, the plot
thickens. BBA Reviews on Cancer Online, 1461: M63-M71 (2001)
Contact Information:
Email: mcmahon@cc.ucsf.edu
Phone: 415/ 502-5829
Address: Box 0128, Room S 329
The University of California, San Francisco, CA 94143, (415) 476-9000
Copyright 2003, The Regents of the University of California.
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