UCSF DIABETES, ENDOCRINOLOGY & METABOLISM TRAINING PROGRAM FACULTY RESEARCH SUMMARIES

Work in my laboratory focuses on the role of oncogenes and tumor suppressors in the aberrant proliferation of cancer cells. The past 25 years have seen enormous progress in the elucidation of the fundamental mechanisms by which normal cells are converted to a tumorigenic phenotype. The general consensus is that activation of oncogenes such as Ras, accompanied by loss of function of tumor suppressor genes such as p53, promotes the conversion of normal cells to a neoplastic phenotype. Alterations in oncogenes and tumor suppressor genes leads to subversion of the machinery that controls the cell division cycle, cell senescence and the process of programmed cell death (apoptosis).

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. 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. 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 and angiogenesis (VEGF).

Although the molecular genetics of pancreatic cancer and melanoma have been explored in some detail, there is a large gap in our understanding of how mutations in oncogenes and tumor suppressors influence the aberrant behavior of these tumors. Furthermore, both pancreatic cancer and melanoma are diseases for which there is an urgent need for new diagnostic and therapeutic tools. Consequently, we have initiated a series of new projects to understand the role of oncogenes and tumor suppressors in the initiation and progression of both pancreatic cancer and melanoma in more detail. We are taking three main approaches to explore fundamental aspects of the cell and molecular biology of these diseases.

Understanding the Cell of Origin
To understand the aberrant properties of the cancer cell, we need to know something about the properties of the normal cells from which the cancer cells are derived. To do this we have established conditions that allow the in vitro culture of human pancreatic ductal epithelial cells (PDEC) and melanocytes, the cells from which pancreatic cancer and melanoma arise respectively. By expression of telomerase or other “immortalizing” genes, we are attempting to isolate long-term cultures of these cells. Such cell lines 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 will use these cell lines as recipients in gene transfer experiments to explore the effects of activated Ras and Raf on human cells.

Understanding the Altered Properties of the Cancer Cell
Using human pancreatic cancer and melanoma cell lines, we are using high-throughput profiling techniques to explore the genetic alterations and gene expression changes that occur in the initiation and progression of pancreatic cancer and melanoma. We are also assessing the effects of inhibitors of various cell signaling pathways on cancer cell physiology. Moreover, as new techniques become available to scan the proteome of human cancer cells, 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 these cells. Although the goal of this research is to understand the biology of the cancer cell, we anticipate that this analysis may ultimately lead to the identification of candidate diagnostic and therapeutic targets to aid in the management of these diseases

Mouse Models of Pancreatic Cancer and Melanoma
To explore the initiation and progression of pancreatic cancer and melanoma in a true in vivo setting, we are deriving transgenic mice with tissue specific expression of oncogenes that will confer an inherited pre-disposition either to pancreatic cancer or melanoma. These mice will then be bred to other transgenic/knock-out mice to explore the role of specific oncogenes and tumor suppressors in the genesis and progression of pancreatic cancer and melanoma. These studies will focus in particular on genes that regulate the cell division cycle, apoptosis and senescence. Clearly, transgenic mouse models that accurately recapitulate the features of human cancer will be very useful tools in understanding the earliest stages of the initiation and progression of these diseases. Such mice may also be useful in testing new therapies to target these diseases.

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