Maria F. Czyzyk-Krzeska Studies

Maria F. Czyzyk-Krzeska, MD, PhD of the University of Cincinnati, won a $932,919 three-year grant from the Defense Department’s (DoD) Peer Review Medical Research Program to identify genes in kidney cancer oncogenesis. This grant, which commenced in early 2007, will run until early 2010, is the first grant ever awarded by DoD for kidney cancer research and is the result of a lobbying campaign by ACKC that requested a Congressional appropriation for kidney cancer research at the Department of Defense. We congratulate Dr. Czyzyk-Krzeska on her grant.

Dr. Czyzyk-Krzeska started working on kidney cancer research itself only three years ago. Prior to that she worked on hypoxic (lack of oxygen) regulation of gene expression. This led to her working on the hypoxia-inducible factor (HIF), a transcription factor controlled by the von Hippel-Lindau gene (VHL) – see below. Aside from her research, Dr. Czyzyk-Krzeska is a full professor at the Genome Research Institute of the University of Cincinnati where she teaches cancer biology. She has been at the university for the past 14 years.

The Public Abstract of Dr. Czyzyk-Krzeska’s project is available at the Department of Defense’s website http://tinyurl.com/3y6qkr. What follows is our attempt to describe her project. With Dr. Czyzyk-Krzeska’s approval, we will refer to her by her first name, Maria.

It has been known for approximately 15 years that a high percentage of patients with sporadic (non-inherited) clear cell carcinoma have a mutation of the VHL gene. This fact was discovered by the National Cancer Institute as a result of their work on von Hippel-Lindau Disease, which is an inheritable genetic disease. People with VHL disease develop both renal tumors and tumors in other areas of the body. Further study of the VHL gene’s function uncovered the fact that the VHL protein, pVHL, has ubiquitin ligase E3 activity, which causes another protein called HIF-1 alpha to degrade. In this natural process of ubiquitylation, or degradation, one can think of it as pVHL generating a scout (ubiquitin), that seeks out and finds HIF which it binds or attaches itself to and then turns off its function. HIF-1 alpha normally produces other proteins, among them, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). VEGF promotes blood vessel growth (angiogenesis) and PDGF promotes tumor growth.

In the embryonic stage, we need blood vessel growth to support expansion of our body, but when we reach the adult stage, although cells die and are replaced by new ones, blood vessel growth is not as rapid. If normal cells are damaged, then they stimulate new blood vessel growth to heal the wound. Once healed, new blood vessel growth is turned off. If a tumor develops and its cells begin to rapidly divide and multiply, without an adequate blood supply, the tumor cells will die. But if something in the tumor cells has the ability to generate new blood vessel growth, the tumor will thrive. The VHL gene is such a candidate. Under normal conditions, the VHL gene controls HIF and inhibits new blood vessel development. If the VHL gene is mutated, it loses its capacity to send out a ubiquitin to turn off HIF-1 alpha, which itself then multiplies and sets off a rapid growth of VEGF and blood vessel growth. This doesn’t happen all over the body but only where the VHL gene is mutated, that is, in the RCC tumor area.

The new targeted therapy drugs such as Sutent and Nexavar are anti-angiogenic drugs in that they are molecules that bind to the receptors on the surface of the VEGF and PDGF cells and immobilize them to prevent new blood vessel formation and tumor growth. The theory is that the rapidly growing tumor is voracious and requires new blood vessels to feed it nutrients and oxygen to survive and proliferate. If one can stop angiogenesis, then, according to the theories of the late Judah Folkman, one can starve the tumor and shrink it. In other words, these drugs don’t attack the tumor itself, but fight a rear guard action hoping to stem its growth. VEGF and PDGF are often called “downstream” targets, i.e., downstream from the “upstream” VHL protein.

VHL has other functions as well. In a paper written in 2004 (Czyzyk-Krzeska MF, Meller J
von Hippel-Lindau tumor suppressor: not only HIF’s executioner. Trends Mol Med. 2004 Apr;10(4):146-9.), Maria delineated some of these. Her current study, for which she received the DoD grant, focuses on RNA Polymerase II, a protein that transcribes genetic information. Rpb1 is the largest subunit of RNA Polymerase II. Normally, pVHL’s ubiquitin ligase E3, which degrades HIF-1 alpha, also binds to Rpb1 (note: Rpb1 makes RNA). In order for pVHL to bind to Rpb1, a short sequence of amino acids (called a proline) on the protein Rpb1 must first be hydroxylated (that is, an -OH group must be added to its structure). Then the protein pVHL can bind to Rpb1. When it does, pVHL doesn’t degrade or destroy Rpb1 as it does with HIF, but changes its function.

Maria

At this time, the function of Rpb1 and Rpb1 interaction with pVHL in kidney cancer are subjects of research. The research will utilize human cell lines with different levels and mutations of pVHL and human renal clear cell carcinoma tumors where the status of pVHL will be evaluated. In the first project the lab will “Identify genes regulated by the loss of Rpb1 hydroxylation and pVHL-dependent ubiquitylation using microarray screening” and to “Identify protein factors regulated by loss of Rpb1 hydroxylation and pVHL-dependent ubiquitylation using proteomic approaches”. She and her colleagues are trying to find out what the role of hydroxylation is in the oncogenic process. To do that, they will analyze the genes and their protein derivatives that are affected by hydroxylation of Rpb1. From this analysis, they hope to learn if hydroxylation of Rpb1 is a tumor promoting or tumor suppressing activity. That will hopefully lead to a better understanding the effect of VHL mutation on the role of Rpb1 in the pathogenesis of renal cell carcinoma for clear cell pathology. Working with microarrays can lead to an investigation of thousands of genes, but in the last year, Maria has narrowed down her focus to 10-20 genes that fit her criteria.

The third project is “To determine the functional role of identified target genes in the pathogenesis of renal cancer”. There are two approaches to learn what the function of genes are in the oncogenic setting. One works with human tumor samples to see which genes are over-expressed in clear cell cancer tissues versus in normal kidney tissues. The second approach is to take cell lines where genes are either over-expressed or under-expressed and inject them into the kidneys of immuno-compromised mice and monitor them for tumor development. Genes that promote tumor development are oncogenic and genes that inhibit tumor development are tumor suppressors.

All projects are an early look into an area that could lead to therapy targeted against an RNA Polymerase II component, depending on the results of the genetic analysis. Given the limits of the anti-angiogenic therapies in kidney cancer, we look hopefully toward initiatives in other areas.