Resistance to Therapy: p53 and Chemosensitivity in Gastric Cancer

I am going to be talking about P53 and chemosensitivity. I think I have two challenges. One is being the last speaker in a long session in which all the speakers actually were here. The second is to try to briefly try to summarize the role of P53 in chemosensitivity, which is a very complicated subject. For those of you anxious to get to the airport, I can summarize the talk by saying that, despite truly an enormous literature, the specific role of P53 in clinical chemosensitivity really remains not well understood, it there is any all, regardless of its fairly clear role in carcinogenesis and prognostics.

Nevertheless, the biology and model systems for studying P53 in sensitivity and drug resistance are very provocative. What I am going to try to do this morning is to try to talk about some of our newer understandings of the molecular mechanisms of regulation of P53 and its targets, and a couple models that are provocative in ways to try to exploit P53 in this process.

Let me start by summarizing some of the things you have already heard. P53 in normal cells, normal mammalian systems, is a central player in response to DNA damage of a variety of sorts. And traditionally this has been studied with types of radiation, such as X-irradiation, UV-irradiation, but this is equally applicable to most of our commonly used chemotherapy drugs, which are DNA-damaging agents.

So in a normal cell, P53 at very low levels, usually barely detectable, but following DNA damage is stabilized and activated and the protein level increases, which is the basis for the immuno-peroxidase studies of tumor tissues. And after activation it serves as a transcription factor to regulate a variety of downstream genes which are involved in important biological effects of cell cycle check points, apoptosis and DNA repair. Therefore, in tumors mutant or null for P53, these downstream effects are defective and you can certainly see why this is central to the process of tumor formation and also how this certainly may regulate the response to chemotherapy in cell death.

So why is P53 important? As is often cited, it is mutated in nearly 50 percent of many common tumors, and you can see many of the GI tumors fall into that range, including stomach cancer, as in this graph.

More close studies have confirmed this. Indeed, in most esophageal, GE-junction and gastric cancers, more than 50 percent of them over-express protein.

The region where the majority of known mutations occur is, in fact, the region critical for transcription factor activity and DNA binding activity, suggesting that the effect of these mutations, therefore, is to disrupt that activity. But it is important to note that P53 has other activities in terms of binding other important proteins, though very few mutations occur in those regions.

This is a larger schema of the regulation of P53 both upstream and downstream, and I think it is one of the major points I want to make today, which is that simply looking at P53 status in isolation is not surprisingly confusing in terms of trying to make predictions, given this very complicated regulation. So in response to a variety of stresses important for today, types of DNA damage, but also many other stresses such as oncogene activation, hypoxia, P53 is activated through a huge number of upstream factors that I am not even showing on this slide, and don’t want to get into today, but serve to phosphorylate, acetylate, regulate P53. As we well know, P21 and other genes are involved in cell cycle. There is quite a list of genes involved in P53-dependent apoptosis. We’ve heard of newer genes, such as PERP and pig genes, P53-inducible genes. And what I will tell you about briefly, since this is not as known information, are several genes directly involved in DNA repair and some of the processes that you have just heard about.

So I want to spend just a few minutes talking about DNA repair since we have heard this brought up with respect to chemotherapy agents, particularly cisplatin, and how P53 can regulate this. The first point is that there are as many DNA repair processes as there are types of DNA damage. This slide just simply demonstrates some of the many types of DNA damage and strand breaks from x-ray, various alkylation and adduct damage, oxidative-induced DNA damage.
The mechanism of DNA repair that I am going to talk about today is nucleotide excision repair, which has been historically studied in response to UV irradiation, which produces dimers, but is also relevant to the drug cisplatin and various alkylator agents.

So the commonly known substrates for the repair process, and I will show you mostly data involved with UV, but importantly also inter-strand cross-links, alkylation and oxidative damage. The regulation of nucleotide excision repair therefore may very well affect the cellular response to these types of chemotherapeutic drugs.

The process for repair is simply shown here, which involves several steps. One is recognition of a DNA-damaged adduct, which can be caused by a drug such as cisplatin, excision of this whole piece of DNA that contains that damage, and then re-synthesis of the DNA to replace it based on the template strand and restore the normal DNA sequence.

In humans this enzymatic process is very complicated and I just want to point out a few of the enzymes important in this. Several of them have been discussed already and I will discuss several more today. The initial recognition step is activated by two complexes, one called XPE and one called XPC, and this contains a gene called P48. Following this recognition, this complex recruits a number of other genes and complexes, which you can see here, including this whole TF2H complex and I just want to point out ERCC-1, which you were just hearing about as a predictive factor for platinum sensitivity. As you can see, ERCC-1 is just but one of a large and highly regulated complex, and so how this one gene plays a role in this larger one still remains to be sorted out. What my lab and others now have identified is that both of these very upstream recognition complexes are transcriptionally regulated by P53. So XPE and XPC are DNA-damage inducible, and that is through a P53-dependent process, and in P53 mutant cells, the expression of these is deficient and those cells are actually deficient in nucleotide excision repair.