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- [James Cleaver] I'd like to thank very much to the organizers of this conference
for inviting me. I just want to, as I frequently speak to clinical audiences, I
have to make my declaration of disclaim. I essentially work in borrowed space, use
borrowed money, and I'm living on borrowed time. Hence, I like as my theme Thomas
Huxley's remark that, "Scientists over 60 do more harm than good."
Now I want to direct you to a traveling story. Skin is our major defense against
an onslaught of one of the strongest carcinogens that we experience,
ultraviolet light. And some years ago I was directed to this village in Northern
Guatemala were the missionary's orders that the children had enormous amount of
skin cancer, and they were dying at 10 years of age.
Now, commonly, you would think skin cancer as an older person's problem. These are
dramatic examples of how, if we lose the genetic defense against sun exposure,
cancer can be an enormously disastrous problem at the skin level. So they
directed me to this village and it had the phenotype of a disease that I'd work for
many years, xeroderma pigmentosum.
That's normally a recessive disease. In then U.. S population, it occurs about one
to four per million of the population. Twenty-five percent of the children had
this disease. They lived under extreme poverty. When we had a look at the
patients, you could see the enormous sun damage of their face, and the painful
sunburns. When we brought cells back, you can see here and exposed these cells to
ultraviolet in the lab where the normal skin has excellent survival, their cells
were killed at very, very low doses of UV. So they had a genetic predisposition in
which they could not handle sun exposure, solar exposure. We sequenced, and we found
that they lacked what you call now the global genome repair pathway. The founder
effect in this village was a mutation which caused...which removed one cytosine
from a gene we call XPC which is one of the main damage recognition proteins in
the skin.
When we did...even a staining of a section of the skin, the black staining here,
indications of high expression of the XPC protein in the epidermis of normal skin.
From the Guatemalan village, there is no expression of this gene. As a result,
their cells fail to recognize DNA damage and it goes on to develop into cancer.
I will show you some of the ramifications of this. Now, xeroderma pigmentosum, XP,
is one of a family of some sensitive diseases involving different components of
the nucleotide excision repair pathway. As an aside, I'd just like to mention that
you may have seen that the Nobel prize for chemistry this year was given to Tom
Lindahl, Z. Sancar, and the mismatch repair pathway for unraveling the
biochemistry of this pathway.
Now, xeroderma pigmentosum is unique from among the repair systems for solar UV in
that they have very high cancer. These other diseases are also components of the
same repair pathway, but they essentially are photosensitive but without skin
cancer. And especially a disease here known as the
UVS syndrome has acute sunburn, but no neurological disorders, none of the
developmental disorders or the current Cockayne syndrome, and a very mild
version. So there's an extremely wide variation in the genetic response to sun
exposure.
Now the main photoproducts caused by UV occur between two adjacent pyrimidines in
the DNA. The major photoproduct is the dimer in which two covalent bonds are
formed. An alternative photoproduct, the 6-4 photoproduct. Now if you do not remove
these photoproducts, either of these, if you don't remove them bio-chemically from
the DNA, they can go on to interfere with DNA replication. And a cytosine and a TC
photoproduct can deaminate, code as an A, you know, codes as a T and then opposite
an A is placed. After two cycles of replication, a TC sequence becomes a TT.
So, a C to a T transition mutation in a TC sequence is an absolute fingerprint for UV
exposure and a UV-induced mutation. Now, the excision repair pathway for UV damage
has two major routes. If the DNA is unexpressed, which is a large majority of
our DNA, damage is recognized by a dedicated damage recognition protein of
which XPC, the Guatemalan population, has a mutation. So they failed to recognize.
An alternative mechanism by which damage is recognize is an arrested transcription.
If cells cannot recover from this, they have the disease called Cockayne syndrome.
And then, these two pathways converge on an extensive remodeling system to repair
the DNA. If we look more closely at the transcription process, there are two
strands, the coding strands and a template strand. The template strand is the one
which is transcribed into RNA. The repair of these two strands is different. The XPC
system repairs this strand. The transcribed strand is repaired by a
complex of proteins that are associated with a transcription complex. The
difference between these two strands have a major impact on carcinogenesis.
First, if you look at repair in terms of the removal of a number of photoproducts,
with time, in a highly expressed gene like P53 or Dhfr, the photoproducts were remove
rapidly. In unexpressed or lower expressed genes, the photoproducts were removed to a
much lower extent. So the rate of repair depends upon which strand the damage lies.
We then collaborated recently with a group in Seattle, Laura Loeb and Kev Bayliss, in
which they use a high throughput sequencing method to determine exactly
what are the sequence changes in a UV-rated population.
Now, those of you...those of us who have worked in a million [sp] cell biology and
tissue culture immunogenesis realize that over the years, we've only had one or two
genes that are capable of studying mutagenesis in culture because of the
number...the rarity of having a drug-selectable locus. With high
throughput sequencing, we can get by that completely. And so, here is the result of
measuring the mutation frequency in nucleotide changes per 10 to the 7th
nucleotides. High resolution sequencing averaged over a large number of genes that
are relevant for skin cancer. And you can see that a wild-type normal cell has a low
increase in mutations or function of dose, but in a XPC such as we see in Guatemala
village, enormous increase in mutation rates.
Going up from 0.5 up to 5. A tenfold or more increase in mutation rates.
We then decided once we had that system working, we went back to Guatemala and
with some help of a clinical service team that goes there from San Diego regularly
to treat the patients and give them some clinical help, we brought back five
squamous carcinomas from this population. Sequenced three million single nucleotide
mutations by exome sequencing. Transition mutations were a majority in XPC.
Now since it was a small population size, we didn't concentrate on which genes were
mutated, but we asked, "What is the frequency of mutations per kilobase across
the whole genome?” and asked, “What's the mutation density?" When we do that...this
is a very interesting situation to illustrate how the genome structure and
function have a major impact on mutation rates.
In a normal population...see in here, normal squamous carcinomas from a normal
population. There is a protein in the histone that methylates a region and
suppresses access for other enzymes. As a result, the mutation density goes up
because those regions of the chromatin are inaccessible to repair. As transcription
increases across the genome, we find that this is...removes the repression, and as a
result mutation frequency declines. But still, if you compare the non-transcribed
strand to the transcribed strand in the tumor, there are more mutations in the
non-transcribed strand. When we move to the XPC tumors from the Guatemalan
patients, we find no removal on mutagenic lesions right across the genome
irrespective of transcription level, but in the transcribed strand, very efficient
removal. Now these are striking illustration of the
fact that it depends critically which strand of the DNA is damaged, which one
gets repaired. If the cells fail to repair the non-transcribed strand, mutation rate
is very high when replication comes along there. On the other strand, the
transcribed strand, it doesn't matter how well that's repaired in the Guatemalan
population. Because they can't repair the other strand, cancer is high. So that the
repair of these transcribed strand could be very efficient, but if confers
absolutely no protection against UV-induced mutations and carcinogenesis.
Now, if we can turn to another response of the skin to ultraviolet exposure, sunburn.
We often think and it's established more or less that the risk for melanoma from
the sun is related to how many sunburns you get. Well in squamous carcinoma
there's a very different situation. This is the spectrum of xeroderma pigmentosum
patients from sun exposure, you can have a patient with very high erythema and
sunburn. You can have patients with freckling. Patients that develop severe
skin cancer. So you're going to have this range, and this is under genetic control.
If you look, and this is data published recently from England where they
have...bring in all the XPC patients from the country, and treat them and follow
them in one single location. And this was reported a couple of years ago. If you
look at sunburn versus which gene in the XP repair pathway is mutated. If you look
at the patients with mutations in XPC, XPE, or XPV which are genes that code for
proteins involved in the processing of DNA damage in the non-expressed regions of the
genome. They get cancer but they get no sunburn.
However, if the genes are involved in repair and in transcription, sunburn is
very high. If you now look at the probability of cancer, and then look at
patients that are involved...in which the genes involved in transcription, cancer
appears slowly over time. And the 50% onset is out...almost out as late as the
normal population. If we look at XPC and E patients which are involved in the repair
of the non-transcribed regions and they don't get sunburn, the cancers are
appearing more rapidly. Their 50% onset is about the age of 20. So an acute sunburn
in this disease is actually delays squamous carcinoma. It's the converse of
what you get with melanoma. As an aside, it's interesting for me to note that the
50% onset here is in the early 20s. In American xeroderma patients, it occurs
below the age of 10 which is reminiscent for my childhood that England is a pretty
cloudy, damp, rainy place.
So let me just summarize. Xeroderma pigmentosum and related nucleotide repair
diseases highlight mechanisms of sunburn and cancer. Repair is more rapid in more
highly expressed genes. Presumably because an expressed gene has to open up the
chromatin to allow transcription to work whereupon repair can work. Now deficient
repair of solar UV products in the non-transcribed strands, and in
unexpressed DNA results in a high mutation rate and cancer, but deficient repair in
the transcribed strand results in increased sunburn.
Cancer is delayed or not increased. And in fact, in Cockayne Syndrome which is
uniquely involved in the repair of the transcribed strands, those patients with
Cockayne Syndrome have severe developmental disorders, but as far as we
know they never get cancer.
So, my question that I leave you with is, "Can we increase repair in otherwise
healthy persons to protect against skin cancer including melanoma and other
cancers?" And alternatively, since the nucleotide excision repair pathway repairs
UV-type damage which is large modifications of DNA very similar to what
occurs when you use chemotherapy like cisplatins. Since it's the same repair
pathway, "Can we inhibit repair, and then sensitize cancers to chemotherapy with
very few side effects?" So I leave you with that. Thank you.
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