Monday, July 30, 2012

Melanoma, Sun Damage and Pathways


There is extensive epidemiological data indicating that melanoma is caused by UV radiation. Now there has been, up to this date, little information relating specific effects of UV radiation to specific causative gene changes. In a recent article in Cell by Hodis etal, the authors relate the impact of sun damage on melanocytes and the initiation of melanoma[1].This is an interesting paper and the approach is quite innovative and worth examining.

The authors summarize their work as follows:

Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure.

We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis.

Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which—RAC1, PPP6C, and STK19—harbored recurrent and potentially targetable mutations.

Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations.

The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.

In a release from MD Anderson Cancer Center they state[2]:

By creating a method to spot the drivers in a sea of passengers, scientists at the Broad Institute of MIT and Harvard, the Dana-Farber Cancer Institute and The University of Texas MD Anderson Cancer Center have identified six genes with driving mutations in melanoma, three of which have recurrent 'hotspot' mutations as a result of damage inflicted by UV light. Their findings are reported in the July 20 issue of the journal Cell.

"Those three mutations are the first 'smoking gun' genomic evidence directly linking damage from UV light to melanoma," said co-senior author Lynda Chin, M.D., Professor and Chair of MD Anderson's Department of Genomic Medicine. "Until now, that link has been based on epidemiological evidence and experimental data."

"This study also is exciting because many of the recent large-scale genomic studies have not discovered new cancer genes with recurrent hot-spot mutations, a pattern strongly indicative of biological importance," said Chin, who also is scientific director of MD Anderson's Institute for Applied Cancer Science.

The six new melanoma genes identified by the team are all significantly mutated and provide potential targets for new treatments.

Let us first detail several of these genes.

1. RAC1

From NCI we have RAC1 located at 7p22 and described as follows[3]:

The protein encoded by this gene is a GTPase which belongs to the RAS superfamily of small GTP-binding proteins. Members of this superfamily appear to regulate a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases.

From the NCI Pathway database we have a complex set of pathway interactions[4]. In a similar manner we can examine the pathways from the MMMP data base[5]. In all cases of this gene and the others recenbctly elucidated, the pathways are partially informative and need additional investigation.

2. PPP6C

From NCI we have PPP6C located at 9q33.3 and described as follows[6]:

This gene encodes the catalytic subunit of protein phosphatase, a component of a signaling pathway regulating cell cycle progression. Splice variants encoding different protein isoforms exist.

3. STK19

From the NCI database this gene is located at 6q21.3 and functions as follows[7]:

This gene encodes a serine/threonine kinase which localizes predominantly to the nucleus. Its specific function is unknown; it is possible that phosphorylation of this protein is involved in transcriptional regulation. This gene localizes to the major histocompatibility complex (MHC) class III region on chromosome 6 and expresses two transcript variants

Thus the genes perform a broad and generally non-correlative set of functions. The authors have argued that the genes are targetable as say with BRAF but a more complete understanding of full pathway interactions would be essential.

Counting UV Hits

The authors discuss the fact that m UV mutations convert C (cytidine) to T (thymidine). Now as Watson et al have shown pp 204-209[8]) when cytidine is methylated as shown below the uridine product is converted to thymidine and this there is a mis-reading of the DNA. These C to T transitions are caused in the case of melanoma often by UV. We have also argued that they may equally be caused by backscatter X-rays which have enough energy to break bonds to cause a methylation as well. 


Now the principle the authors employed was a presumptive one based upon the following:

1. C to T transition is random across the DNA sequences.

2. The average number of such transitions should be the same in both Introns and Exons.

3. Exons express genes which do things.

4. If there are a statically larger number of C to T transitions in a specific Exon, i.e. a gene say Gene X, then that gene most likely is causative of the melanoma.

We demonstrate this concept in the diagram below:

 Now it was through a process of this type which allowed the authors to identify a collection of twelve genes, six known to be related to melanome, and six not previously known to be related, to be presumptively causitive of the malignancy.

From an article in Science Daily they state, using a somewhat less than precise metaphor, the following[9]:

By creating a method to spot the drivers in a sea of passengers, scientists at the Broad Institute of MIT and Harvard, the Dana-Farber Cancer Institute and The University of Texas MD Anderson Cancer Center have identified six genes with driving mutations in melanoma, three of which have recurrent 'hotspot' mutations as a result of damage inflicted by UV light. Their findings are reported in the July 20 issue of the journal Cell.

"Those three mutations are the first 'smoking gun' genomic evidence directly linking damage from UV light to melanoma," said co-senior author Lynda Chin, M.D., Professor and Chair of MD Anderson's Department of Genomic Medicine. "Until now, that link has been based on epidemiological evidence and experimental data."

"This study also is exciting because many of the recent large-scale genomic studies have not discovered new cancer genes with recurrent hot-spot mutations, a pattern strongly indicative of biological importance," said Chin, who also is scientific director of MD Anderson's Institute for Applied Cancer Science.

The six new melanoma genes identified by the team are all significantly mutated and provide potential targets for new treatments.

Puzzle has thousands of potential pieces, but only requires a few dozen. A number of important mutations had previously been identified as melanoma drivers. These include BRAF (V600) mutations, present in half of all melanomas, and NRAS (Q61) mutations. However, the vast majority of these mutations do not appear to be caused by direct damage from UV light exposure.

UV light causes many mutations of genes in melanocytes. The mutations occur in both introns and exons. The question is which of these mutations is significant and for example is there a level at which they become malignant. An interesting question can be asked about melanoma in situ, the early stage of melanoma where the melanocytes have enlarged nucleoli and express a loss of localization. It is well known histologically that MIS is often discovered in sun damaged areas. Thus one would suspect that at this early stage many of this methylation like changes doe to UV radiation is present.

The article continues:

To counter this effect, the researchers turned to parts of the genome that don't code for proteins, called introns, and other inactive DNA segments that flank exons. By comparing the frequency of mutations in the inactive segments to the frequency of mutations in the exons, the researchers built a framework for assessing the statistical significance of functional mutations.

Approach identifies six known cancer genes, six new ones.

The analysis identified functional mutations in the well-known cancer genes BRAF, NRAS, PTEN, TP53, CDKN2A and MAP2K1.

It also uncovered five new genes, RAC1, PPP6C, STK19, SNX31, and TACC1.

Most are associated with molecular pathways involved in cancer but had not been previously recognized as significantly mutated in melanoma. Their presence in the tumor samples ranged from 3 percent to 9 percent.

The sixth new gene tied to melanoma was ARID2, an apparent tumor-suppressor gene possessing a significant number of loss-of-function mutations found in 7% of patient samples.

"Six new melanoma genes have been picked out from thousands of mutated genes," said Eran Hodis, co-lead author who is a computational biologist in the Garraway lab at the Broad Institute and an M.D.-Ph.D. student at Harvard and MIT. "The same approach may bring clarity to genome sequencing studies of other cancers plagued by high passenger mutation rates, for example lung cancer." ...


Most exciting, three of the discovered genes possessed 'hotspot' mutations found in the exact same position in multiple patients providing another line of evidence indicating these mutations contribute to melanoma.

"We have now discovered the third most common hotspot mutation found in melanoma is present in a gene called RAC1, and unlike BRAF and NRAS mutations, this activating mutation is attributable solely to characteristic damage inflicted by sunlight exposure" said Ian R. Watson, Ph.D.,...

Observations

This is a significant contribution in my opinion. It also, in my opinion, raises some very interesting questions.

1. How many hits are required to make the change?

2. What are the pathway effects that result in malignancy?

3. How does MIS fit within this model?

4. If UV radiation can do this then we would expect that X rays would have equal effects and if so then backscatter X rays which penetrate just enough would be of significance. If that is correct how much radiation would be required?

5. If we have these putative genes and there are targets, then how easy would it be to develop anti-cancer drugs for these targets?

6. If we see BRAF failure and return of the malignancy then is it possibly from these new genes, if so which ones, and if some of them in what order of importance?

7. When performing biopsies on melanomas, should examination for these genes be a common practice?

This paper raises many more such questions.
 
References

Hodis, E., et al, A Landscape of Driver Mutations in Melanoma, Cell, Volume 150, Issue 2, 251-263, 20 July 2012.

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