Loss of Gene Islands May Promote a Cancer Cell’s Survival, Proliferation and Evolution: A new Hypothesis (and second paper validating model) on Oncogenesis from the Elledge Laboratory
Writer, Curator: Stephen J. Williams, Ph.D.
It is well established that a critical event in the transformation of a cell to the malignant state involves the mutation of hosts of oncogenes and tumor suppressor genes, which in turn, confer on a cell the inability to properly control its proliferation. On a genomic scale, these mutations can result in gene amplifications, loss of heterozygosity (LOH), and epigenetic changes resulting in tumorigenesis. The “two hit hypothesis”, proposed by Dr. Al Knudson of Fox Chase Cancer Center[1], proposes that two mutations in the same gene are required for tumorigenesis, initially proposed to explain the progression of retinoblastoma in children, indicating a recessive disease.
(Excerpts from a great article explaining the two-hit-hypothesis is given at the end of this post).
And, although many tumor genomes display haploinsufficeint tumor suppressor genes, and fit the two hit model quite nicely, recent data show that most tumors display hemizygous recurrent deletions within their genomes. Tumors display numerous recurrent hemizygous focal deletions that seem to contain no known tumor suppressor genes. For instance a recent analysis of over three thousand tumors including breast, bladder, pancreatic, ovarian and gastric cancers averaged greater than 10 deletions/tumor and 82 regions of recurrent focal deletions,
It has been proposed these great number of hemizygous deletions may be a result of:
- a recessive tumor suppressor gene requiring mutation or silencing of second allele
- the mutation may recur as they are located in fragile sites (unstable genomic regions)
- single-copy loss may provide selective advantage regardless of the other allele
Note: some definitions of hemizygosity are given below. In general at any locus, each parental chromosome can have 3 deletion states:
- wild type
- large deletion
- small deletion
Hemizygous deletions only involve one allele, not both alleles which is unlike the classic tumor suppressor like TP53
To see if it is possible that only one mutated allele of a tumor suppressor gene may be a casual event for tumorigenesis, Dr. Nicole Solimini and colleagues, from Dr. Stephen Elledge’s lab at Harvard, proposed a hypothesis they termed the cancer gene island model, after analyzing the regions of these hemizygous deletions for cancer related genes[2]. Dr. Soliin and colleagues analyzed whole-genome sequence data for 526 tumors in the COSMIC database comparing to a list generated from the Cancer Gene Census for homozygous loss-of-function mutations (mutations which result in a termination codon or frame-shift mutation: {this produces a premature stop in the protein or an altered sequence leading to a nonfunctional protein}.
Results of this analysis revealed:
- although tumors have a wide range of deletions per tumor (most epithelial high grade like ovarian, bladder, pancreatic, and esophageal adenocarcinomas had 10-14 deletions per tumor
- and although tumors exhibited a wide range (2- 16 ) loss of function mutations
- ONLY 14 of 82 recurrent deletions contained a known tumor suppressor gene and was a low frequency event
- Most recurrent cancer deletions do not contain putative tumor suppressor genes.
Therefore, as the authors suggest, an alternate method to the two-hit hypothesis may account for a selective growth advantage for these types of deletions, defining these low frequency hemizygous mutations in two general classes
- STOP genes: suppressors of tumor growth and proliferation
- GO genes: growth enhancers and oncogenes
Identifying potential STOP genes
To identify the STOP and GO genes the authors performed a primary screen of an shRNA library in telomerase (hTERT) immortalized human mammary epithelial cells using increased PROLIFERATION as a screening endpoint to determine STOP genes and decreased proliferation and lethality (essential genes) to determine possible GO genes. An initial screen identified 3582 possible STOP genes. Using further screens and higher stringency criteria which focused on:
- Only genes which increased proliferation in independent triplicate screens
- Validated by competition assays
- Were enriched more than four fold in three independent shRNA screens
the authors were able to focus on and validate 878 genes to determine the molecular pathways involved in proliferation.
These genes were involved in cell cycle regulation, apoptosis, and autophagy (which will be discussed in further posts).
To further validate that these putative STOP genes are relevant in human cancer, the list of validated STOP genes found in the screen was compared to the list of loss-of-function mutations in the 526 tumors in the COSMIC database. Surprisingly, the validated STOP gene list were significantly enriched for known and possibly NOVEL tumor suppressor genes and especially loss of function and deletion mutations but also clustered in gene deletions in cancer. This not only validated the authors’ model system and method but suggests that hemizygous deletions in multiple STOP genes may contribute to tumorigenesis
as the function of the majority of STOP genes is to restrain tumorigenesis
A few key conclusions from this study offer strength to an alternative view of oncogenesis NAMELY:
- Loss of multiple STOP genes per deletion optimize a cancer cell’s proliferative capacity
- Cancer cells display an insignificant loss of GO genes, minimizing negative impacts on cellular fitness
- Haploinsufficiency in multiple STOP genes can result in similar alteration of function similar to complete loss of both alleles of
- Cancer evolution may result from selection of hemizygous loss of high number of STOP and low number of GO genes
- Leads to a CANCER GENE ISLAND model where there is a clonal evolution of transformed cells due to selective pressures
A link to the supplemental data containing STOP and GO genes found in validation screens and KEGG analysis can be found at the following link:
http://www.sciencemag.org/content/337/6090/104/suppl/DC1#
A link to an interview with the authors, originally posted on Harvard’s site can be found here.
http://www.cell.com/retrieve/pii/S0092867413012877
A concern of the authors was the extent to which gene silencing could have on their model in tumors. The validation of the model was performed in cancer cell lines and compared to tumor genome sequence in publicly available databases however a followup paper by the same group shows that haploinsufficiency contributes a greater impact on the cancer genome than these studies have suggested.
In a follow-up paper by the Elledge group in the journal Cell[3], Theresa Davoli and colleagues, after analyzing 8,200 tumor-normal pairs, show there are many more cancer driver genes than once had been predicted. In addition, the distribution and potency of STOP genes, oncogenes, and essential genes (GO) contribute to the complex picture of aneuploidy seen in many sporadic tumors. The authors proposed that, together with these and their previous findings, that haploinsufficiency plays a crucial role in shaping the cancer genome.
Hemizygosity and Haploinsufficiency
Below are a few definitions from Wikipedia:
Zygosity is the degree of similarity of the alleles for a trait in an organism.
Most eukaryotes have two matching sets of chromosomes; that is, they are diploid. Diploid organisms have the same loci on each of their two sets of homologous chromosomes, except that the sequences at these loci may differ between the two chromosomes in a matching pair and that a few chromosomes may be mismatched as part of a chromosomal sex-determination system. If both alleles of a diploid organism are the same, the organism is homozygous at that locus. If they are different, the organism is heterozygous at that locus. If one allele is missing, it is hemizygous, and, if both alleles are missing, it is nullizygous.
Haploinsufficiency occurs when a diploid organism has only a single functional copy of a gene (with the other copy inactivated by mutation) and the single functional copy does not produce enough of a gene product (typically a protein) to bring about a wild-type condition, leading to an abnormal or diseased state. It is responsible for some but not all autosomal dominant disorders.
Al Knudsen and The “Two-Hit Hypothesis” of Cancer
Excerpt from a Scientist article by Eugene Russo about Dr. Knudson’s Two hit Hypothesis;
for full article please follow the link http://www.the-scientist.com/?articles.view/articleNo/19649/title/-Two-Hit–Hypothesis/
“
The “two-hit” hypothesis was, according to many, among the more significant milestones in that rapid evolution of biomedical science. The theory explains the relationship between the hereditary and nonhereditary, or sporadic, forms of retinoblastoma, a rare cancer affecting one in 20,000 children. Years prior to the age of gene cloning, Knudson’s 1971 paper proposed that individuals will develop cancer of the retina if they either inherit one mutated retinoblastoma (Rb) gene and incur a second mutation (possibly environmentally induced) after conception, or if they incur two mutations or hits after conception.3 If only one Rb gene functions normally, the cancer is suppressed. Knudson dubbed these preventive genes anti-oncogenes; other scientists renamed them tumor suppressors.
When first introduced, the “two-hit” hypothesis garnered more interest from geneticists than from cancer researchers. Cancer researchers thought “even if it’s right, it may not have much significance for the world of cancer,” Knudson recalls. “But I had been taught from the early days that very often we learn fundamental things from unusual cases.” Knudson’s initial motivation for the model: a desire to understand the relationship between nonhereditary forms of cancer and the much rarer hereditary forms. He also hoped to elucidate the mechanism by which common cancers, such as those of the breast, stomach, and colon, become more prevalent with age.
According to the then-accepted somatic mutation theory, the more mutations, the greater the risk of cancer. But this didn’t jibe with Knudson’s own studies on childhood cancers, which suggested that, in the case of cancers such as retinoblastoma, disease onset peaks in early childhood. Knudson set out to determine the smallest number of cancer-inducing events necessary to cause cancer and the role of these events in hereditary vs. nonhereditary cancers. Based on existing data on cancer cases and some mathematical deduction, Knudson came up with the “two-hit” hypothesis.
Not until 1986, when researchers at the Whitehead Institute for Biomedical Research in Cambridge, Mass., cloned the Rb gene, would there be solid evidence to back up Knudson’s pathogenesis paradigm.4 “Even with the cloning of the gene, it wasn’t clear how general it would be,” says Knudson. There are, it turns out, several two-hit lesions, including polyposis, neurofibromitosis, and basal cell carcinoma syndrome. Other cancers show only some correspondence with the two-hit model. In the case of Wilm’s tumor, for example, the model accounts for about 15 percent of the cancer incidence; the remaining cases seem to be more complicated.
”
His seminal paper on the two-hit hypothesis[1]
A.G. Knudson, “Mutation and cancer: statistical study of retinoblastoma,” Proceedings of the National Academy of Sciences, 68:820-3, 1971.
The two hit hypothesis proposed by A.G. Knudson. A description with video of Dr. Knudson talk at AACR can be found at the following link (photo creditied to A.G. Knudson and Fox Chase Cancer Center at the following link:http://www.fccc.edu/research/research-awards/knudson/index.html
Sources
1. Knudson AG, Jr.: Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Sciences of the United States of America 1971, 68(4):820-823.
2. Solimini NL, Xu Q, Mermel CH, Liang AC, Schlabach MR, Luo J, Burrows AE, Anselmo AN, Bredemeyer AL, Li MZ et al: Recurrent hemizygous deletions in cancers may optimize proliferative potential. Science 2012, 337(6090):104-109.
3. Davoli T, Xu Andrew W, Mengwasser Kristen E, Sack Laura M, Yoon John C, Park Peter J, Elledge Stephen J: Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns and Shape the Cancer Genome. Cell 2013, 155(4):948-962.
Other papers on this site on CANCER and MUTATION include:
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