Epigenetics in Retinoblastoma

Table of Contents

1. Abstract

2. Introduction

3. Stability of the Retinoblastoma genome

4. Epigenetic Investigations

5. Testing SYK

6. Discussion

7. References

8. Abbreviations and notation

1. Abstract

Today's DNA sequencing technologies are becoming more and more impressive. That it is not only important to focus on mutations in the sequence of DNA, however, but also on differences that occur on the level above the genes, the epigenetic modifications of the DNA, has been found out by the researchers of Michael Dyer's group.

The quickly developing childhood cancer retinoblastoma is directly related to the inactivation of the tumor suppressor gene RB1. With RB1 having different roles relevant for understanding this cancer, the process leading to advancing retinoblastoma after its inactivation is not yet understood.

After analysis of the genome and the epigenome of different retinoblastomas, the genome was detected as relatively stable. A newly proposed part of the chain of events leading to the outbreak of this severe cancer is as follows.

Biallelic loss of RB1 leads to the deregulation of epigenetic mechanisms and thereby also of cancer pathways. This is followed by cells acquiring the characteristics of cancer cells. With the experiments conducted by Dyer et al., the proto-oncogene SYK (spleen tyrosine kinase) was found to be upregulated in retinoblastoma and also necessary for the survival of the tumor cells. With SYK inhibited, increasing apoptosis of the cancer cells could be observed. The conclusion seems likely that SYK promises to be a new target for treating retinoblastoma patients and a broader approach in research, including not only genomic data, but also epigenetic analyses, is necessary for better understanding of cancer in the future.

2. Introduction

Historically noteworthy, the Retinoblastoma gene (RB1) was the first human tumor suppressor gene discovered (Houston, 2011). In most cases, biallelic loss of the RB1 gene is considered to be the critical factor leading to the outbreak of cancer. The quick progress of the disease after that step, however, has long been a mystery and still is not completely figured out.

With 9000 new cases every year worldwide, corresponding to one affected child per 15 000-20 000 live births (Dimaras, 2012), retinoblastoma is a rare disease. It is, however, the most frequent intraocular malignancy in children, representing 3% of all pediatric cancers (Mehta, 2012).

Untreated, this disease of the developing retina, which can occur sporadically (mostly unilateral) as well as in a familial form (often bilateral), leads to death (Willerding, 2008). Diagnosis of retinoblastoma often follows the observation of strabism or leukocoria, a white reflection in the eye (Fig. 1).

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Figure 1 Child with retinoblastoma visible as Leukocoria in the left eye. Diagnosis of retinoblastoma often follows noticing this white reflection. From: Houston SK, Murray TG, Wolfe SQ, Fernandes CE (2011) Current Update on Retinoblastoma. International Ophthalmology Clinics 51 : 77-91.

In general, it is widely accepted, that the crucial characteristics of cancer cells consist of the activation of cell-growth-promoting pathways, avoiding senescence and differentiation, evasion of limits in proliferation, ignorance of internal and external signals for cell proliferation regulation, the tendency to avoid apoptosis, continuous The path to retinoblastoma Sophia Stahl angiogenesis, invasion, survival and proliferation in other tissues (metastasis) (Alberts, 2008). These characteristics are acquired by cells on their way to becoming cancer cells by genetic lesions and/or epigenetic mechanisms.

According to the results of the research done by Dyer et al., though, the often mentioned hallmark of genetic instability is not a hallmark of retinoblastoma cells. Instead, the epigenetic changes in the retinoblastoma genome are much more relevant. One currently proposed definition for an epigenetic trait is that it describes changes in a chromosome without altering the DNA sequence, such as methylation, resulting in a stably inherited phenotype (Berger, 2009).

The critical gene on chromosome 13 RB1 has two known roles relevant for the understanding of this cancer. On the one hand, it is necessary for chromosomal stability, thus, if lost, genetic instability in key cancer pathways may be the result. This option would mean that other mutations can go together with RB1 loss. The genomes tested by Dyer et al., however, proved to be relatively stable. On the other hand, RB1 is important in the regulation of most significant epigenetic processes, therefore inactivation may lead to changes in the DNA modifications that inevitably result in different levels of gene regulation.

Knudson's “two-hit”-hypothesis states that the biallelic loss of RB1 is necessary for retinoblastoma development (Houston, 2011). The impact of this incident that leads to the alterations in the appearance and cell constitution of the eye, though, has not yet been identified. Thus, treatment of retinoblastoma has been similar to that of other cancer types, involving chemotherapy or, in already further progressed cases, operational enucleation (eye removal) (Willerding, 2008). Now a new therapeutic target, the gene SYK, is proposed by Dyer et al.

3. Stability of the Retinoblastoma genome

Wondering what the cause of the quick progress of the disease after RB1 inactivation could be, Dyer and his group, part of the St Jude Children's Research Hospital (SJCRH) - Washington University Pediatric Cancer Genome Project, took a closer look at the retinoblastomas and their germline DNA of four different patients. They performed whole-genome sequencing (WGS) and found out that the genomes were not significantly instable in comparison to other adult tumors that also have undergone WGS. In fact, the mean mutation rate of the analyzed retinoblastomas was, with only 6.7·10-⁸, even 15-fold lower than in the common adult malignancies it was compared to. Loss of heterozygosity (LOH) of RB1 on chromosome 13 and a gain of chromosome 6p were the only found structural variations. This is a surprising result, since genomic instability including chromosomal aberrations as well as many mutations were expected to be crucial for cancer cell development (Murphree, 2012).

42 additional retinoblastomas from SJCRH were used for further studies to support the findings. The BCL-6-co-repressor gene BCOR was the only gene that was noticed to be mutated significantly (13% out of the 42 samples).

Dyer's team used a paired-end sequencing approach and provided supplementary coverage by sequencing the transcriptomes of each primary tumor. Orthotopic xenografts were also generated of two of the samples (SJRB001X and SJRB002X) in order to come to further conclusions in vivo by having similar features of the originals represented in living organisms; immunocompromised mice got primary tumor cells implanted into the vitreous humor of their eyes.

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Figure 2 Kinetochore structure analysis in RB1-deficient retinoblastoma cells. c) Representative images of anaphase cells from control (BJ) and retinoblastoma (SJRB001X) cell lines. The two white arrows on the bottom right indicate lagging chromosomes. d) Defective anaphases in control and retinoblastoma cells in percent. From: Zhang J, Benavente CA, McEvoy J, Flores-Otero J, Ding L, Chen X, Ulyanov A, Wu G, Wilson M, Wang J, Brennan R, Rusch M, Manning AL, Ma J, Easton J, Shurtleff S, Mullighan C, Pounds S, Mukatira S, Gupta P, Neale G, Zhao D, Lu C, Fulton RS, Fulton LL, Hong X, Dooling DJ, Ochoa K, Naeve C, Dyson NJ, Mardis ER, Bahrami A, Ellison D, Wilson RK, Downing JR, Dyer MA (2012) A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature 481 : 329-334. Supplementary Information Fig. 17.

Since other studies had found a connection between RB1 inactivation and incorrect chromosome segregation (Manning, 2010), some amount of aneuploidy and chromosome instability was expected to occur in these xenografts. The results of genomic analyses of the parts of tumor that were growing in the mice for more than nine months, however, were not that different from the originals. All alterations from the original retinoblastoma were still apparent. The few differences found (67 new SNVs and four structural variations) were not only present in none of the affected annotated genes, but also at a subclonal level. Additionally, even though there was evidence of greater distances between sister chromatids during anaphase of mitosis (Figure 2), the expected aneuploidy did not eventuate. The ploidy was rather similar to that of wild-type cells and in comparison to cancers that are known to have an unstable genome, there were a lot less anomalies in copy number variation (Figure 3). Overall, these results are suggesting very few genetic lesions to be necessary for the progression of retinoblastoma.

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Figure 3 Chromosome instability and aneuploidy analyses in retinoblastoma. a) RPE, retinal pigment epithelium, red plot traces chromosomal mis-segregation of SJRB001X cells after at least 21 rounds of cell division. The proportion of cells with chromosomal gains/losses is higher with inactivated RB1, but not significantly. b) Representative spectral karyotype image of SJRB0001X after the third passage in mice. c) Compared to 153 high-grade serious ovarian cancer (OV) cases from The Cancer Genome Atlas, alterations in the 46 Rb cases are shown. The median percentage of the genome involved in copy number variations (CNVs) was 1,5% for the retinoblastoma and 27,7% for OV. Red circles, samples used for WGS. From: Zhang J, Benavente CA, McEvoy J, Flores-Otero J, Ding L, Chen X, Ulyanov A, Wu G, Wilson M, Wang J, Brennan R, Rusch M, Manning AL, Ma J, Easton J, Shurtleff S, Mullighan C, Pounds S, Mukatira S, Gupta P, Neale G, Zhao D, Lu C, Fulton RS, Fulton LL, Hong X, Dooling DJ, Ochoa K, Naeve C, Dyson NJ, Mardis ER, Bahrami A, Ellison D, Wilson RK, Downing JR, Dyer MA (2012) A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature 481 : 329-334.

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