Replication stress links structural and numerical cancer chromosomal instability

At the recent "Tumor heterogeneity: Implications for targeted Therapy" conference, Charles Swanton (London Research Institute) presented how kidney, colon and lung tumors change and evolve over time. This work was published in a series of high-profile papers, including two back-to-back articles in Science this week. Here, I review a part of the talk with results published in Burrell et al in Nature in 2013.

In 2013, Swanton and co-workers examined why many tumors display chromosomal instability (CIN). While normal human cells are diploid and carry two copies of each gene, cancer cells from many solid tumors often accumulate specific regions and loose others. This can e.g. lead to the loss of tumor suppressor genes and provides genetic variation that can fuel the evolution of sub-clones.

The balanced inheritance of genetic material is tightly controlled in normal cells, but seems to be error-prone in many human cancers. For example, colorectal cancer can broadly be categorized into chromosomal-stable (CIN-) and -unstable (CIN+) subtypes. Swanton an co-workers set out to understand the mechanisms specifically destabilizing CIN+ colorectal tumors.

Through careful microscopy imaging of dividing cells, they documented a high frequency of DNA replication artifacts in CIN+ cell lines. These cells seemed unable to duplicate their genome correctly before cell division and produced e.g. chromosomal fragments without centromers, which were randomly distributed to the daughter cells. The root of the problems appeared to be a disruption of the DNA replication process itself, as the authors noticed that the replication forks in CIN+ cells progressed at a slower pace than in their CIN- counterparts. This is a sign of "replication stress", which was previously shown to cause DNA damage and chromosomal aberrations.

What could cause replication stress in colorectal cancers ? To formulate specific hypotheses, Swanton and co-workers compared the cancer genomes of CIN+ and CIN- tumors. First, they checked known oncogenes and tumor suppressors. While the TP53 gene, which is frequently deactivated in human cancers, appeared to be more often mutated in CIN+ cases, its biological function did not explain the observed chromosomal instability.

Next, the scientists enumerated copy number variants (CNVs), looking for regions lost or gained in CIN+ but not CIN- tumors, and found a promising candidate: loss of a specific region of chromosome 18 (region 18q) was observed in 88% of aneuploid tumours and 80% of CIN+ cell lines. The researchers had detected a statistically significant correlation between 18q loss and chromosomal instability - but had they really identified a causal relationship ?

If region 18 really contained genes important for the correct execution of replication, its loss should precede the onset of chromosomal instability. During colon cancer development, cells typically progress through a precursor stage, called adenoma, before progressing into malignant carcinomas.

A genetic model for colorectal tumorigenesis, Fearon & Vogelstein, Cell, 1990; Image source: Wikimedia Commons

Both 18q loss and chromosomal instability were found to be less frequent in adenomas than carcinoma samples from the same patients, consistent with a causal relationship between these two observations (but not proving it).

To elucidate the molecular consequences of 18q loss, the researchers systematically deactivated all of the protein-coding genes contained in this region of the genome. Targeting any one of three genes - PIGN, MEX3C or ZNF516 - produced a CIN+ phenotype in cell lines including acentric chromosomes and anaphase bridges, activation of the DNA damage response and reduced replication fork speed. In addition, just like the naturally observed CIN+ phenotype, the consequences of inactivating these genes could be prevented by supplying additiona DNA building blocks in the form of nucleosides to the cells.

Conclusions:
Recurrent loss of a specific genomic region, 18q, in colorectal cancers may be responsible for disrupting the normal replication process in cancer cells. This could trigger a cascade of subsequent losses or gains, increasing the genetic heterogeneity in the following generations of cancer cells and accelerate the emergence of resistance.

References

• Intratumor heterogeneity and branched evolution revealed by multiregion sequencing; Gerlinger et al, N Engl J Med. 2012 Mar 8;366(10):883-92;  doi: 10.1056/NEJMoa1113205.

• Replication stress links structural and numerical cancer chromosomal instability.; Burrell et al, Nature. 2013 Feb 28;494(7438):492-6. doi: 10.1038/nature11935.

• Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing.; Gerlinger et al, Nat Genet. 2014 Mar;46(3):225-33. doi: 10.1038/ng.2891. Epub 2014 Feb 2.

• Spatial and temporal diversity in genomic instability processes defines lung cancer evolution.; de Bruin et al, Science. 2014 Oct 10;346(6206):251-6. doi: 10.1126/science.1253462.

• Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing; Zhang et al, Science. 2014 Oct 10;346(6206):256-9. doi: 10.1126/science.1256930.