Evolutionary success of cancers due to their induction of failed DNA repair and errors during DNA replication ???

How a cancer evolves and how mutations are generated — are highly intertwined processes, and nearly impossible to observe directly (instead, scientists are usually restricted to making inferences about them, using data from a single snapshot in time after a cancer has formed). However, when the two DNA strands (that form the double helix) are considered independently, authors [see attached article & editorial] showed that, for a cell that has undergone DNA damage, such a snapshot provides intriguing information. Analysis of cancer genomes has led to identification of many driver mutations and mutation signatures, which illustrate how environmental mutagens cause genetic damage and increase cancer risk. The numerous patterns of mutations identified in cancer genomes reflects both temporal and spatial heterogeneity of exogenous and endogenous exposures, mutational processes, and germline variation among patients. A previous study of diverse human cancers had identified 49 distinct single-base-substitution signatures — with almost all tumors showing evidence of at least three such signatures.

This is a complicated story, … so bear with me. 😊 Authors reasoned that a more controlled and genetically uniform cancer model system would overcome some of the limitations as to how a cancer evolves and how mutations are generated. By effectively re-running cancer evolution hundreds of times, authors aimed to explore oncogenesis and mutation patterns at high resolution (and with good statistical power). Using a single dose of diethylnitrosamine (DEN) in postnatal-day-15 (P15) male inbred C3H mice, authors chemically induced liver tumors. For comparison and validation, authors replicated the study in the divergent CAST mouse strain.

DNA resembles a ladder, with the ‘Watson’ and the ‘Crick’ strands; these are fused together by ‘rungs’ of two complementary nucleotide base-pairs [either cytosine (C) paired with guanine (G), or adenine (A) paired with thymine (T)]. When a cell divides, each daughter cell inherits either the Watson or the Crick strand from the parent cell; this provides the template from which the other (complementary) strand is then replicated. Damage to a base can trigger a repair process — but, if repair is not swift enough, the damaged base might be mispaired with an incorrect base during the next round of DNA replication. At the next round of cell division, when a daughter cell with such a mispaired base prepares to divide, the base complementary to the mispaired base will be added to the newly synthesized strand. This leads to a double-stranded mutation at the base-pair corresponding to the original damaged base [see this in the fantastic illustration shown in the editorial].

Standard practice for genome sequencing is to consider mutations — without paying attention to which of the strands was originally damaged. However, when a chemical change occurs that damages a base, creating a site referred to as a “lesion”, this lesion is on only one of the two DNA strands of the affected base-pair. Authors were clever enough to see that (because the ‘parental’ Watson and Crick strands of an original cell that underwent DNA damage are separated into different daughter cells) when the cell divides, two cell lineages can be tracked individually by following the unique pattern of mutations, on each of the parental strands, that the lesions generate.

Whole-genome sequencing (WGS) of 371 independently-evolved tumors from 104 C3H mice— revealed that each

genome had ~60,000 (approximately 13 per Mb) somatic point mutations (which is a level similar to that found in human cancers caused by exogenous mutagens — such as tobacco or UV exposure). [Insertion–deletion (indel) mutations and larger segmental changes were rare.] Point mutations were predominantly (76%) T→N, or their complement A→N, changes (where N represents any other nucleotide; this is consistent with the long-lived thymine adduct O4-ethyl-deoxythymidine being the principal mutagenic lesion). Known driver mutations were in the EGFR–RAS–RAF (epidermal growth factor receptor/GTPase-activating protein/proto-oncogene, serine-threonine kinase) pathway — and usually mutually exclusive; similar results were replicated in CAST mice.

Authors found that most mutagenic DNA lesions are not resolved into a mutated DNA base-pair during a single cell cycle. Instead — DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations — resulting in the

chromosome-scale phasing of subsequent mutations. Authors showed that DNA replication, across persisting lesions, can produce multiple alternative alleles in successive cell divisions, thereby generating both multi-allelic and combinatorial genetic diversity. The phasing of lesions — enabled the authors to accurately measure strand-biased repair processes,

quantifying oncogenic selection, and fine mapping of sister-chromatid-exchange events. Lastly, authors showed that, in human cells and human tumors as well, lesion segregation is a unifying property of exogenous mutagens — including UV light and chemotherapy agents. This study has profound implications for understanding the evolution and adaptation of cancer genomes. 😊

DwN

Nature 9 Jul 2020; 583: 256-270 & editorial pp 207-209

COMMENT:

Thank you, Dave. Pondering this question for a while, my first thought was to do the study in an established cell line in culture. Or freshly prepared trypsinized hepatocytes which are then grown in culture. However, I always worry about artifacts in “established cell lines” and when cells are placed in culture — even for very short periods of time.

Consequently, the best experiment I can think of — is to treat the intact mouse with whatever (e.g. dietary substance, chemotherapeutic drug, or DMN, etc.). Although “liver tumors” would arise many weeks after the tumor initiator is given, any initial “distinct tumor mutational pattern” is likely to appear within the first few days after the tumor initiator is given. Hence, one would need to sac mice at several time-points (e.g. 0, 12, 24, 48, etc. hrs after exposure to tumor initiator), isolate a substantial number of single hepatocytes, and perform whole-genome sequencing (WGS) on each single-cell sample (yes, this is not trivial; this would be a very expensive and time-consuming project). ☹ If anyone has a better idea, speak now or forever hold your peace. 😊

DwN

From: Dave
Sent: Friday, August 7, 2020 6:39 PM

Fascinating study — and an excellent summary of this very complex paper. Do you think it will be possible some day (maybe soon) to distinguish a tumor mutational pattern that would allow an assessment of a specific ‘environmental chemical’ contribution, separating this from a random ‘background’ mutation [e.g. mutations from dietary substances vs chemotherapeutic agents vs dimethylnitrosamine (DMN) mutations]?

David

This entry was posted in Center for Environmental Genetics. Bookmark the permalink.