Potential biomarkers of DNA replication stress in cancer

Abstract

Oncogene activation is an established driver of tumorigenesis. An apparently inevitable consequence of oncogene activation is the generation of DNA replication stress (RS), a feature common to most cancer cells. RS, in turn, is a causal factor in the development of chromosome instability (CIN), a near universal feature of solid tumors. It is likely that CIN and RS are mutually reinforcing drivers that not only accelerate tumorigenesis, but also permit cancer cells to adapt to diverse and hostile environments. This article reviews the genetic changes present in cancer cells that influence oncogene-induced RS and CIN, with a particular emphasis on regions of the human genome that show enhanced sensitivity to the destabilizing effects of RS, such as common fragile sites. Because RS exists in a wide range of cancer types, we propose that the proteins involved counteracting this stress are potential biomarkers for indicating the degree of RS in cancer specimens. To test this hypothesis, we conducted a pilot study to validate whether some of proteins that are known from in vitro studies to play an essential role in the RS pathway could be suitable as a biomarker. Our results indicated that this is possible. With this review and pilot study, we aim to accelerate the development of a biomarker for analysis of RS in tumor biopsy specimens, which could ultimately help to stratify patients for different forms of therapy such as the RS inhibitors already undergoing clinical trials.

Keywords: Chromosome Section; MiDAS; cancer biomarker; chromosome instability; common fragile sites; replication stress.

Figure 1. Representative karyotypes of a virally transformed lymphocyte from a normal individual (GM06865) (A), and of an osteosarcoma cell line (U2OS) with CIN (B)

Unlike the diploid normal lymphocyte, the aneuploidy U2OS cell karyotype is highly abnormal with many chromosomes showing such an abnormal G-banding pattern that they could not be reliably assigned.

Figure 2. Representative IHC images of the expression of Ki-67, Cyclin E, POLD3, γH2AX, and FANCD2 in colon cancer FFPE specimens

Figure 3. Representative images of the cellular response to RS (induced by a low dose of hydroxyurea; HU) in U2OS cells, as determined by IF staining for γH2AX and FANCD2

A selected cell is defined by the yellow arrow, and is enlarged in the bottom panel. Scale bars are indicated.

Figure 4. A summary of the underlying mechanisms and detection of RS and CIN in cancer samples

Top panel: An illustration of increasing levels of RS and CIN during multi-step carcinogenesis using colorectal cancer as an example. During this process, multiple tumor suppressor genes (in green below the diagram) lose their function in the maintenance of genome stability, while multiple oncogenes (in red below the diagram) become activated and stimulate cells to proliferate. The RS and CIN thus formed become mutually reinforcing events that allow the cancer cells to ‘evolve’ and develop metastatic potential and drug resistance. Lower panel: A summary of the methods that can be applied to the detection of RS and CIN in laboratory or clinic settings. RS: replication stress. CIN: chromosome instability. UFB: ultra-fine anaphase DNA bridge. IF: immunofluorescence. IHC: Immunohistochemistry.