Ribosomal DNA copy number loss and sequence variation in cancer

Abstract

Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

Human rDNA Copy Number Is Unstable in Metastatic Breast Cancers

“…Instead, we find these data most easy to reconcile as a result of destabilization of repeat copy number in general. This assertion, and our data, are consistent with those recently published by Xu and colleagues (Xu et al 2017), and by Wang and Lemos (Wang and Lemos 2017), although both of those studies analyze their data in the context of a cancer adaptive phenotype for rDNA copy number changes. These researchers showed that in tumors or samples derived from leukemias and lymphomas, medulloblastomas, osteosarcomas, and esophageal adenocarcinomas, although the average rDNA copy number was reduced, there were clear cases of individuals with increased copy numbers.…”

“…In breast cancer, de novo mutations and copy number variations are known to exist but have been difficult to quantify or monitor because of the heterogeneity of typical tumors in situ, thus much of the mutation and copy number mutational analyses have been investigated using cell cultures which can be made clonal and grown to large numbers (Xu et al 2017). Studies investigating copy number changes in cancer have so-far analyzed such changes in the context of adaptive advantage by the cancer phenotype Wang and Lemos 2017), but find them to be small and variable in scope, and without any phenotypic consequence.…”

“…The large variation in natural and presumably-healthy human rDNA copy number (Long and Dawid 1980) also raises the possibility that the "aberrant" copy numbers in these cell lines are merely captured isolates from within natural human population variation ). Although copy number variation analysis is progressing with the advent of new low-copynumber sequencing technologies (Xu et al 2017), repetitious regions of the genome remain difficult to analyze. Repeated DNAs including the rDNA continue to be under-reported in databases or under-investigated in the literature, either because they do not have a tradition of being considered as mutagenic or capable or regulatory function, or because they are refractory to sequencing technologies and assemblies.…”

Nucleolus and chromatin

“…Indeed, both of these conditions were identified in different human cancer types (Udugama et al 2018; Wang and Lemos 2017; Xu et al 2017). Increased rRNA transcription is a ubiquitous and well-known hallmark of cancer cells and the resulting increased nucleolar sizes were used as diagnostic marker by pathologists.…”

Promotion of hyperthermic-induced rDNA hypercondensation inSaccharomyces cerevisiae

“…In yeast, changes in rDNA homeostasis impacts cellular aging and replicative lifespan in which extrachromosomal rDNA circles (ERCs), that arise through recombination, deplete the remaining genome of critical regulatory factors (Lewinska et al, 2014; Park et al, 1999; Sinclair and Guarente, 1997; Sinclair et al, 1997; Shcheprova et al, 2008). Clinically, disruption of rDNA function in humans results in neurodegeneration, tumorigenesis and severe developmental defects that include Treacher-Collins Syndrome, Blackfan Anemia, CHARGE Syndrome and several others (Danilova and Gazda, 2015; Udugama et al, 2018; Wang and Lemos, 2017; Xu et al, 2014; Xu et al, 2017; Hallgren et al, 2014). Given that a rather surprisingly small percentage of nucleolar proteins function in ribosome biogenesis (Tiku and Antebi, 2018; Schöfer and Weipoltshammer, 2018), it becomes critical to explore the regulatory mechanisms through which rDNA responds to the many challenges imposed on the cell to ensure proper development and cell cycle regulation.…”