SnapShot: Tumor evolution

Understanding how tumors grow and evolve over time is crucial to help shed light on the underlying reasons why treatments fail and tumors metastasize. This SnapShot provides a brief introduction into the main concepts of tumor evolution

The effect of age on the acquisition and selection of cancer driver mutations in sun-exposed normal skin

BACKGROUND: The accumulation of somatic mutations contributes to ageing and cancer. Sunlight is the principal aetiological factor associated with skin cancer development. However, genetic and phenotypic factors also contribute to skin cancer risk. This study aimed at exploring the role of photoaging, as well as other well-known epidemiological risk factors, in the accumulation of somatic mutations in cancer-free human epidermis. MATERIAL AND METHODS: We deeply sequenced 46 genes in normal skin biopsies from 127 healthy donors, from which phenotypic data (including age, pigmentation-related genotype and phenotype) and sun exposure habits were collected. We determined the somatic mutational burden, mutational signatures, clonal selection and frequency of driver mutations in all samples. RESULTS: Our results reveal an exponential accumulation of UV-related somatic mutations with age, matching skin cancer incidence. The increase of mutational burden is in turn modified by an individual's skin phototype. Somatic mutations preferentially accumulated in cutaneous squamous cell carcinoma (cSCC) cancer genes and clonally expanded with age, with distinct mutational processes underpinning different age groups. Our results suggest loss of fidelity in transcription-coupled repair later in life. CONCLUSION: Our findings reveal that aging is not only associated with an exponential increase in the number of somatic mutations accumulated in normal epidermis, but also with selection and expansion of cancer-associated mutations. Aged, sun-exposed normal skin is thus an extended mosaic of multiple clones with driver mutations, poised for the acquisition of transforming events

DNA replication stress mediates APOBEC3 family mutagenesis in breast cancer

BACKGROUND: The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. RESULTS: HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo. HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo. Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro. APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. CONCLUSION: These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically

Clinical aspects of short-chain acyl-CoA dehydrogenase deficiency

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation. SCADD is biochemically characterized by increased C4-carnitine in plasma and ethylmalonic acid in urine. The diagnosis of SCADD is confirmed by DNA analysis showing SCAD gene mutations and/or variants. SCAD gene variants are present in homozygous form in approximately 6% of the general population and considered to confer susceptibility to development of clinical disease. Clinically, SCADD generally appears to present early in life and to be most frequently associated with developmental delay, hypotonia, epilepsy, behavioral disorders, and hypoglycemia. However, these symptoms often ameliorate and even disappear spontaneously during follow-up and were found to be unrelated to the SCAD genotype. In addition, in some cases, symptoms initially attributed to SCADD could later be explained by other causes. Finally, SCADD relatives of SCADD patients as well as almost all SCADD individuals diagnosed by neonatal screening remained asymptomatic during follow-up. This potential lack of clinical consequences of SCADD has several implications. First, the diagnosis of SCADD should never preclude extension of the diagnostic workup for other potential causes of the observed symptoms. Second, patients and parents should be clearly informed about the potential lack of relevance of the disorder to avoid unfounded anxiety. Furthermore, to date, SCADD is not an optimal candidate for inclusion in newborn screening programs. More studies are needed to fully establish the relevance of SCADD and solve the question as to whether SCADD is involved in a multifactorial disease or represents a nondisease

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast DCIS, and in pre-invasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx pre-invasive to invasive NSCLC lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models, revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in pre-invasive disease, providing fuel for selection early in cancer evolution

ELECTROENCEPHALOGRAPHY (EEG) AND ITS USE IN MOTOR LEARNING AND CONTROL

Electroencephalography (EEG) is a non-invasive technique of measuring electric currents generated from active brain regions and is a useful tool for researchers interested in motor control. The study of motor learning and control seeks to understand the way the brain understands, plans and executes movement both physical and imagined. Thus, the purpose of this study was to better understand the ways in which electroencephalography can be used to measure regions of the brain involved with motor control and learning. For this purpose, two independent studies were completed using EEG to monitor brain activity during both executed and imagined actions. The first study sought to understand the cognitive demand of altering a running gait and provides EEG evidence of motor learning. 13 young healthy runners participated in a 6-week in-field gait-retraining program that altered running gait by increasing step rate (steps per minute) by 5-10%. EEG was collected while participants ran on a treadmill with their original gait as a baseline measurement. After the baseline collection, participants ran for one minute at the same speed with a 5-10% step rate increase while EEG was collected. Participants then participated in a 6-week in-field gait-retraining program in which they received bandwith feedback while running in order to learn the new gait. After completing the 6-week training protocol, participants returned to the lab for post training EEG collection while running with the new step rate. Power spectral density plots were generated to measure frequency band power in all gait-retraining phases. Results in the right prefrontal cortex showed a significant increase in beta (13-30 Hz) while initially running with the new gait compared to the baseline step rate. Previous work suggests the right prefrontal cortex is involved with the inhibition of a previously learned behavior and thus, our results suggest an increase in cognitive load to inhibit the previous full stride motion. After training, this increase in beta over the right prefrontal cortex decreased, suggesting motor adaptations had occurred as a result of motor learning. These results give promising evidence for a new method of ensuring permanent changes in performance that will benefit rehabilitation and athletic performance training programs. The second study in this project sought to understand differences in right and left-handers as they mentally simulate movement. 24 right and left-handed individuals (12 right-handers, 12 left-handers) were shown pictures of individual hands on a screen while EEG was collected. Previous research has shown than while solving this task, participants mentally rotate a mental representation of their own hand to determine the handedness of the image. Event-related potential results showed that right-handers had an earlier and greater activation in the parietal regions than left-handers, whereas left-handers had a later and greater activation in the motor related brain regions compared to right-handers. These results suggest differing strategies while mentally solving motor related tasks between right and left-handers. We speculate this is a result of left-handers' need to adapt to a majorly right-hand dominant environment. Both these studies show the benefits of using EEG to understand the motor system in physically executed and imagined actions

Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths1. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relaps

The evolution of lung cancer and impact of subclonal selection in TRACERx

Lung cancer is the leading cause of cancer-associated mortality worldwide1. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource