Wills, J. | bioRxiv Science

bioRxiv2018

Molecular changes during extended neoadjuvant letrozole treatment of breast cancer: distinguishing acquired resistance from dormant tumours

BackgroundThe risk of recurrence for endocrine-treated breast cancer patients persists for many years or even decades following surgery and apparently successful adjuvant therapy. This period of dormancy and acquired resistance is inherently difficult to investigate, previous efforts have been limited to in vitro or in vivo approaches. In this study, sequential tumour samples from patients receiving extended neoadjuvant endocrine treatment were characterised as a novel clinical model.\n\nMethodsConsecutive tumour samples from 62 patients undergoing extended (4-45 months) neoadjuvant aromatase inhibitor, letrozole, therapy were subjected to transcriptomic and proteomic analysis, representing before ([≤]0), early-on (13-120 days) and long-term (>120 days) neoadjuvant letrozole treatment. Patients with at least a 40% initial reduction in tumour size by 4 months of treatment were included. Of these, 42 patients with no subsequent progression were classified as \"dormant\", and the remaining 20 patients as \"acquired resistant\".\n\nResultsChanges in gene expression in dormant tumours begin early and become more pronounced at later timepoints. Therapy-induced changes in resistant tumours were common features of treatment, rather than being specific to resistant phenotype. Comparative analysis of long-term treated dormant and resistant tumours highlighted changes in epigenetics pathways including DNA methylation and histone acetylation. DNA methylation marks 5-methylcytosine and 5-hydroxymethylcytosine were significantly reduced in resistant tumours compared to dormant tissues after extended letrozole treatment.\n\nConclusionsThis is the first patient-matched gene expression study investigating long-term aromatase inhibitor-induced dormancy and acquired resistance in breast cancer. Dormant tumours continue to change during treatment whereas acquired resistant tumours more closely resemble their diagnostic samples. Global loss of DNA methylation was observed in resistant tumours under extended treatment. Epigenetic alterations may lead to escape from dormancy and drive acquired resistance in a subset of patients supporting a potential role for therapy targeted at these epigenetic alterations in the management of endocrine resistant breast cancer.

cancer biology↗

bioRxiv2018

ITPase Deficiency Causes Martsolf Syndrome With a Lethal Infantile Dilated Cardiomyopathy

Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Through genetic analysis of 85 unrelated \"mutation negative\" probands referred with Martsolf syndrome we identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). The probands reported here each presented with a lethal and highly distinctive disorder; Martsolf syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rl/dl) into RNA and DNA. In Itpa-null cells, dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA but this was not associated with detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in lymphoblastoid RNA from an affected individual. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA - and by implication rI production - correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.\n\nAuthor SummaryNucleotide triphosphate bases containing inosine, ITP and dITP, are continually produced within the cell as a consequence of various essential biosynthetic reactions. The enzyme inosine triphosphate pyrophosphatase (ITPase) scavenges ITP and dITP to prevent their incorporation into RNA and DNA. Here we describe two unrelated families with complete loss of ITPase function as a consequence of disruptive mutations affecting both alleles of ITPA, the gene that encodes this protein. Both of the families have a very distinctive and severe combination of clinical problems, most notably a failure of heart muscle that was lethal in infancy or early childhood. They also have features of another rare genetic disorder affecting the brain and the eyes called Martsolf syndrome. We could not detect any evidence of dITP accumulation in genomic DNA from the affected individuals. A low but detectable level of inosine was present in the mitochondrial DNA but this did not have any obvious detrimental effect. The inosine accumulation in RNA was detectable in the patient cells. We made both cellular and animal models that were completely deficient in ITPase. Using these reagents we could show that the highest level of inosine accumulation into RNA was seen in the embryonic mouse heart. In this tissue more than 1 in 400 bases in all RNA in the cell was inosine. In normal tissues inosine is almost undetectable using very sensitive assays. The inosine accumulation did not seem to be having a global effect on the balance of RNA molecules or proteins.

genetics↗