Biology subjects

Coombes, C.

Publications and source records attributed to Coombes, C..

6 recordsLinked to original sources

bioRxiv2023

Circulating neutrophils from patients with early breast cancer have distinct subtype-dependent phenotypes

PurposeA high number of circulating neutrophils is a poor prognostic factor for breast cancer, where evidence of bone marrow cancer-dependent priming is found. However, how early this priming is detectable remains unclear. Patients and MethodsHere, we investigate changes in circulating neutrophils from newly diagnosed breast cancer patients before any therapeutic interventions. To do this, we assessed their lifespan and their broader intracellular kinase network activation states by using the Pamgene Kinome assay which measures the activity of neutrophil kinases. ResultsWe found sub-type specific L-selectin (CD62L) changes in circulating neutrophils as well as perturbations in their overall global kinase activity. Strikingly, breast cancer patients of different subtypes (HR+, HER2+, triple negative) exhibited distinct neutrophil kinase activity patterns indicating that quantifiable perturbations can be detected in circulating neutrophils from early breast cancer patients, that are sensitive to both hormonal and HER-2 status. We also detected an increase in neutrophils lifespan in cancer patients, independently of tumour subtype. ConclusionsOur results suggest that the tumour-specific kinase activation patterns in circulating neutrophils may be used in conjunction with other markers to identify patients with cancer from those harbouring only benign lesions of the breast. Given the important role neutrophil in breast cancer progression, the significance of this sub-type of specific priming warrants further investigation. Clinical RelevanceThe current study aims to investigate cancer-specific changes in circulating neutrophils in patients with newly diagnosed early breast cancer before any therapeutic intervention. We found L-selectin (CD62L) changes in circulating neutrophils from patients with early-stage breast cancer compared to healthy volunteers, which is an indication of an early phenotypical change. Moreover, these changes in CD62L were dependent on the breast cancer sub-type, showing opposing trends according to the hormonal receptor status of the tumour. Importantly, this subtype dependent phenotypic alteration was reflected in broader intracellular signalling perturbation when measuring intracellular kinase activity. Moreover, those cancer perturbed neutrophils, show expanded life span when cultured ex vivo, suggesting an alteration in their physiologic state. The tumour-specific kinase activation patterns in circulating neutrophils may be useful in conjunction with other markers to distinguish patients with cancer from those with benign lesions of the breast.

cancer biology↗

bioRxiv2022

A comparison between Mini-loop mediated isothermal amplification and polymerase spiral reaction for selective amplification of short template DNA

Isothermal amplification of circulating tumour-derived DNA (ctDNA) in the blood plasma could provide a rapid and cost effective alternative to PCR and NGS approaches for real-time cancer monitoring. Several variations of isothermal technologies exist, typically designed over unconstrained template length. Here, we compared the amplification efficiency of a compact loop mediated isothermal amplification reaction (termed AS-Mini-LAMP) with polymerase spiral reaction (PSR) suitable for analysis of ctDNA. Utilising 4-primers and targeting a 155 bp template encompassing the estrogen receptor (ESR1) c.1138G>C (p.E380Q) missense mutation. Using synthetic E380Q template DNA and Bst2.0 polymerase, results demonstrate that AS-Mini-LAMP was capable of selective mutant allele DNA amplification to a limit of 1,000 mutant copies, whereas no specific amplification was observed by PSR. The alternative use of Bst3.0 polymerase for either AS-Mini-LAMP or PSR revealed non-canonical events that underpin potentially misleading results when employing isothermal chemistries. In conclusion, AS-Mini-LAMP is more suited to mutation detection than PSR.

molecular biology↗

bioRxiv2020

VGLL1-directed TEAD activation drives endocrine therapy resistance in estrogen receptor positive breast cancer

Resistance to endocrine therapies (ET) is common in estrogen receptor (ER) positive breast cancer and most relapsed patients die with ET-resistant disease. While genetic mutations provide explanations for some relapsed patients1, mechanisms of resistance remain undefined in many cases. Drug-induced epigenetic reprogramming provides possible routes to resistance2. By analysing histone H3 lysine 27 acetylation (H3K27ac) profiles in models of ET resistance, we discovered that selective ER down-regulators (SERDs) such as fulvestrant promote epigenetic activation of VGLL1, a co-activator for TEAD transcription factors. We show that VGLL1, acting via TEADs, promotes expression of genes that drive growth of fulvestrant-resistant breast cancer cells. Pharmacological disruption of VGLL1/TEAD4 interaction inhibits VGLL1/TEAD transcriptional programmes to block growth of the resistant cells and prevents growth. Among the VGLL1/TEAD-regulated genes, we identify EGFR, whereby VGLL1-directed EGFR upregulation sensitises fulvestrant-resistant breast cancer cells to EGFR inhibitors. Taken together, our findings identify VGLL1 as a transcriptional driver in ET resistance and advance new therapeutic possibilities for relapsed ER+ breast cancer patients.

cancer biology↗

bioRxiv2020

UNC-45A Destabilizes MTs Independent of its Effect on Non-Muscle Myosin II

In invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only and UNC-45A, expressed in all cells and implicated in regulating both Non-Muscle Myosin II (NMII)- and microtubule (MT)-associated functions. Here we show for the first time that: a) in vitro UNC-45A binds to the MT lattice and weakens its integrity leading to MT bending, breakage and depolymerization, b) in cells, UNC-45A overexpression causes loss of MT mass and increase in MT breakages, c) both in vitro and in cells, UNC-45A destabilizes MTs independent of its NMII C-terminal binding domain and destabilization occurs even in presence of the NMII inhibitor blebbistatin. These findings are consistent with a not mutually exclusive but rather dual role of UNC-45A in regulating NMII activity and MT stability. Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability our findings have profound implications in both, the biology of MTs as well as the biology of human diseases and possible therapeutic implications for their treatment.

cell biology↗

bioRxiv2019

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a [~]6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the conversion of growing microtubule ends from a tapered into a blunt configuration resulted in reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

cell biology↗

bioRxiv2017

Enhancers mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer.

The degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumour evolution is poorly understood. Phenotypic divergence can be achieved via the inheritance of alternative transcriptional programs1,2. Cell-type specific transcription is maintained through the activation of epigenetically-defined regulatory regions including promoters and enhancers1,3,4. In this work, we annotated the epigenome of 47 primary and metastatic oestrogen-receptor (ER)-positive breast cancer specimens from clinical samples, and developed strategies to deduce phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Highly shared regions contain a unique set of regulatory information including the motif for the transcription factor YY1. In vitro work shows that YY1 is essential for ER transcriptional activity and defines the critical subset of functional ER binding sites driving tumor growth in most luminal patients. YY1 also control the expression of genes that mediate resistance to endocrine treatment. Finally, we show that H3K27ac levels at active enhancer elements can be used as a surrogate of intra-tumor phenotypic heterogeneity, and to track expansion and contraction of phenotypic subpopulations throughout breast cancer progression. Tracking YY1 and SLC9A3R1 positive clones in primary and metastatic lesions, we show that endocrine therapies drive the expansion of phenotypic clones originally underrepresented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.

cancer biology↗