Transcriptomic analysis of the tumour vasculature and its clinical relevance

The vasculature of many solid tumours is highly distinct from that of its host tissue, both structurally and in terms of functional protein expression. These differences offer an opportunity for specific targeting of therapeutics against the tumour vasculature. This thesis describes the investigation of this tumour vascular profile, in clinical samples from renal cell carcinoma, colorectal cancer and colorectal liver metastases, as well as murine breast tumours resistant to the antiangiogenic drug, sunitinib. This analysis allowed the identification of three tumour endothelial markers in renal and colorectal malignancies, MCAM, LAMA4 and GRIN2D. The expression of each of these markers was linked with patient survival with these malignancies, suggesting their utility for prognostication. MCAM and GRIN2D showed highly tumour specific expression profiles in multi-organ tissue array analysis, highlighting them as promising candidates for the targeting of therapies to the tumour vasculature. The specific localisation of monoclonal anti-MCAM antibodies to renal tumour vasculature was demonstrated, further supporting this suggestion. Putative vascular markers of tumour resistance to antiangiogenic therapy were also identified. Aquaporin-1 (AQP1) was found to be up-regulated in cases of acquired resistance, mammalian target of rapamycin (mTOR) in innate resistance and pleiotrophin (PTN) in both, highlighting their potential as diagnostic candidates for predicting therapy response, or as targets to circumvent resistance. The need for effective diagnostic tests in this indication, was demonstrated by the finding that metastasis is enhanced by sunitinib therapy, in innately resistant tumours

Sunitinib treatment enhances metastasis of innately drug resistant breast tumors

Anti-angiogenic therapies have failed to confer survival benefits in patients with metastatic breast cancer (mBC). However, to date there has not been an inquiry into roles for acquired versus innate drug resistance in this setting. In this study, we report roles for these distinct phenotypes in determining therapeutic response in a murine model of mBC resistance to the anti-angiogenic tyrosine kinase inhibitor sunitinib. Using tumor measurement and vascular patterning approaches, we differentiated tumors displaying innate versus acquired resistance. Bioluminescent imaging of tumor metastases to the liver, lungs and spleen revealed that sunitinib administration enhances metastasis, but only in tumors displaying innate resistance to therapy. Transcriptomic analysis of tumors displaying acquired versus innate resistance allowed the identification of specific biomarkers, many of which have a role in angiogenesis. In particular, aquaporin-1 upregulation occurred in acquired resistance, mTOR in innate resistance, and pleiotrophin in both settings, suggesting their utility as candidate diagnostics to predict drug response or to design tactics to circumvent resistance. Our results unravel specific features of antiangiogenic resistance, with potential therapeutic implications

Supporting the uptake of low cost resilience: Final report (FD2682)

Executive SummaryThe Defra research project FD2682 examined the technical, social and behavioural aspects of supporting low cost flood repairable measures designed to limit damage to buildings during and after flood events. Flood repairable measures (sometimes called ‘flood resilient measures’) applied to buildings are designed to limit damage, or speed up recovery where water has entered a property. They include strategies to keep water away from building elements (such as raising power sockets) and the internal use of waterproof or water resistant materials, including those capable of retaining their integrity and recovering quickly after inundation. These measures have traditionally been regarded as most useful when water exclusion approaches(measures to keep water out of the building, sometimes called ‘resistant measures’)are not practical or cost effective.The investigation took an action research approach, consulting widely and reflecting on findings on an ongoing basis. The research comprised the following stages:1. A rapid evidence assessment (REA) including a review of relevant academic and grey literature; consultation with a panel of experts; interviews with flood reinstatement and property protection professionals; and interviews with occupants of properties where flood repairable measures have been adopted.2. An assessment of the costs and benefits of selected low cost flood repairable measures, and illustrative packages of measures.3. A demonstration project to explore innovative approaches that could be used by local agencies and businesses to address some of the barriers to the use of flood repairable measures. This made use of a co-design process, via the formation of the Tewkesbury ‘Learning and Action Alliance’ (LAA).The REA concluded that (in contrast to previous perceptions of repairable measures as a last resort for properties at highest risk) low cost repairable measures are widely applicable as part of an integrated approach to limiting the residual risk to individual properties that may also include water exclusion measures. Interviews as part of theREA showed repairability to be a pragmatic approach that can be applied incrementally at various windows of opportunity with lower financial barriers to implementation than alternative strategies. The assessment of costs and benefits of selected low cost flood repairable measures, and illustrative packages of measures, confirmed their potential cost effectiveness in limiting flood damage.The REA concluded that the weight of evidence supports the effectiveness of an ever expanding list of low cost resilience measures in limiting flood damage.However, there are also major gaps in evidence, and in communication and sharing of available evidence, reducing the confidence in implementation of measures within relevant trades and professionals, as well as by owners and occupiers directly. Key areas in urgent need of additional scientific evidence include: the implications ofdebris and contaminants in floodwater; the effect of hydrodynamic and hydrostatic pressure on ‘waterproof’ materials; and durability of resilient measures afterprolonged flood exposure. However, attention should also be directed towards further understanding the real performance of flood repairable measures in a variety of types of building before, during and after flooding.The REA and demonstration project both concluded that, in order for the potential benefits of repairable measures to be realised in practice, there will need to be a shiftin the repair and reinstatement process. Improved protocols (and incentives) are required that include clarity regarding the autonomy and responsibility of differentactors within the repair process to recommend adoption of repairable measures. The inception of Flood Re offers both a challenge and an opportunity in this regard. The research finds that there could be benefits to placing the specification of negligible cost and cost neutral measures within the professional remit of surveyors and contractors on the ground. To support this, improved technical guidance and training is needed to raise levels of understanding and awareness within the industry. The surveyors’ checklist, designed within the project, was seen as a useful contribution to this requirement. Improved confidence in appropriate measures could also be fostered through provision of exemplars and factsheets.The REA and demonstration project highlighted the potential importance of other windows of opportunity (outside the recovery period) in the take up of low cost floodrepairable measures. Insurance renewal and property transfer represent opportunities to raise awareness of measures at very low cost with minimal upskillingof professionals and may provide direct triggers to action. Other building work and redecoration opportunities are harder to target in terms of awareness raising,therefore a well-informed and up-skilled local ‘property support network’ (PSN) is needed, in order to spot opportunities to support uptake on an individual basis.Evaluation of the demonstration project innovations indicated that implementation was most successful in those innovations driven by members of the LAA, or hadsignificant input from members of the local PSN. Increased awareness of low cost flood resilience measures amongst LAA members was also achieved. Therefore the LAA model was seen as a potential platform to engage relevant local propertyexperts and agencies, and to empower them to encourage property level approaches.However, the REA evidence and that from the LAA meetings together with the evaluation of the surveyor’s checklist suggest that emotional barriers to implementation of low cost resilience are important. Use of repairable measures is a difficult concept, as it requires an acceptance that water might enter the property (home or business) and changes within the living space that might feel abnormal.Interviews with practitioner experts, together with an assessment of current regulations, suggest that making small adjustments to building regulations, relevantto passive avoidance and resilience, could aid normalisation of such measures. A greater focus on design and aesthetics aspects, and clearer guidance on the ways todeal with perceived contamination is also seen as important by professionals, the PSN and in the demonstration project. Finally, a wider framing of property level flooddamage reduction, with suggested schemes including both water entry and water exclusion measures was indicated by the interviews with homeowners and professionals and discussed by the LAA as helpful in addressing emotional barriers

Proline-Rich Homeodomain protein (PRH/HHEX) is a suppressor of breast tumour growth

Breast tumours progress from hyperplasia to ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). PRH/HHEX (Proline Rich Homeodomain/Haematopoietically expressed homeobox) is a transcription factor that displays both tumour suppressor and oncogenic activity in different disease contexts however, the role of PRH in breast cancer is poorly understood. Here we show that nuclear localisation of the PRH protein is decreased in DCIS and IBC compared to normal breast. Our previous work has shown that PRH phosphorylation by protein kinase CK2 prevents PRH from binding to DNA and regulating the transcription of multiple genes encoding growth factors and growth factor receptors. Here we show that transcriptionally inactive phosphorylated PRH is elevated in DCIS and IBC compared to normal breast. To determine the consequences of PRH loss of function in breast cancer cells we generated inducible PRH depletion in MCF-7 cells. We show that PRH depletion results in increased MCF-7 cell proliferation in part at least due to increased vascular endothelial growth factor signaling. Moreover we demonstrate that PRH depletion increases the formation of breast cancer cells with cancer stem cell-like properties. Finally, and in keeping with these findings, we show that PRH over-expression inhibits the growth of mammary tumours in mice. Collectively these data indicate that PRH plays a tumour suppressive role in the breast and they provide an explanation for the finding that low PRH mRNA levels are associated with a poor prognosis in breast cancer

Intra-promoter switch of transcription initiation sites in proliferation signaling-dependent RNA metabolism

Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5' terminal oligopyrimidine (5'TOP) sequence, dramatically increasing the range of genes potentially subjected to 5'TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K-Akt-mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.</p

Dual-initiation promoters with intertwined canonical and TCT/TOP transcription start sites diversify transcript processing

Variations in transcription start site (TSS) selection reflect diversity of preinitiation complexes and can impact on post-transcriptional RNA fates. Most metazoan polymerase II-transcribed genes carry canonical initiation with pyrimidine/purine (YR) dinucleotide, while translation machinery-associated genes carry polypyrimidine initiator (5’-TOP or TCT). By addressing the developmental regulation of TSS selection in zebrafish we uncovered a class of dual-initiation promoters in thousands of genes, including snoRNA host genes. 5’-TOP/TCT initiation is intertwined with canonical initiation and used divergently in hundreds of dual-initiation promoters during maternal to zygotic transition. Dual-initiation in snoRNA host genes selectively generates host and snoRNA with often different spatio-temporal expression. Dual-initiation promoters are pervasive in human and fruit fly, reflecting evolutionary conservation. We propose that dual-initiation on shared promoters represents a composite promoter architecture, which can function both coordinately and divergently to diversify RNAs

MCAM and LAMA4 are highly enriched in tumor blood vessels of renal cell carcinoma and predict patient outcome

The structure and molecular signature of tumor-associated vasculature are distinct from those of the host tissue, offering an opportunity to selectively target the tumor blood vessels. To identify tumor-specific endothelial markers, we performed a microarray on tumor-associated and nonmalignant endothelium collected from patients with renal cell carcinoma (RCC), colorectal carcinoma (CRC), or colorectal liver metastasis (CRM). We identified a panel of genes consistently upregulated by tumor blood vessels of which melanoma cell adhesion molecule (MCAM) and its extracellular matrix interaction partner laminin alpha 4 (LAMA4) emerged as the most consistently expressed genes. This result was subsequently confirmed by immunohistochemical analysis of MCAM and LAMA4 expression in RCC and CRC blood vessels. Strong MCAM and LAMA4 expression was also shown to predict poor survival in RCC, but not in CRC. Notably, MCAM and LAMA4 were enhanced in locally advanced tumors as well as both the primary tumor and secondary metastases. Expression analysis in 18 different cancers and matched healthy tissues revealed vascular MCAM as highly specific in RCC, where it was induced strongly by VEGF, which is highly abundant in this disease. Lastly, MCAM monoclonal antibodies specifically localized to vessels in a murine model of RCC, offering an opportunity for endothelial-specific targeting of anticancer agents. Overall, our findings highlight MCAM and LAMA4 as prime candidates for RCC prognosis and therapeutic targeting

Suboptimal SARS-CoV-2-specific CD8+ T cell response associated with the prominent HLA-A*02:01 phenotype

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2−specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269–277 and A2/Orf1ab3183–3191. Using peptide−HLA tetramer enrichment, direct ex vivo assessment of A2/S269+CD8+ and A2/Orf1ab3183+CD8+ populations indicated that A2/S269+CD8+ T cells were detected at comparable frequencies (∼1.3 × 10−5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10−6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein–Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10−4) populations. Phenotyping A2/S269+CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269+CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269+CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19