Trends in International Cancer Research Investment 2006-2018.

The International Cancer Research Partnership (ICRP) is an active network of cancer research funding organizations, sharing information about funded research projects in a common database. Data are publicly available to enable the cancer research community to find potential collaborators and avoid duplication. This study presents an aggregated analysis of projects funded by 120 partner organizations and institutes in 2006-2018, to highlight trends in cancer research funding. Overall, the partners' funding for cancer research increased from $5.562 billion (bn) US dollars (USD) in 2006 to $8.511bn USD in 2018, an above-inflation increase in funding. Analysis by the main research focus of projects using Common Scientific Outline categories showed that Treatment was the largest investment category in 2018, followed by Early Detection, Diagnosis, and Prognosis; Cancer Biology; Etiology; Control, Survivorship, and Outcomes; and Prevention. Over the 13 years covered by this analysis, research funding into Treatment and Early Detection, Diagnosis, and Prognosis had increased in terms of absolute investment and as a proportion of the portfolio. Research funding in Cancer Biology and Etiology declined as a percentage of the portfolio, and funding for Prevention and Control, Survivorship and Outcomes remained static. In terms of cancer site-specific research, funding for breast cancer and colorectal cancer had increased in absolute terms but declined as a percentage of the portfolio. By contrast, investment for brain cancer, lung cancer, leukemia, melanoma, and pancreatic cancer increased both in absolute terms and as a percentage of the portfolio.

A new role for Hedgehogs in juxtacrine signaling.

The Hedgehog pathway plays important roles in embryonic development, adult stem cell maintenance and tumorigenesis. In mammals these effects are mediated by Sonic, Desert and Indian Hedgehog (Shh, Dhh and Ihh). Shh undergoes autocatalytic cleavage and dual lipidation prior to secretion and forming a response gradient. Post-translational processing and secretion of Dhh and Ihh ligands has not previously been investigated. This study reports on the synthesis, processing, secretion and signaling activities of SHH, IHH and DHH preproteins expressed in cultured cells, providing unexpected evidence that DHH does not undergo substantial autoprocessing or secretion, and does not function in paracrine signaling. Rather, DHH functions as a juxtacrine signaling ligand to activate a cell contact-mediated HH signaling response, consistent with its localised signaling in vivo. Further, the LnCAP prostate cancer cell, when induced to express endogenous DHH and SHH, is active only in juxtacrine signaling. Domain swap studies reveal that the C-terminal domain of HH regulates its processing and secretion. These findings establish a new regulatory role for HHs in cell-mediated juxtacrine signaling in development and cancer.

DUOX enzyme activity promotes AKT signalling in prostate cancer cells.

Reactive oxygen species (ROS) and oxidative stress are related to tumour progression, and high levels of ROS have been observed in prostate tumours compared to normal prostate. ROS can positively influence AKT signalling and thereby promote cell survival. The aim of this project was to establish whether the ROS generated in prostate cancer cells positively regulate AKT signalling and enable resistance to apoptotic stimuli. In PC3 cells, dual oxidase (DUOX) enzymes actively generate ROS, which inactivate phosphatases, thereby maintaining AKT phosphorylation. Inhibition of DUOX by diphenylene iodium (DPI), intracellular calcium chelation and small-interfering RNA (siRNA) resulted in lower ROS levels, lower AKT and glycogen synthase kinase 3ß (GSK3ß) phosphorylation, as well as reduced cell viability and increased susceptibility to apoptosis stimulating fragment (FAS) induced apoptosis. This report shows that ROS levels in PC3 cells are constitutively maintained by DUOX enzymes, and these ROS positively regulate AKT signalling through inactivating phosphatases, leading to increased resistance to apoptosis.

Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2 transcripts.

BACKGROUND: Genetic screening of breast cancer patients and their families have identified a number of variants of unknown clinical significance in the breast cancer susceptibility genes, BRCA1 and BRCA2. Evaluation of such unclassified variants may be assisted by web-based bioinformatic prediction tools, although accurate prediction of aberrant splicing by unclassified variants affecting exonic splice enhancers (ESEs) remains a challenge. METHODS: This study used a combination of RT-PCR analysis and splicing reporter minigene assays to assess five unclassified variants in the BRCA2 gene that we had previously predicted to disrupt an ESE using bioinformatic approaches. RESULTS: Analysis of BRCA2 c.8308 G > A (p.Ala2770Thr) by mRNA analysis, and BRCA2 c.8962A > G (p.Ser2988Gly), BRCA2 c.8972G > A (p.Arg2991His), BRCA2 c.9172A > G (p.Ser3058Gly), and BRCA2 c.9213G > T (p.Glu3071Asp) by a minigene assay, revealed no evidence for aberrant splicing. CONCLUSIONS: These results illustrate the need for improved methods for predicting functional ESEs and the potential consequences of sequence variants contained therein.

Identification and functional analysis of novel BRCA1 transcripts, including mouse Brca1-Iris and human pseudo-BRCA1.

Recent characterization of the mammalian transcriptome has confirmed its predicted complexity, with many loci encoding multiple splice variants and pseudogenes. The breast cancer susceptibility gene BRCA1 is a tumour suppressor gene that produces multiple functional transcripts. For example, BRCA1-IRIS is a splice variant of BRCA1, which encodes a protein that is functionally distinct from BRCA1. Here we describe the identification of ten novel Brca1 splice variants including Brca1-Iris, the mouse orthologue of human BRCA1-IRIS. We show that Brca1-Iris is differentially expressed during mammary epithelial differentiation and regulates survival of mammary epithelial cells. Another transcript, Brca1-Delta22, expressed in both mouse and human cells, was found to be defective in transcriptional activation capacity. Finally, we show that the human BRCA1 pseudogene produces a spliced pseudoBRCA1 transcript. The identification of these transcripts has implications for the understanding of the role of BRCA1 in biology and disease and for the interpretation of mouse knockout models.

Deregulation of cell death (apoptosis): implications for tumor development.

The first descriptions of apoptosis were made over 150 years ago, although the implications for tumor development were not appreciated until the 1970s. Natural cell death is a critical part of development of multicellular organisms, and also counter-balances the cell generating effects of mitosis. Disruptions in the highly regulated apoptotic pathway can lead to disease, such as tumors, due to the accumulation of excessive numbers of cells. Restoring normal apoptosis in cancer cells is one of the current challenges of cancer research.

Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide.

Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H(2)O(2), can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non-mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H(2)O(2). This study demonstrated that 661W cells, a retina-derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well-established regulators. The roles of Nox2 and Nox4 in producing pro-survival H(2)O(2) were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time-lapse imaging of dichlorofluorescein fluorescence (generated upon H(2)O(2) production) in retinal explants was established and it showed that explants also produce a burst of H(2)O(2). The increase in H(2)O(2) production was partly blocked by an inhibitor of Nox proteins. Overall, this study demonstrates a pro-survival role of Nox2 and Nox4 in retina-derived cells, elucidates some of the regulatory mechanisms and reveals that a similar phenomenon exists in retinal tissue as a whole.

Colocalisation of predicted exonic splicing enhancers in BRCA2 with reported sequence variants.

Disruption of the breast cancer susceptibility gene BRCA2 is associated with increased risk of developing breast and ovarian cancer. Over 1800 sequence changes in BRCA2 have been reported, although for many the pathogenicity is unclear. Classifying these changes remains a challenge, as they may disrupt regulatory sequences as well as the primary protein coding sequence. Sequence changes located in the splice site consensus sequences often disrupt splicing, however sequence changes located within exons are also able to alter splicing patterns. Unfortunately, the presence of these exonic splicing enhancers (ESEs) and the functional effect of variants within ESEs it is currently difficult to predict. We have previously developed a method of predicting which sequence changes within exons are likely to affect splicing, using BRCA1 as an example. In this paper, we have predicted ESEs in BRCA2 using the web-based tool ESEfinder and incorporated the same series of filters (increased threshold, 125 nt limit and evolutionary conservation of the motif) in order to identify predicted ESEs that are more likely to be functional. Initially 1114 ESEs were predicted for BRCA2, however after all the filters were included, this figure was reduced to 31, 3% of the original number of predicted ESEs. Reported unclassified sequence variants in BRCA2 were found to colocalise to 55% (17/31) of these conserved ESEs, while polymorphisms colocalised to 0 of the conserved ESEs. In summary, we have identified a subset of unclassified sequence variants in BRCA2 that may adversely affect splicing and thereby contribute to BRCA2 disruption.

Pre-mRNA splicing aberrations and cancer.

Splicing requires the accurate recognition of exonic sequences from the surrounding thousands of nucleotides of intronic sequence and is achieved by the coordinate interplay of splicing regulatory elements in genes and the trans-acting RNA and protein molecules to which they bind. Infidelity in this process can have dramatic consequences for protein production, with an errors resulting in mRNA instability or the production of aberrant protein products. It is therefore not surprising that disruptions of splicing processes have been associated with a wide range of diseases, including cancer. This review looks at some of the mechanisms that regulate splicing and how disruption of such mechanisms can contribute to cancer susceptibility and progression.