A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice, however until recently a major limitation to performing screens on this scale has been the cost effective isolation and sequencing of insertion sites. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA isolation, DNA digestion, linker or splinkerette ligation, primary and secondary PCR amplification, and sequencing. This method, which takes about 1 week to perform, has allowed us to isolate hundreds of thousands of insertion sites from mouse tumours and, unlike other methods, has been specifically optimised for the isolation of insertion sites generated with the murine leukaemia virus (MuLV), and can easily be performed in 96 well plate format for the efficient multiplex isolation of insertion sites

Correction:How the COVID-19 pandemic highlights the necessity of animal research (vol 30, pg R1014, 2020)

(Current Biology 30, R1014–R1018; September 21, 2020) As a result of an author oversight in the originally published version of this article, a number of errors were introduced in the author list and affiliations. First, the middle initials were omitted from the names of several authors. Second, the surname of Dr. van Dam was mistakenly written as “Dam.” Third, the first name of author Bernhard Englitz was misspelled as “Bernard” and the surname of author B.J.A. Pollux was misspelled as “Pullox.” Finally, Dr. Keijer's first name was abbreviated rather than written in full. These errors, as well as various errors in the author affiliations, have now been corrected online

Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis

The evolution of colorectal cancer suggests the involvement of many genes. We performed insertional mutagenesis with the Sleeping Beauty (SB) transposon system in mice carrying germline or somatic Apc mutation. Analysis of common insertion sites (CISs) isolated from 446 tumors revealed many hundreds of candidate cancer drivers. Comparison to human datasets suggested that 234 CIS genes are also deregulated in human colorectal cancers. 183 CIS genes are candidate Wnt targets, and 20 are shown to be novel modifiers of canonical Wnt signaling. We also identified gene mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included members of the FGF signaling cascade. Some 70 genes showed pairwise co-occurrence clustering into 38 sub-networks that may regulate tumor development

Selective resistance to the PARP inhibitor olaparib in a mouse model for BRCA1-deficient metaplastic breast cancer

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics

Strategies to decrease inequalities in cancer therapeutics, care and prevention: Proceedings on a conference organized by the Pontifical Academy of Sciences and the European Academy of Cancer Sciences, Vatican City, February 23–24, 2023

Analyses of inequalities related to prevention and cancer therapeutics/care show disparities between countries with different economic standing, and within countries with high Gross Domestic Product. The development of basic technological and biological research provides clinical and prevention opportunities that make their implementation into healthcare systems more complex, mainly due to the growth of Personalized/Precision Cancer Medicine (PCM). Initiatives like the USA-Cancer Moonshot and the EU-Mission on Cancer and Europe's Beating Cancer Plan are initiated to boost cancer prevention and therapeutics/care innovation and to mitigate present inequalities. The conference organized by the Pontifical Academy of Sciences in collaboration with the European Academy of Cancer Sciences discussed the inequality problem, dependent on the economic status of a country, the increasing demands for infrastructure supportive of innovative research and its implementation in healthcare and prevention programs. Establishing translational research defined as a coherent cancer research continuum is still a challenge. Research has to cover the entire continuum from basic to outcomes research for clinical and prevention modalities. Comprehensive Cancer Centres (CCCs) are of critical importance for integrating research innovations to preclinical and clinical research, as for ensuring state-of-the-art patient care within healthcare systems. International collaborative networks between CCCs are necessary to reach the critical mass of infrastructures and patients for PCM research, and for introducing prevention modalities and new treatments effectively. Outcomes and health economics research are required to assess the cost-effectiveness of new interventions, currently a missing element in the research portfolio. Data sharing and critical mass are essential for innovative research to develop PCM. Despite advances in cancer research, cancer incidence and prevalence is growing. Making cancer research infrastructures accessible for all patients, considering the increasing inequalities, requires science policy actions incentivizing research aimed at prevention and cancer therapeutics/care with an increased focus on patients' needs and cost-effective healthcare