Malignant melanoma is frequently driven by mutational activation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) accompanied by silencing of the phosphatase and tensin homology (PTEN) tumor suppressor. Despite the implied importance of PI3K signaling in PTEN Null melanomas, mutational activation of the gene encoding the catalytic subunit of PI3Kα (PIK3CA), is rarely detected. Since PTEN has both PI3-lipid phosphatase-dependent and -independent tumor suppressor activities, we investigated the contribution of PI3K signaling to BRAF V600E -induced melanomagenesis using mouse models, cultured melanoma cells, and PI3K pathway-targeted inhibitors. These experiments revealed that mutationally activated PIK3CA H1047R cooperates with BRAF V600E for melanomagenesis in mice. Moreover, pharmacological inhibition of PI3Ks prevented growth of BRAF V600E /PTEN Null melanomas in vivo and in tissue culture. Combined inhibition of BRAF V600E and PI3K had more potent effects on the regression of established BRAF V600E /PTEN Null melanomas and cultured melanoma cells than individual blockade of either pathway. Surprisingly, growth of BRAF V600E /PIK3CA H1047R melanomas was dependent on the protein kinase AKT; however, AKT inhibition had no effect on growth of BRAF V600E /PTEN Null melanomas. These data indicate that PTEN silencing contributes a PI3K-dependent, but AKT-independent, function in melanomagenesis. Our findings enhance our knowledge of how BRAF V600E and PI3K signaling cooperate in melanomagenesis and provide preclinical validation for combined pathway-targeted inhibition of PI3K and BRAF V600E in the therapeutic management of BRAF V600E /PTEN Null melanomas.

Phosphatidylinositide 3′ (PI3′)-lipid signaling cooperates with oncogenic BRAFV600E to promote melanomagenesis. Sustained PI3′-lipid production commonly occurs via silencing of the PI3′-lipid phosphatase PTEN or, less commonly, through mutational activation of PIK3CA, encoding the 110kDa catalytic subunit of PI3′-kinase-α (PI3Kα). To define the PI3K catalytic isoform dependency of BRAF-mutated melanoma, we utilized pharmacologic, isoform-selective PI3K inhibitors in conjunction with melanoma-derived cell lines and genetically engineered mouse (GEM) models. While BRAFV600E/PIK3CAH1047R melanomas were sensitive to the anti-proliferative effects of selective PI3Kα blockade, inhibition of BRAFV600E/PTENNull melanoma proliferation required combined blockade of PI3Kα, δ and γ, and was insensitive to PI3Kβ blockade. In GEM models, isoform-selective PI3K inhibition elicited cytostatic effects, but significantly potentiated melanoma regression in response to BRAFV600E pathway-targeted inhibition. Interestingly, PI3K inhibition forestalled the onset of MEK inhibitor resistance in two independent GEM models of BRAFV600E-driven melanoma. These results suggest that combination therapy with PI3K inhibitors may be a useful strategy to extend the duration of clinical response of BRAF-mutated melanoma patients to BRAFV600E pathway-targeted therapies. (Words: 165)

Mutation or loss of the genes PTEN and KRAS have been implicated in human colorectal cancer (CRC), and have been shown to co-occur despite both playing a role in the PI3' kinase (PI3'K) pathway. We investigated the role of these genes in intestinal tumour progression in vivo, using genetically engineered mouse models, with the aim of generating more representative models of human CRC. Intestinal-specific deletion of Pten and activation of an oncogenic allele of Kras was induced in wild-type (WT) mice and mice with a predisposition to adenoma development (Apc(fl/+) ). The animals were euthanized when they became symptomatic of a high tumour burden. Histopathological examination of the tissues was carried out, and immunohistochemistry used to characterize signalling pathway activation. Mutation of Pten and Kras resulted in a significant life-span reduction of mice predisposed to adenomas. Invasive adenocarcinoma was observed in these animals, with evidence of activation of the PI3'K pathway but no metastasis. However, mutation of Pten and Kras in WT animals not predisposed to adenomas led to perturbed homeostasis of the intestinal epithelium and the development of hyperplastic polyps, dysplastic sessile serrated adenomas and metastasizing adenocarcinomas with serrated features. These studies demonstrate synergism between Pten and Kras mutations in intestinal tumour progression, in an autochthonous and immunocompetent murine model, with potential application to preclinical drug testing. In particular, they show that Pten and Kras mutations alone predispose mice to the spectrum of serrated lesions that reflect the serrated pathway of CRC progression in humans.

Cowden syndrome (CS) is a rare autosomal dominant cancer-prone disorder caused by germ-line mutation of the phosphatase and tensin homolog mutated on chromosome 10 (PTEN) tumor-suppressor gene. Affected patients commonly develop juvenile polyps, and show an elevated risk of developing colorectal cancers. The etiology of these peculiar polyps remains unclear, although previous work has suggested somatic PTEN alterations in the stroma of juvenile polyps. After a long latency period, we find epithelial-specific PTEN deletion to cause formation of juvenile polyps in the colorectum without stromal PTEN loss. More important, we find that these lesions closely recapitulate all of the characteristic histopathological features of juvenile polyps seen in patients with CS, including stromal alterations and dysplastic transformation to colorectal carcinoma. The stromal alterations we identify after epithelial-specific PTEN loss suggest that PTEN may be involved in altered epithelial-mesenchymal cross talk, which, in turn, predisposes to colorectal neoplasia and polyposis. Our transgenic model is the first to recapitulate colorectal juvenile polyposis in patients with CS. We conclude that stromal PTEN loss is not a prerequisite for the formation of juvenile polyps, and that colorectal juvenile polyps in CS are bona fide neoplastic precursor lesions.

Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH)2D3, the active form of vitamin D. CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ (phosphatase and tensin homolog, cell division cycle 42 and protein kinase C zeta). Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted “Swiss cheese-like” cribriform morphology (CM) comprising multiple abnormal “back to back” lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked 1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors.

Over the past 20 years, huge advances have been made in modelling human diseases such as cancer using genetically modified mice. Accurate in vivo models are essential to examine the complex interaction between cancer cells, surrounding stromal cells, tumour associated inflammatory cells, fibroblast and blood vessels, and to recapitulate all the steps involved in metastasis. Elucidating these interactions in vitro has inherent limitations, and thus animal models are a powerful tool to enable researchers to gain insight into the complex interactions between signalling pathways and different cells types. This review will focus on how advances in in vivo models have shed light on many aspects of cancer biology including the identification of oncogenes, tumour suppressors and stem cells, epigenetics, cell death and context dependent cell signalling. SummaryThis review highlights the achievements of the late Professor Alan Clarke in developing the first knockout mice and sets his work in the general context of the scientific field he contributed to, namely using mouse models to understand cancer biology.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

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