The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphatase dephosphorylates PIP3, the lipid product of the class I PI 3-kinases, and suppresses the growth and proliferation of many cell types. It has been heavily studied, in large part due to its status as a tumour suppressor, the loss of function of which is observed through diverse mechanisms in many tumour types. Here we present a concise review of our understanding of the PTEN protein and highlight recent advances, particularly in our understanding of its localization and regulation by ubiquitination and SUMOylation.

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The lipid and protein tyrosine phosphatase, PTEN, is one of the most frequently mutated tumor suppressors in human cancers and is essential for regulating the oncogenic pro-survival PI3K/AKT signaling pathway. Because of its diverse physiological functions, PTEN has attracted great interest from researchers in multiple research fields. The functional diversity of PTEN demands a collection of delicate regulatory mechanisms, including transcriptional control and posttranslational mechanisms that include ubiquitination. Addition of ubiquitin to PTEN can have several effects on PTEN function, potentially regulating its stability, localization, and activity. In cell and in vitro ubiquitination assays are employed to study the ubiquitination-mediated regulation of PTEN. However, PTEN ubiquitination assays are challenging to perform and the data published from these assays has been of mixed quality. Here we describe protocols to detect PTEN ubiquitination in cultured cells expressing epitope tagged ubiquitin (in cell PTEN ubiquitination assay) and also using purified proteins (in vitro PTEN ubiquitination assay).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-κB; signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

Rearrangements of the actin cytoskeleton are regulated in part by dynamic localised activation and inactivation of Rho family small GTPases. SWAP70 binds to and activates the small GTPase RAC1 as well as binding to filamentous actin and PIP3. We have developed an encoded biosensor, which uses Forster resonance energy transfer to reveal conformational changes in SWAP70 in live cells. SWAP70 adopts a distinct conformation at the plasma membrane, which in migrating glioma cells is enriched at the leading edge but does not always associate with its PIP3‐dependent translocation to the membrane. This supports a role for SWAP70 in positive feedback activation of RAC1 at sites of filamentous actin, PIP3 and active RAC1.

Despite the vast increase in research activity in the coronavirus field over the past two years, researchers are still heavily reliant on non-human cells, for example Vero E6, highly heterogeneous or not fully differentiated cells, such as Calu-3, or not naturally susceptible cell lines overexpressing receptor ACE2 and other accessory factors, such as TMPRSS2. Complex cell models, such as primary cell-derived air-liquid interface epithelial models are highly representative of human tissues but are expensive and time-consuming to develop and maintain and have limited suitability for high-throughput analysis. In vitro investigations of host-pathogen interactions of viruses is highly reliant on suitable cell and tissue culture models and results are only as good as the model they have been validated in. Here, we show the use of a highly characterized human kidney cell line, Caki-1, for infection with three human coronaviruses: Betacoronaviruses severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and Alphacoronavirus human coronavirus 229E (hCoV-229E). Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cells lines for the cultivation of the respective virus. Furthermore, we used a panel of antibodies generated against 21 SARS-CoV-2-encoded proteins to identify their location in the infected Caki-1 cells using immunocytochemistry. Most importantly, Caki-1 cells are also susceptible to two other respiratory viruses, Influenza A virus and RSV, making them an ideal model for cross-comparison of not only a broad range of coronaviruses but respiratory viruses in general.

Loss PTEN function is one of the most common events driving aggressive prostate cancers and biochemically, PTEN is a lipid phosphatase which opposes the activation of the oncogenic PI3K-AKT signalling network. However, PTEN also has additional potential mechanisms of action, including protein phosphatase activity. Using a mutant enzyme, PTEN Y138L, which selectively lacks protein phosphatase activity, we characterised genetically modified mice lacking either the full function of PTEN in the prostate gland or only lacking protein phosphatase activity. The phenotypes of mice carrying a single allele of either wild-type Pten or PtenY138L in the prostate were similar, with common prostatic intraepithelial neoplasia (PIN) and similar gene expression profiles. However, the latter group, lacking PTEN protein phosphatase activity additionally showed lymphocyte infiltration around PIN and an increased immune cell gene expression signature. Prostate adenocarcinoma, elevated proliferation and AKT activation were only frequently observed when PTEN was fully deleted. We also identify a common gene expression signature of PTEN loss conserved in other studies (including Nkx3.1, Tnf and Cd44). We provide further insight into tumour development in the prostate driven by loss of PTEN function and show that PTEN protein phosphatase activity is not required for tumour suppression.

PTEN negatively regulates the Class I PI 3-kinases by metabolizing phosphatidylinositol(3,4,5)trisphosphate (PIP3) and acts as a tumor suppressor. Heterozygous germline PTEN mutations in humans manifest into a complex multiorgan disorder known as PTEN hamartoma tumor syndrome (PHTS). Patients present with hamartomatous skin lesions, benign GI polyps, have an increased risk of developing certain types of cancer (breast, colon, endometrium, thyroid, GI tract and kidney) and have macrocephaly and autism spectrum disorders (ASD). There is huge variability in disease symptoms in PHTS patients, the cause of which is unclear. It has been proposed that this correlates with the nature of the aberration in PTEN and its impact on PTEN expression and function, allowing to discriminate the following cancer-risk groups in PHTS patients: 1. High cancer risk: Patients expressing a catalytically inactive but stable PTEN protein (mis-sense PTEN mutations such as the C124S, G129R, R130Q etc.) have a severe phenotype, with benign tumors and aggressive malignant cancers at a young age of one of more tissues. 2. Medium risk: Patients with complete loss of PTEN expression due to insertions, deletions and truncations in the PTEN gene present with an intermediate severity with benign and malignant tumors. 3. Low risk: Patients with PTEN protein characteristics that do not fall into Group 1 or 2 but with some detectable suppression of AKT signaling. This covers a range of PTEN mutants, with often a mix of characteristics such as being unstable but active or even overactive, stable but partially inactive or no apparent activity defect. These patients have a mild phenotype, mainly related to a broad range of ASD symptoms such as macrocephaly, developmental delay and mental retardation. Some patients are severely autistic. Patients may have benign tumors and skin hamartomas, but malignant cancer is rare. Here we expand our analysis by characterizing a range of additional PHTS PTEN mutations using biochemical methods. Of the several mutations characterized, the R173C mutation leads to a PTEN enzyme that retains its activity but becomes partially unstable, putting it in Group 3. This mutation was of particular interest because the R173 site in PTEN is also the third most commonly somatically mutated PTEN site in cancer (COSMIC database; accessed September 2018). Mouse models such as the heterozygous PTEN heterozygous mice (PTEN +/- mice) and PTEN +/C124S represent Groups 1 and 2, and their characteristics coincide with the predicted phenotypes. However, no clinically relevant mouse models for the low-risk PHTS group have been reported. We have generated and characterized a PTEN +/R173C PHTS mouse model. We show that the PTEN +/R173C mice have macrocephaly, fewer tumors and a significantly longer tumor-free survival compared to the PTEN +/-, thus providing further evidence that the above-proposed PTEN genotype-phenotype correlation holds true in PHTS. Acknowledgment: This work has been supported by PTEN Research. Citation Format: Wayne Pearce, Nicoletta Kessaris, Nicholas R. Leslie, Bart Vanhaesebroeck, Priyanka Tibarewal, Gala Classen, Virginia A Garcia, Victoria Rathbone, Nisha Kriplani, Georgia Constantinou. Investigation of PTEN genotype-phenotype correlations in the PTEN hamartoma tumor syndrome (PHTS) using in vitro and in vivo approaches [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B22.