Rebecca L. Ross

Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical signal transduction pathway that regulates multiple cellular functions. Aberrant activation of this pathway has been identified in a wide range of cancers. Several pathway components including AKT, PI3K and mTOR represent potential therapeutic targets and many small molecule inhibitors are in development or early clinical trials. The complex regulation of the pathway, together with the multiple mechanisms by which it can be activated, make this a highly challenging pathway to target. For successful inhibition, detailed molecular information on individual tumours will be required and it is already clear that different tumour types show distinct combinations of alterations. Recent results have identified alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer, some of which are significantly related to tumour phenotype and clinical behaviour. Co-existence of alterations to several PI3K pathway genes in some bladder tumours indicates that these proteins may have functions that are not related solely to the known canonical pathway.

PIK3CA mutation spectrum in urothelial carcinoma reflects cell context-dependent signaling and phenotypic outputs

Although activating mutations of PIK3CA are frequent in urothelial carcinoma (UC), no information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum, which has a large excess of helical domain mutations. We investigated the phenotypic and signaling consequences of hotspot and UC-specific rare PIK3CA mutations in immortalized normal human urothelial cells (NHUC) and mouse fibroblasts (NIH3T3). Our results indicate that in NHUC, rare mutant forms and all three hotspot mutant forms of PIK3CA can activate the PI3K/AKT pathway. The relative frequency at which helical domain and kinase domain mutations are found in UC is related to their potency in inducing signaling downstream of AKT and to the phenotypic effects induced in this cell type (E545K>E542K>H1047R). Helical domain mutations E542K and E545K conferred a significant proliferative advantage at confluence and under conditions of nutrient depletion, and increased cellular resistance to anoikis. Both helical and kinase domain mutants induced increased NHUC cell motility and migration towards a chemoattractant, though no significant differences were found between the mutant forms. In NIH3T3 cells, the kinase domain mutant H1047R induced high levels of AKT activation, but helical domain mutants were significantly less potent and this was reflected in their relative abilities to confer anchorage-independent growth. Our findings indicate that the effects of mutant PIK3CA are both cell type- and mutation-specific. Helical domain mutations in PIK3CA may confer a selective advantage in the urothelium in vivo by overcoming normal contact-mediated inhibitory signals and allowing proliferation in nutrient-limiting conditions. Mutant forms of PIK3CA may also stimulate intraepithelial cell movement, which could contribute to spread of cells within the urothelium.

Affimer proteins are versatile and renewable affinity reagents

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001

NG2 proteoglycan as a pericyte target for anticancer therapy by tumor vessel infarction with retargeted tissue factor

tTF-TAA and tTF-LTL are fusion proteins consisting of the extracellular domain of tissue factor (TF) and the peptides TAASGVRSMH and LTLRWVGLMS, respectively. These peptides represent ligands of NG2, a surface proteoglycan expressed on angiogenic pericytes and some tumor cells. Here we have expressed the model compound tTF-NGR, tTF-TAA, and tTF-LTL with different lengths in the TF domain in E. coli and used these fusion proteins for functional studies in anticancer therapy. We aimed to retarget TF to tumor vessels leading to tumor vessel infarction with two barriers of selectivity, a) the leaky endothelial lining in tumor vessels with the target NG2 being expressed on pericytes on the abluminal side of the endothelial cell barrier and b) the preferential expression of NG2 on angiogenic vessels such as in tumors. Chromatography-purified tTF-TAA showed identical Factor X (FX)-activating procoagulatory activity as the model compound tTF-NGR with Km values of approx. 0.15 nM in Michaelis-Menten kinetics. The procoagulatory activity of tTF-LTL varied with the chosen length of the TF part of the fusion protein. Flow cytometry revealed specific binding of tTF-TAA to NG2-expressing pericytes and tumor cells with low affinity and dissociation KD in the high nM range. In vivo and ex vivo fluorescence imaging of tumor xenograft-carrying animals and of the explanted tumors showed reduction of tumor blood flow upon tTF-TAA application. Therapeutic experiments showed a reproducible antitumor activity of tTF-TAA against NG2-expressing A549-tumor xenografts, however, with a rather small therapeutic window (active/toxic dose in mg/kg body weight).

Identification of Mutations in Distinct Regions of p85 Alpha in Urothelial Cancer

Bladder cancers commonly show genetic aberrations in the phosphatidylinositol 3-kinase signaling pathway. Here we have screened for mutations in PIK3R1, which encodes p85α, one of the regulatory subunits of PI3K. Two hundred and sixty-four bladder tumours and 41 bladder tumour cell lines were screened and 18 mutations were detected. Thirteen mutations were in C-terminal domains and are predicted to interfere with the interaction between p85α and p110α. Five mutations were in the BH domain of PIK3R1. This region has been implicated in p110α-independent roles of p85α, such as binding to and altering the activities of PTEN, Rab4 and Rab5. Expression of these mutant BH-p85α forms in mouse embryonic fibroblasts with p85α knockout indicated that all forms, except the truncation mutants, could bind and stabilize p110α but did not increase AKT phosphorylation, suggesting that BH mutations function independently of p110α. In a panel of 44 bladder tumour cell lines, 80% had reduced PIK3R1 mRNA expression relative to normal urothelial cells. This, along with mutation of PIK3R1, may alter BH domain functioning. Our findings suggest that mutant forms of p85α may play an oncogenic role in bladder cancer, not only via loss of ability to regulate p110α but also via altered function of the BH domain.

Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

Deregulation of proliferation and differentiation-dependent signalling pathways is a hallmark of human papillomavirus (HPV) infection. Although the manipulation of these pathways by E6 and E7 has been extensively studied, controversies surround the role of the E5 oncoprotein during a productive virus life cycle. By integrating primary keratinocytes harbouring wild type or E5 knockout HPV18 genomes with pharmacological and gain/loss of function models, this study aimed to provide molecular information about the role of E5 in epithelial proliferation and differentiation. We show that E5 contributes to cell cycle progression and unscheduled host DNA synthesis in differentiating keratinocytes. E5 function correlates with increased EGFR activation in differentiating cells and blockade of this pathway impairs differentiation-dependent cell cycle progression of HPV18 containing cells. Our findings provide a functional requirement of enhanced EGFR signalling for suprabasal cellular DNA synthesis during the virus life cycle. They also reveal an unrecognised contribution of E5 towards the impaired keratinocyte differentiation observed during a productive HPV infection. E5 suppresses a signalling axis consisting of the keratinocyte growth factor receptor (KGFR) pathway. Inhibition of this pathway compensates for the loss of E5 in knockout cells and re-instates the delay in differentiation. The negative regulation of KGFR involves suppression by the EGFR pathway. Thus our data reveal an unappreciated role for E5-mediated EGFR signalling in orchestrating the balance between proliferation and differentiation in suprabasal cells.

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma

BackgroundMany urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases.MethodsWe used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway.ResultsSpecific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance.ConclusionsMutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.

Transforming Growth Factor β Activation Primes Canonical Wnt Signaling Through Down-Regulation of Axin-2

Aberrant activation of Wnt signaling has been observed in tissues from patients with systemic sclerosis (SSc). This study aimed to determine the role of transforming growth factor β (TGFβ) in driving the increased Wnt signaling, through modulation of axis inhibition protein 2 (Axin‐2), a critical regulator of the Wnt canonical pathway.

Abstract LB-152: PIK3CA mutation spectrum in urothelial carcinoma reflects cell context-dependent signalling and phenotypic outputs

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Activating p110α mutations are frequently observed in urothelial carcinoma (UC). Their specific effects in urothelial cells and the reason for the large excess of helical domain mutations relative to kinase domain mutations have not been studied. Thus we investigated the phenotypic consequences of hotspot and UC-specific rare PIK3CA mutations in immortalized normal human urothelial cells (NHUC) and mouse fibroblasts (NIH3T3). Our results show that in NHUC, all PIK3CA mutants activate the AKT pathway. The potency of AKT downstream activation by helical and kinase domain mutants was related to the frequency of mutation occurrence in UC (E545K>E542K>H1047R). In NIH3T3, the H1047R kinase domain mutant induced significantly higher levels of AKT activation relative to helical domain mutant forms and this was reflected in their abilities to promote anchorage-independent growth. In NHUC, only E542K and E545K helical domain mutant forms conferred a significant proliferative advantage at confluence and in nutrient limiting conditions. Both types of mutant forms induced NHUC cell motility, though no significant differences were found between helical and kinase domain mutant forms. Our findings indicate that the cellular consequences of mutant PIK3CA are both cell type- and mutation-specific. This provides a rationale for the observed PIK3CA mutation spectrum in UC and suggests that helical domain mutations may confer a selective advantage in the urothelium in vivo by overcoming normal contact-mediated inhibitory signals and allowing nutrient-independent proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-152. doi:1538-7445.AM2012-LB-152