Jon M. Askham

AKT1 mutations in bladder cancer: identification of a novel oncogenic mutation that can co-operate with E17K

The phosphatidylinositol-3-kinase (PI3 kinase)-AKT pathway is frequently activated in cancer. Recent reports have identified a transforming mutation of AKT1 in breast, colorectal, ovarian and lung cancers. We report here the occurrence of this mutation in bladder tumours. The AKT1 G49A (E17K) mutation was found in 2/44 (4.8%) bladder cancer cell lines and 5/184 (2.7%) bladder tumours. Cell lines expressing mutant AKT1 show constitutive AKT1 activation under conditions of growth factor withdrawal. We also detected a novel AKT1 mutation G145A (E49K). This mutation also enhances AKT activation and shows transforming activity in NIH3T3 cells, though activity is weaker than that of E17K. Enhanced activation of AKT1 when E17K and E49K mutations are in tandem suggests that they can co-operate.

MCAK associates with EB1

The microtubule (MT)-associated protein EB1 localizes to and promotes growth at MT plus ends. The MT depolymerizing kinesin MCAK has also been reported to track growing MT plus ends. Here, we confirm that human MCAK colocalizes with EB1 at growing MT ends when expressed as a GFP fusion protein in transfected cells. We show that MCAK associates with the C-terminus of EB1 and EB3 but much less efficiently with RP1. EB1 associates with the N-terminal localization and regulatory domain in MCAK but not with the motor domain of the protein. The interaction is competitive with the binding of other EB1 ligands and does not require MTs. Knockdown of EB1 expression using siRNA impaired the ability of GFP-MCAK to localize to MT tips in transfected cells. We propose that MCAK is targeted to growing MT ends by EB1, that MCAK is held in an inactive conformation when associated with EB1 and that this could provide the basis for a mechanism that facilitates rapid switching between phases of MT growth and depolymerization.

Bladder tumour-derived somatic TSC1 missense mutations cause loss of function via distinct mechanisms

More than 50% of transitional cell carcinomas of the bladder show loss of heterozygosity of a region spanning the TSC1 locus at 9q34 and mutations of TSC1 have been identified in 14.5% of tumours. These comprise nonsense mutations, splicing mutations, small deletions and missense mutations. Missense mutations are only rarely found in the germline in TSC disease. Therefore, we have examined six somatic missense mutations found in bladder cancer to determine whether these result in loss of function. We describe loss of function via distinct mechanisms. Five mutations caused mutually exclusive defects at mRNA and protein levels. Of these, two mutations caused pre-mRNA splicing errors that were predicted to result in premature protein truncation and three resulted in markedly reduced stability of exogenous TSC1 protein. Primary tumours with aberrant TSC1 pre-mRNA splicing were confirmed as negative for TSC1 expression by immunohistochemistry. Expression was also significantly reduced in a tumour with a TSC1 missense mutation resulting in diminished protein half-life. A single TSC1 missense mutation identified in a tumour with retained heterozygosity of the TSC1 region on chromosome 9 caused an apparently TSC2- and mTOR-independent localization defect of the mutant protein. We conclude that although TSC1 missense mutations do not play a major role in causation of TSC disease, they represent a significant proportion of somatic loss of function mutations in bladder cancer.

Examination of actin and microtubule dependent APC localisations in living mammalian cells

BackgroundThe trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented.ResultsUsing live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC.ConclusionOur data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer.

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.

EB1 immunofluorescence reveals an increase in growing astral microtubule length and number during anaphase in NRK-52E cells

Spindle positioning in animal cells is thought to rely upon the interaction of astral microtubules with the cell cortex. Information on the dynamics of astral microtubules during this process is scarce, in part because of the difficulty in visualising these microtubules by light microscopy. EB1 is a protein which specifically localises to growing microtubule distal tips. Immunostaining for EB1 therefore represents a powerful method for visualising the distribution of growing microtubule tips within cells. In this study we used EB1 immunostaining in mitotic NRK-52E cells to quantitatively analyse the length and number of growing astral microtubules during metaphase and anaphase. We observed a dramatic increase in growing astral microtubule length and number during anaphase. Furthermore, drug treatments which specifically destroyed astral microtubules resulted in an increase in misaligned anaphase but not metaphase spindles. We suggest that an anaphase-specific increase in growing astral microtubule length and number facilitates the maintenance of a correctly aligned spindle in mitotic NRK-52E cells.

EB1 is required for spindle symmetry in mammalian mitosis.

Most information about the roles of the adenomatous polyposis coli protein (APC) and its binding partner EB1 in mitotic cells has come from siRNA studies. These suggest functions in chromosomal segregation and spindle positioning whose loss might contribute to tumourigenesis in cancers initiated by APC mutation. However, siRNA-based approaches have drawbacks associated with the time taken to achieve significant expression knockdown and the pleiotropic effects of EB1 and APC gene knockdown. Here we describe the effects of microinjecting APC- or EB1- specific monoclonal antibodies and a dominant-negative EB1 protein fragment into mammalian mitotic cells. The phenotypes observed were consistent with the roles proposed for EB1 and APC in chromosomal segregation in previous work. However, EB1 antibody injection also revealed two novel mitotic phenotypes, anaphase-specific cortical blebbing and asymmetric spindle pole movement. The daughters of microinjected cells displayed inequalities in microtubule content, with the greatest differences seen in the products of mitoses that showed the severest asymmetry in spindle pole movement. Daughters that inherited the least mobile pole contained the fewest microtubules, consistent with a role for EB1 in processes that promote equality of astral microtubule function at both poles in a spindle. We propose that these novel phenotypes represent APC-independent roles for EB1 in spindle pole function and the regulation of cortical contractility in the later stages of mitosis. Our work confirms that EB1 and APC have important mitotic roles, the loss of which could contribute to CIN in colorectal tumour cells.

Phenotypic changes associated with DYNACTIN-2 (DCTN2) over expression characterise SJSA-1 osteosarcoma cells.

DYNACTIN‐2 (DCTN2) localises to chromosome 12q13‐q15, a region prone to stable amplification in several cancers. Transient DCTN2 overexpression has a significant impact on cellular phenotype primarily due to disruption of the DYNEIN‐dynactin motor. Changes reported include alterations of microtubule‐directed movement of molecular (e.g. TP53) and organelle (e.g. Golgi) cargoes towards the nucleus, centrosome biology, cellular movement and mitosis with a potential predisposition to mitotic block and polyploidy. These changes would be expected to be of relevance to carcinogenesis. To investigate this, we report the first study of DCTN2 genomic amplification and sustained DCTN2 overexpression in cancer cells. QFMPCR was employed to characterise the extent of chromosome 12q13‐q15 amplicons in SJSA‐1, SJRH30, U373MG and CCF‐STTG1 cancer cells. DCTN2 amplification was present in SJSA‐1, U373MG and SJRH30 cells, yet was incomplete at the 5′‐end in SJRH30 cells. Only SJSA‐1 cells were characterised by DCTN2 overexpression on Western blot analyses. Microscopy studies distinguished SJSA‐1 cells by greater DCTN2 immunofluorescence and diminished centrosome and 58K protein Golgi‐marker focus compared to SJRH30 cells. Indirect evidence derived from the published work of others indicated that TP53 transport into the nucleus was unimpaired. Furthermore, we observed that SJSA‐1 cells were easy to propagate. In conclusion, persistent DCTN2 overexpression can be tolerated in SJSA‐1 cancer cells despite phenotypic abnormalities predicted from transient overexpression studies. This preliminary study does not support a major role for DCTN2 overexpression in carcinogenesis, although further studies would be necessary to confirm this.

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