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Cells are eliminated in a variety of physiological settings by apoptosis, a genetically encoded process of cellular suicide. Apoptosis comprises an intrinsic cellular defence against tumorigenesis, which, when suppressed, may contribute to the development of malignancies. The bcl-2 oncogene, which is activated in follicular lymphomas, functions as a potent suppressor of apoptosis under diverse conditions. Here we describe the complementary DNA cloning and functional analysis of a new Bcl-2 homologue, Bak, which promotes cell death and counteracts the protection from apoptosis provided by Bcl-2. Moreover, enforced expression of Bak induces rapid and extensive apoptosis of serum-deprived fibroblasts. This raises the possibility that Bak is directly involved in activating the cell death machinery.

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

Studies suggest that activation of phosphoinositide 3-kinaseAkt may protect against neuronal cell death in Alzheimer's disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho-Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated-Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated-Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3b Ser9 , tau Ser214 , mTOR Ser2448 , and decreased levels of the Akt target, p27 kip1 , were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated-Akt and PTEN-containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.

Using a series of insulin-like growth factor I (IGF-I) receptor mutants, we have attempted to define domains required for transmitting the antiapoptotic signal from the receptor and to compare these domains with those required for mitogenesis or transformation. In FL5.12 cells transfected with wild-type IGF-I receptors, IGF-I affords protection from interleukin 3 withdrawal but is not mitogenic. An IGF-I receptor lacking a functional ATP binding site provided no protection from apoptosis. However, receptors mutated at tyrosine residue 950 or in the tyrosine cluster (1131, 1135, and 1136) within the kinase domain remained capable of suppressing apoptosis, although such mutations are known to inactivate transforming and mitogenic functions. In the C terminus of the IGF-I receptor, two mutations, one at tyrosine 1251 and one which replaced residues histidine 1293 and lysine 1294, abolished the antiapoptotic function, whereas mutation of the four serines at 1280 to 1283 did not. Interestingly, receptors truncated at the C terminus had enhanced antiapoptotic function. In Rat-1/ c-MycER fibroblasts, the Y950F mutant and the tyrosine cluster mutant could still provide protection from c-Myc-induced apoptosis, whereas mutant Y1250/1251F could not. These studies demonstrate that the domains of the IGF-I receptor required for its antiapoptotic function are distinct from those required for its proliferation or transformation functions and suggest that domains of the receptor required for inhibition of apoptosis are necessary but not sufficient for transformation.

The contribution of the insulin-like growth-factor-I receptor (IGF-IR) to tumour progression is well documented. To identify new mediators of IGF-IR function in cancer, we recently isolated genes differentially expressed in cells overexpressing the IGF-IR. Among these was the serine/threonine kinase PBK/TOPK (PDZ-binding kinase/T-LAK cell-originated protein kinase), previously associated with highly proliferative cells and tissues. Here, we show that PBK is expressed at high levels in tumour cell lines compared with non-transformed cells. IGF-I could induce PBK expression only in transformed cells, whereas epidermal growth factor could induce PBK in non-transformed MCF-10A breast epithelial cells. Suppression of PBK expression using small interfering RNA did not prevent progression through the cell cycle, but caused decreased proliferation over time in culture, and reduced clonogenic growth in soft agarose. PBK knockdown impaired p38 activation after long-term stimulation with different growth factors and reduced DU145 cells motility. Suppressed PBK expression also resulted in an impaired response to DNA damage that was evident by the decreased generation of c-H2AX, increased DNA damage and decreased cell survival. Taken together, the data indicate that PBK is necessary for appropriate activation and function of the p38 pathway by growth factors. Thus, enhanced expression of PBK may facilitate tumour growth by mediating p38 activation and by helping cells to overcome DNA damage.

The insulin receptor and insulin-like growth factor 1 receptor (IGF-1R), activated by their ligands, control metabolism, cell survival, and proliferation. Although the signaling pathways activated by these receptors are well characterized, regulation of their activity is poorly understood. To identify regulatory proteins we undertook a two-hybrid screen using the IGF-1R ␤-chain as bait. This screen identified Receptor for Activated C Kinases (RACK1) as an IGF-1R-interacting protein. RACK1 also interacted with the IGF-1R in fibroblasts and MCF-7 cells and with endogenous insulin receptor in COS cells. Interaction with the IGF-1R did not require tyrosine kinase activity or receptor autophosphorylation but did require serine 1248 in the C terminus. Overexpression of RACK1 in either R؉ fibroblasts or MCF-7 cells inhibited IGF-1-induced phosphorylation of Akt, whereas it enhanced phosphorylation of Erks and Jnks. Src, the p85 subunit of phosphatidylinositol 3-kinase, and SHP-2 were all associated with RACK1 in these cells. Interestingly, the proliferation of MCF-7 cells was enhanced by overexpression of RACK1, whereas IGF-1-mediated protection from etoposide killing was greatly reduced. Altogether the data indicate that RACK1 is an IGF-1R-interacting protein that can modulate receptor signaling and suggest that RACK1 has a particular role in regulating Akt activation and cell survival.The insulin and IGF-1 1 receptors (IR and IGF-1R) belong to a family of tyrosine kinase receptors that also includes the insulin-related receptor. They are tetrameric receptors made up of two ␣-subunits that bind the ligands insulin, IGF-1 or IGF-2, and two ␤-subunits that share high homology in their kinase domains (reviewed in Ref. 1). These receptors are homologous to a receptor found in the nematode Caenorhabditis elegans and in Drosophila, and they activate an evolutionarily conserved metabolic and survival signaling pathway that includes insulin-related substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3-K), the serine/threonine kinase Akt, and the Forkhead family of transcription factors (2-5).

By comparing differential gene expression in the insulin-like growth factor (IGF)-IR null cell fibroblast cell line (R؊ cells) with cells overexpressing the IGF-IR (R؉ cells), we identified the INTRODUCTIONInsulin-like growth factor (IGF)-I and IGF-II are ligands for the widely expressed IGF-I receptor tyrosine kinase, which promotes mitogenesis and cell survival (reviewed in Adams et al., 2000). The IGF-IR is essential for normal growth during embryonic development and promotes cell survival and migration. Circulating IGFs and the IGF-IR signaling pathways also have been associated with cancer progression (reviewed in LeRoith and Roberts, 2003). In a mouse model of pancreatic islet cell tumorigenesis, endogenous IGF-IR expression was up-regulated at invasive regions of the tumors, and ectopic IGF-IR expression resulted in the accelerated development of highly invasive and metastatic carcinomas (Lopez and Hanahan, 2002). Conversely, the suppression of IGF-IR expression by antisense strategies (Resnicoff, 1998) or blocking antibodies results in decreased tumor growth and decreased metastatic capacity in tumor cell models (Maloney et al., 2003). Signals from the IGF-IR associated with survival, tumorigenicity, and metastasis are associated with the C terminus of the receptor (O'Connor et al., 1997;Brodt et al., 2001;Baserga et al., 2003).Cell migration and invasion are complex processes that require the coordination of signals from both adhesion and growth factor receptors. Signals from the IGF-IR can interact with those from integrins to initiate the formation of signaling complexes necessary for the formation and disassembly of cell adhesions with the extracellular matrix (ECM) (Doerr and Jones, 1996;Brooks et al., 1997). These signals involve enhancement of Shc phosphorylation (Mauro et al., 1999;Jackson et al., 2000;Kim et al., 2004), regulation of focal adhesion kinase phosphorylation at focal adhesions (Manes et al., 1999), differential regulation of signals by scaffolding proteins such as RACK1 (Hermanto et al., 2002;Kiely et al., 2002), signals from reorganization of the cytoskeleton (Casamassima and Rozengurt, 1998;Kim and Feldman, 1998;Guvakova et al., 2002), expression of angiogenic and invasive factors , regulation of cadherin location (Playford et al., 2000;Pennisi et al., 2002), and transactivation of the epidermal growth factor (EGF) receptor (Burgaud and Baserga, 1996;Roudabush et al., 2000). How all of these events are coordinated during cell migration or invasion, or how some of these signals are enhanced in metastatic cancer, is still poorly understood.IGF-I induces expression of several genes that promote cell migration and cancer progression, including ␤-catenin (Playford et al., 2000), the cadherin complex protein ZO-1 (Mauro et al., 2001), the angiogenic factor vascular endothelial growth factor (Miele et al., 2000), the metalloprotease MT1 MMP , and heparin-binding EGF-like growth factor (Mulligan et al., 2002 refractory to cellular transformation by several oncogenes (Sell et al., 1994), ...