Inhibitor of Apoptosis Proteins: Promising Targets for Cancer Therapy.

Cancer is a disease in which normal physiological processes are imbalanced, leading to tumour formation, metastasis and eventually death. Recent biological advances have led to the advent of targeted therapies to complement traditional chemotherapy and radiotherapy. However, a major problem still facing modern medicine is resistance to therapies, whether targeted or traditional. Therefore, to increase the survival rates of cancer patients, it is critical that we continue to identify molecular targets for therapeutic intervention. The Inhibitor of Apoptosis (IAP) proteins act downstream of a broad range of stimuli, such as cytokines and extracellular matrix interactions, to regulate cell survival, proliferation and migration. These processes are dysregulated during tumourigenesis and are critical to the metastatic spread of the disease. IAPs are commonly upregulated in cancer and have therefore become the focus of much research as both biomarkers and therapeutic targets. Here we discuss the roles that IAPs may play in cancer, and the potential benefits and pitfalls that targeting IAPs could have in the clinic.

Specific ß-containing integrins exert differential control on proliferation and two-dimensional collective cell migration in mammary epithelial cells.

Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the ß1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon ß1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of ß3-integrin in ß1-integrin-null cells, indicating that integrins containing different ß-subunits exert differential control on mammary epithelial proliferation and migration. ß1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in ß1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative ß1-integrin signals. These results show that ß1-integrins promote cell cycle in mammary epithelial cells, whereas ß3-integrins are involved in migration.

Analysis of inhibitor of apoptosis protein family expression during mammary gland development.

BACKGROUND: Inhibitors-of-Apoptosis-Proteins (IAPs) are an evolutionarily conserved family of proteins capable of regulating several facets of apoptosis. IAPs are frequently dysregulated in cancer, but their role in the regulation of apoptosis during developmental processes is not fully understood. Here we examined the expression of IAPs during the post-natal development of the mouse mammary gland, which is a tissue that exhibits a profound induction of apoptosis during involution. RESULTS: Six out of eight mammalian IAP family members are expressed in the mammary gland. Notably, quantitative PCR and immunoblotting revealed that XIAP, c-IAP1 and c-IAP2 are down-regulated in pregnancy and lactation, and prior to the onset of involution. In cultured mammary epithelial cells (MECs), XIAP levels decreased in response to inhibition of growth factor signalling. Maintaining XIAP levels in MECs by expressing exogenous XIAP protected them from all apoptotic stimuli tested. CONCLUSIONS: These data suggest that the developmental regulation of IAP expression in vivo contributes to naturally occurring programmes of cell death.

Role for X-linked Inhibitor of apoptosis protein upstream of mitochondrial permeabilization.

Apoptosis is controlled by a signaling equilibrium between prosurvival and proapoptotic pathways, such that unwanted apoptosis is avoided, but when required it occurs rapidly and efficiently. Many apoptosis regulators display dual roles, depending upon whether a cell has received an apoptotic stimulus or not. Here, we identify a novel and unexpected function for X-linked inhibitor of apoptosis (XIAP) that occurs when apoptosis is triggered under physiological conditions. We show that in response to loss of survival signals provided by cell adhesion, endogenous XIAP translocates from the cytosol into a mitochondrial 400-kDa complex and that this occurs very early in the apoptosis process. Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak. Thus, although XIAP suppresses apoptosis in healthy cells, our data indicate that XIAP may contribute to it in response to a proapoptotic signal such as loss of extracellular matrix-dependent survival signaling. We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified. Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

How adhesion signals reach a mitochondrial conclusion--ECM regulation of apoptosis.

A fundamental aspect in metazoans is the ability of a cell to recognise its positional context within a tissue. This is important in both development and homeostasis, where cell proliferation, differentiation and apoptosis are strictly controlled to form and maintain tissues. Much information has been generated on how cells receive and interpret adhesion-mediated signals. The non-receptor tyrosine kinase, Fak (focal adhesion kinase) has received much attention with regard to adhesion mediated signalling, including its role in survival. Survival signals are required to suppress the default pathway of apoptosis. The ultimate outcome of apoptotic signalling is the release of factors from the mitochondria into the cytosol. How the defined signalling pathways that control apoptosis converge on the mitochondria is an area with many unresolved questions.

Targeting inhibitor of apoptosis proteins in combination with ErbB antagonists in breast cancer.

INTRODUCTION: Inhibitor of apoptosis (IAPs) proteins are a family of proteins that can block apoptosis in normal cells and have been suggested to cause resistance to apoptosis in cancer. Overexpression of oncogenic receptor tyrosine kinases is common in breast cancer; in particular 20% of all cases show elevated Her2. Despite clinical success with the use of targeted therapies, such as Trastuzumab, only up to 35% of Her2-positive patients initially respond. We reasoned that IAP-mediated apoptosis resistance might contribute to this insensitivity to receptor tyrosine kinase therapy, in particular ErbB antagonists. Here we examine the levels of IAPs in breast cancer and evaluate whether targeting IAPs can enhance apoptosis in response to growth factor receptor antagonists and TRAIL. METHODS: IAP levels were examined in a breast cancer cell line panel and in patient samples. IAPs were inhibited using siRNA or cell permeable mimetics of endogenous inhibitors. Cells were then exposed to TRAIL, Trastuzumab, Lapatinib, or Gefitinib for 48 hours. Examining nuclear morphology and staining for cleaved caspase 3 was used to score apoptosis. Proliferation was examined by Ki67 staining. RESULTS: Four members of the IAP family, Survivin, XIAP, cIAP1 and cIAP2, were all expressed to varying extents in breast cancer cell lines or tumours. MDAMB468, BT474 and BT20 cells all expressed XIAP to varying extents. Depleting the cells of XIAP overcame the intrinsic resistance of BT20 and MDAMB468 cells to TRAIL. Moreover, siRNA-based depletion of XIAP or use of a Smac mimetic to target multiple IAPs increased apoptosis in response to the ErbB antagonists, Trastuzumab, Lapatinib or Gefitinib in Her2-overexpressing BT474 cells, or Gefitinib in EGFR-overexpressing MDAMB468 cells. CONCLUSIONS: The novel findings of this study are that multiple IAPs are concomitantly expressed in breast cancers, and that, in combination with clinically relevant Her2 treatments, IAP antagonists promote apoptosis and reduce the cell turnover index of breast cancers. We also show that combination therapy of IAP antagonists with some pro-apoptotic agents (for example, TRAIL) enhances apoptosis of breast cancer cells. In some cases (for example, MDAMB468 cells), the enhanced apoptosis is profound.

Are class II phosphoinositide 3-kinases potential targets for anticancer therapies?

Of the three classes of true phosphoinositide (PI) 3-kinases, the class II subdivision, which consists of three isoforms, PI3K-C2alpha, PI3K-C2beta and PI3K-C2gamma, is the least well understood. There are a number of reasons for this. This class of PI 3-kinase was identified exclusively by PCR and homology cloning approaches and not on the basis of cellular function. Like class I PI 3-kinases, class II PI 3-kinases are activated by diverse receptor types. To complicate the elucidation of class II PI 3-kinase function further, their in vitro substrate specificity is intermediate between the receptor activated class I PI 3-kinases and the housekeeping class III PI 3-kinase. The class II PI 3-kinases are inhibited by the two commonly used PI 3-kinase family selective inhibitors, wortmannin and LY294002, and there are no widely available, specific inhibitors for the individual classes or isoforms. Here the current state of understanding of class II PI 3-kinase function is reviewed, followed by an appraisal as to whether there is enough evidence to suggest that pharmaceutical companies, who are currently targeting the class I PI 3-kinases in an attempt to generate anticancer agents, should also consider targeting the class II PI 3-kinases.

Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration.

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2beta). Both PtdIns-3-P and PI3K-C2beta are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2beta as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2beta-PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.