Anthony J. Valentijn

Activation of BAD by Therapeutic Inhibition of Epidermal Growth Factor Receptor and Transactivation by Insulin-like Growth Factor Receptor

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.

Spatial and temporal changes in Bax subcellular localization during anoikis

Bax, a member of the Bcl-2 family, translocates to mitochondria during apoptosis, where it forms oligomers which are thought to release apoptogenic factors such as cytochrome c. Using anoikis as a model system, we have examined spatial and temporal changes in Bax distribution. Bax translocates to mitochondria within 15 min of detaching cells from extracellular matrix, but mitochondrial permeabilization does not occur for a number of hours. The formation of Bax oligomers and perimitochondrial clusters occurs concomitant with caspase activation and loss of mitochondrial membrane potential, before nuclear condensation. Cells can be rescued from apoptosis if they are replated onto extracellular matrix within an hour, whereas cells detached for longer could not. The loss of ability to rescue cells from anoikis occurs after Bax translocation, but before the formation of clusters and cytochrome c release. Our data suggest that Bax regulation occurs at several levels, with formation of clusters a late event, and with critical changes determining cell fate occurring earlier.

The N-terminal conformation of Bax regulates cell commitment to apoptosis

The Bcl-2 protein Bax normally resides in the cytosol, but during apoptosis it translocates to mitochondria where it is responsible for releasing apoptogenic factors. Using anoikis as a model, we have shown that Bax translocation does not commit cells to apoptosis, and they can be rescued by reattachment to extracellular matrix within a specific time. Bax undergoes an N-terminal conformational change during apoptosis that has been suggested to regulate conversion from its benign, cytosolic form to the active, membrane bound pore. We now show that the Bax N-terminus regulates commitment and mitochondrial permeabilisation, but not the translocation to mitochondria. We identify Proline 13 within the N-terminus of Bax as critical for this regulation. The subcellular distribution of Proline 13 mutant Bax was identical to wild-type Bax in both healthy and apoptotic cells. However, Proline 13 mutant Bax induced rapid progression to commitment, mitochondrial permeabilisation and death. Our data identify changes in Bax controlling commitment to apoptosis that are mechanistically distinct from those controlling its subcellular localisation. Together, they indicate that multiple regulatory steps are required to activate the proapoptotic function of Bax.

A role for the cytoskeleton in prolactin-dependent mammary epithelial cell differentiation.

The function of exocrine glands depends on signals within the extracellular environment. In the mammary gland, integrin-mediated adhesion to the extracellular matrix protein laminin co-operates with soluble factors such as prolactin to regulate tissue-specific gene expression. The mechanism of matrix and prolactin crosstalk and the activation of downstream signals are not fully understood. Because integrins organize the cytoskeleton, we analysed the contribution of the cytoskeleton to prolactin receptor activation and the resultant stimulation of milk protein gene expression. We show that the proximal signalling events initiated by prolactin (i.e. tyrosine phosphorylation of receptor and the associated kinase Jak2) do not depend on an intact actin cytoskeleton. However, actin networks and microtubules are both necessary for continued mammary cell differentiation, because cytoskeletal integrity is required to transduce the signals between prolactin receptor and Stat5, a transcription factor necessary for milk protein gene transcription. The two different cytoskeletal scaffolds regulate prolactin signalling through separate mechanisms that are specific to cellular differentiation but do not affect the general profile of protein synthesis.

Apoptosis commitment and activation of mitochondrial Bax during anoikis is regulated by p38MAPK.

Most cells undergo apoptosis through the intrinsic pathway. This is dependent on mitochondrial outer membrane permeabilisation (MOMP), which is mediated by the pro-apoptotic Bcl-2 family proteins, Bax and Bak. During apoptosis, Bax translocates from the cytosol to the outer mitochondrial membrane (OMM), wherein it contributes to the formation of pores to release cytochrome-c. However, it remains unclear whether Bax translocation is sufficient to bring about MOMP or whether Bax requires further signals on the OMM to be fully activated. We have previously shown that during mammary epithelial cell anoikis, Bax translocation does not commit cells to MOMP and detached cells are rescued if survival signals from the extracellular matrix (ECM) are restored. These findings implied that a second signal is required for mitochondrial Bax to fully activate and cause MOMP. We now identify p38MAPK (mitogen-activated protein kinase) as this necessary signal to activate Bax after its translocation to mitochondria. The inhibition of p38MAPK did not prevent Bax translocation, but its activity was required for mitochondrial Bax to bring about MOMP. p38MAPK was activated and recruited to a high molecular weight mitochondrial complex after loss of ECM attachment. Artificially targeting p38MAPK to the OMM increased the kinetics of anoikis, supporting a requirement for its mitochondrial localisation to regulate Bax activation and drive commitment to apoptosis.

Analysis of endogenous Bax complexes during apoptosis using blue native PAGE: implications for Bax activation and oligomerization.

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.

Bax targeting to mitochondria occurs via both tail anchor-dependent and -independent mechanisms

Bax is a member of the Bcl-2 family that, together with Bak, is required for permeabilisation of the outer mitochondrial membrane (OMM). Bax differs from Bak in that it is predominantly cytosolic in healthy cells and only associates with the OMM after an apoptotic signal. How Bax is targeted to the OMM is still a matter of debate, with both a C-terminal tail anchor and an N-terminal pre-sequence being implicated. We now show definitively that Bax does not contain an N-terminal import sequence, but does have a C-terminal anchor. The isolated N terminus of Bax cannot target a heterologous protein to the OMM, whereas the C terminus can. Furthermore, if the C terminus is blocked, Bax fails to target to mitochondria upon receipt of an apoptotic stimulus. Zebra fish Bax, which shows a high degree of amino-acid homology with mammalian Bax within the C terminus, but not in the N terminus, can rescue the defective cell-death phenotype of Bax/Bak-deficient cells. Interestingly, we find that Bax mutants, which themselves cannot target mitochondria or induce apoptosis, are recruited to clusters of activated wild-type Bax on the OMM of apoptotic cells. This appears to be an amplification of Bax activation during cell death that is independent of the normal tail anchor-mediated targeting.

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.

Extracellular Matrix Controls Insulin Signaling in Mammary Epithelial Cells Through the RhoA/Rok Pathway

Cellular responses are determined by a number of signaling cues in the local microenvironment, such as growth factors and extracellular matrix (ECM). In cultures of mammary epithelial cells (MECs), functional differentiation requires at least two types of signal, lactogenic hormones (i.e., prolactin, insulin, and hydrocortisone) and the specialized ECM, basement membrane (BM). Our previous work has shown that ECM affects insulin signaling in mammary cells. Cell adhesion to BM promotes insulin‐stimulated tyrosine phosphorylation of insulin receptor substrate‐1 (IRS‐1) and association of PI3K with IRS‐1, whereas cells cultured on stromal ECM are inefficient in transducing these post‐receptor events. Here we examine the mechanisms underlying ECM control of IRS phosphorylation. Compared to cells cultured on BM, cells on plastic exhibit higher level of RhoA activity. The amount and the activity of Rho kinase (Rok) associated with IRS‐1 are greater in these cells, leading to serine phosphorylation of IRS‐1. Expression of dominant negative RhoA and the application of Rok inhibitor Y27632 in cells cultured on plastic augment tyrosine phosphorylation of IRS‐1. Conversely, expression of constitutively active RhoA in cells cultured on BM impedes insulin signaling. These data indicate that RhoA/Rok is involved in substratum‐mediated regulation of insulin signaling in MECs, and under the conditions where proper adhesion to BM is missing, such as after wounding and during mammary gland involution, insulin‐mediated cellular differentiation and survival would be defective. J. Cell. Physiol. 220: 476–484, 2009.

Control of apoptosis in breast by growth factors and extracellular matrix: targets for therapeutic intervention

Cell survival is adhesion-dependent in normal breast epithelium. Survival requires the integrin class of extracellular matrix (ECM) receptors. We have demonstrated that specific ECM such as basement membrane promote cell survival, whereas others, including collagen I, do not. Basement membrane proteins are largely absent around invasive breast cancer cells. Thus, cancer cells have lost their specific ECM-dependency, presumably due to inappropriate activation of adhesion-regulated survival enzymes. Such enzymes represent potential targets for cancer intervention, particularly where there is sufficient redundancy of signalling on basement membrane to provide reduced or no dependency in normal cells. We have shown that pp125FAK mediates integrin survival signals in breast epithelia, and phosphatidylinositol 3-kinase overcomes apoptosis induced by dominant negative pp125FAK. Signals downstream of pp125FAK regulate apoptosis through a control on the activity of the proapoptotic protein Bax. Signal transduction through growth factor receptors can be regulated by adhesive interactions via integrins. We have discovered that pharmacological inhibition of epidermal growth factor receptor signalling strongly induces apoptosis in breast epithelia. The mechanism of apoptosis induction appears not to be through Bax activation, but rather through dephosphorylation of the proapoptotic protein Bad. Thus, different classes of potent survival regulators (ie adhesion and soluble factors) determine apoptotic cell fate within the same cells through independent control of different mitochondrial acting proapoptotic proteins. Our results broaden the scope for future strategies of cancer intervention.