Andrew E. Aplin

Integrin signaling and cell growth control

Integrins contribute to cell growth by providing a physical linkage between cytoskeletal structures and the extracellular matrix, and also by participating in various signal transduction processes. The interaction of integrins with matrix ligands can generate signals in and of itself, and can also modulate signals instigated by soluble factors such as peptide mitogens. Cellular events affected by integrin-mediated signaling include motility, cell division, differentiation and programmed cell death. Elucidation of how integrin-mediated cell adhesion controls cell growth is likely to be of fundamental importance in understanding complex biological processes, such as tissue morphogenesis and tumor progression.

Cell adhesion molecules, signal transduction and cell growth.

Signals from dynamic cellular interactions between the extracellular matrix and neighboring cells ultimately input into the cellular decision-making process. These interactions form the basis of anchorage-dependent growth. Recent advances have provided the mechanistic details behind the ability of integrins, and other cell adhesion molecules (CAMs), to regulate both early signal transduction events initiated by soluble factors and downstream events more proximally involved in cell cycle progression. These actions appear to depend on the ability of CAMs to initiate the formation of organized structures that permit the efficient flow of information.

Anchorage-dependent ERK signaling - Mechanisms and consequences

Integrin-mediated adhesion to the extracellular matrix regulates the cellular response to mitogens. Anchorage-dependent growth factor activation of the extracellular signal-regulated kinase (ERK) is an intensely studied example of this regulation. Given the central role of ERK in mediating cell migration, division, and survival, it is also an extremely important example. Recent work has demonstrated that cell adhesion can regulate ERK signaling at several checkpoints and has begun to define the mechanism and consequences associated with anchorage-dependent effects on the ERK cascade.

Mitochondrial H2O2 regulates the angiogenic phenotype Via PTEN oxidation

Recent studies have demonstrated that the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10), the antagonist of the phosphosphoinositol-3-kinase (PI3K) signaling cascade, is susceptible to H2O2-dependent oxidative inactivation. This study describes the use of redox-engineered cell lines to identify PTEN as sensitive to oxidative inactivation by mitochondrial H2O2. Increases in the steady state production of mitochondrial derived H2O2, as a result of manganese superoxide dismutase (Sod2) overexpression, led to PTEN oxidation that was reversed by the coexpression of the H2O2-detoxifying enzyme catalase. The accumulation of an oxidized inactive fraction of PTEN favored the formation of phosphatidylinositol 3,4,5-triphosphate at the plasma membrane, resulting in increased activation of Akt and modulation of its downstream targets. PTEN oxidation in response to mitochondrial H2O2 enhanced PI3K signaling, leading to increased expression of the key regulator of angiogenesis, vascular endothelial growth factor. Overexpression of PTEN prevented the H2O2-dependent increase in vascular endothelial growth factor promoter activity and immunoreactive protein, whereas a mutant PTEN (G129R), lacking phosphatase activity, did not. Furthermore, mitochondrial generation of H2O2 by Sod2 promoted endothelial cell sprouting in a three-dimensional in vitro angiogenesis assay that was attenuated by catalase coexpression or the PI3K inhibitor LY2949002. Moreover, Sod2 overexpression resulted in increased in vivo blood vessel formation that was H2O2-dependent as assessed by the chicken chorioallantoic membrane assay. Our findings provide the first evidence for the involvement of mitochondrial H2O2 in regulating PTEN function and the angiogenic switch, indicating that Sod2 can serve as an alternative physiological source of the potent signaling molecule, H2O2.

Akt3-Mediated Resistance to Apoptosis in B-RAF–Targeted Melanoma Cells

Melanoma cells are highly resistant to anoikis, a form of apoptosis induced in nonadherent/inappropriate adhesion conditions. Depleting B-RAF or the prosurvival Bcl-2 family protein Mcl-1 renders mutant B-RAF melanoma cells susceptible to anoikis. In this study, we examined the effect of targeting B-RAF on the survival of primary stage melanoma cells cultured in three-dimensional type I collagen gels, which partially mimics the dermal microenvironment. Depletion/inhibition of B-RAF with small interfering RNA or the mutant B-RAF inhibitor, PLX4720, induced apoptosis of mutant B-RAF melanoma cells in three-dimensional collagen. Apoptosis was dependent on two upregulated BH3-only proteins, Bim-EL and Bmf, and was inhibited by ectopic Mcl-1 expression. Akt3 activation has been associated with the survival of melanoma cells. Mutant B-RAF melanoma cells ectopically expressing a constitutively activated form of Akt3 or endogenously expressing mutant Akt3 were protected from apoptosis induced by B-RAF knockdown or PLX4720 treatment. Furthermore, intrinsically resistant metastatic melanoma cells displayed elevated Akt phosphorylation in three-dimensional collagen and were rendered susceptible to PLX4720 by Akt3 knockdown. Importantly, myristylated Akt3 prevented B-RAF targeting-induced upregulation of Bim-EL and Bmf in three-dimensional collagen and partially protected Mcl-1-depleted cells from apoptosis. These findings delineate how mutant B-RAF protects melanoma cells from apoptosis and provide insight into possible resistance mechanisms to B-RAF inhibitors.

Integrin regulation of cell signalling and motility

Integrins clearly play a key role in regulating both mitogenic signalling and cell migration. Thus integrins modulate the efficiency of the Erk (extracellular-signal-regulated kinase)/MAP kinase (mitogen-activated protein kinase) pathway, acting at several distinct levels. We have shown that both cAMP-dependent protein kinase and PAKs (p21-activated kinases) play a role in integrin regulation of the Erk pathway, acting primarily at the level of Raf-1. Integrins and PAKs also play a role in the control of cell migration. Thus we have discovered a novel protein that links the alpha5beta1 integrin to migration controlled by Rho-family GTPases. This protein, termed Nischarin, is a large cytosolic macromolecule that is not related to well-known protein families. The N-terminus of Nischarin interacts with a short segment of the cytoplasmic domain of the alpha5 integrin subunit. Overexpression of Nischarin alters actin organization and inhibits Rac-driven cell migration and tumour cell invasion. Use of effector domain mutants of Rac suggest that Nischarin acts downstream of Rac, probably at the level of PAK-family kinases. These studies emphasize the intricate connection between integrins and Rho-family GTPases and their effectors in controlling both mitogenesis and migration.

Adhesion control of cyclin D1 and p27Kip1 levels is deregulated in melanoma cells through BRAF-MEK-ERK signaling.

Mutations in BRAF, a component of extracellular signal-regulated kinases 1 and 2 (ERK) cascade, are frequent in melanoma. It is important to understand how BRAF mutations contribute to malignant traits including anchorage- and growth factor-independence. We have previously shown that efficient activation of ERK in normal human epidermal melanocytes (NHEM) requires both adhesion to the extracellular matrix and growth factors. Mutant V599E BRAF is sufficient to promote ERK activation independent of adhesion and growth factors. Here, we analysed regulation of G1 cell cycle events in NHEM and human melanoma cells. We show that S phase entry in NHEM requires both adhesion and growth factor signaling through the MEK-ERK pathway. This control correlates with induction of cyclin D1 and downregulation of p27Kip1, two key G1 cell cycle events. In melanoma cells expressing V599E BRAF, cyclin D1 was constitutively expressed independent of adhesion but dependent upon MEK activation and nuclear accumulation of ERK. Reduction of cyclin D1 levels by RNA interference inhibited S phase entry in melanoma cells. Importantly, expression of V599E BRAF in NHEM was sufficient to promote cyclin D1 promoter activity in the absence of adhesion. Additionally, p27Kip1 levels were downregulated in V599E BRAF-expressing melanoma cells and active BRAF was sufficient to downregulate p27Kip1 in serum-starved NHEM. Thus, adhesion-growth factor cooperation, leading to efficient activation of ERK, regulates cyclin D1 and p27Kip1 levels in human melanocytes and mutant BRAF overrides adhesion-growth factor control of these two G1 cell cycle proteins in melanomas. These findings provide important insight into how BRAF mutations contribute to aberrant human melanocyte proliferation.

Melanoma adapts to RAF/MEK inhibitors through FOXD3-mediated upregulation of ERBB3

The mechanisms underlying adaptive resistance of melanoma to targeted therapies remain unclear. By combining ChIP sequencing with microarray-based gene profiling, we determined that ERBB3 is upregulated by FOXD3, a transcription factor that promotes resistance to RAF inhibitors in melanoma. Enhanced ERBB3 signaling promoted resistance to RAF pathway inhibitors in cultured melanoma cell lines and in mouse xenograft models. ERBB3 signaling was dependent on ERBB2; targeting ERBB2 with lapatinib in combination with the RAF inhibitor PLX4720 reduced tumor burden and extended latency of tumor regrowth in vivo versus PLX4720 alone. These results suggest that enhanced ERBB3 signaling may serve as a mechanism of adaptive resistance to RAF and MEK inhibitors in melanoma and that cotargeting this pathway may enhance the clinical efficacy and extend the therapeutic duration of RAF inhibitors.

Hyperactivation of MEK-ERK1/2 signaling and resistance to apoptosis induced by the oncogenic B-RAF inhibitor, PLX4720, in mutant N-RAS melanoma cells.

Activating mutations in B-RAF and N-RAS occur in ∼60 and ∼15% of melanomas, respectively. The most common mutation in B-RAF is V600E, which activates B-RAF and the downstream MEK–ERK1/2 pathway. Thus, B-RAFV600E is a viable therapeutic target. PLX4720 is a selective inhibitor of mutant B-RAF and its analog, PLX4032, is currently undergoing clinical trials in melanoma. However, the effects of PLX4720 across the genotypic spectrum in melanoma remain unclear. Here, we describe that PLX4720 treatment rapidly induces hyperactivation of the MEK–ERK1/2 pathway in mutant N-RAS melanoma cells. Furthermore, we demonstrate that C-RAF is the major RAF isoform involved in this process. Importantly, PLX4720-induced hyperactivation of the MEK–ERK1/2 pathway promotes resistance to apoptosis in both non-invasive and invasive mutant N-RAS melanoma cells but does not enhance cell cycle properties. These findings underscore the need to genotypically stratify melanoma patients before enrollment on a mutant B-RAF inhibitor trial.

Mcl-1 Is Required for Melanoma Cell Resistance to Anoikis

Melanoma is a particularly aggressive tumor type that exhibits a high level of resistance to apoptosis. The serine/threonine kinase B-RAF is mutated in 50% to 70% of melanomas and protects melanoma cells from anoikis, a form of apoptosis induced by lack of adhesion or adhesion to an inappropriate matrix. Mutant B-RAF down-regulates two BH3-only proapoptotic proteins, BimEL and Bad. BH3-only proteins act, at least in part, by sequestering prosurvival Bcl-2 family proteins and preventing them from inhibiting the mitochondrial apoptotic pathway. Several Bcl-2 proteins are up-regulated in melanoma; however, the mechanisms of up-regulation and their role in melanoma resistance to anoikis remain unclear. Using RNA interference, we show that depletion of Mcl-1 renders mutant B-RAF melanoma cells sensitive to anoikis. By contrast, minor effects were observed following depletion of either Bcl-2 or Bcl-XL. Mcl-1 expression is enhanced in melanoma cell lines compared with melanocytes and up-regulated by the B-RAF-MEK-extracellular signal-regulated kinase 1/2 pathway through control of Mcl-1 protein turnover. Similar to B-RAF knockdown cells, adhesion to fibronectin protected Mcl-1 knockdown cells from apoptosis. Finally, expression of Bad, which does not sequester Mcl-1, further augmented apoptosis in nonadherent Mcl-1 knockdown cells. Together, these data support the notion that BH3 mimetic compounds that target Mcl-1 may be effective for the treatment of melanoma in combinatorial strategies with agents that disrupt fibronectin-integrin signaling. (Mol Caner Res 2009;7(4):549–56)