Marianne Broome Powell

Notch1 is an effector of Akt and hypoxia in melanoma development

Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-kappaB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1alpha. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment.

Topical Treatment with Inhibitors of the Phosphatidylinositol 3′-Kinase/Akt and Raf/Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Pathways Reduces Melanoma Development in Severe Combined Immunodeficient Mice

Topical treatment with inhibitors of the phosphatidylinositol 3′-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways inhibited the growth of TPras transgenic melanomas in severe combined immunodeficient mice, blocked invasive behavior, and reduced angiogenesis. The inhibitor Ly294002, which is specific for phosphatidylinositol 3′-kinase, effectively reduced melanoma cell growth both in vitro and in vivo. Both Ly294002 and U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, reduced invasion, which correlated with reduction of the metalloproteinase matrix metalloproteinase 2. Tumor angiogenesis was disrupted through inhibition of vascular endothelial growth factor production from the tumor cells and antiangiogenic effects on endothelial cells. Observations with TPras melanoma cells that express dominant negative Δp85 or kinase-inactive Raf301 supported the specificity of the phenomena observed with the chemical inhibitors. These studies demonstrate that topical treatment targeting Ras effectors is efficacious, without systemic toxicities, and may prove to be useful in treating and preventing the progression of cutaneous melanoma.

Inhibition of phosphatidylinositol-3-kinase and mitogen-activated protein kinase kinase 1/2 prevents melanoma development and promotes melanoma regression in the transgenic TPRas mouse model

A number of human melanomas show hyperactivation of the Ras pathway due to mutations of the molecule or alteration of upstream or downstream effectors. In this study, we evaluated the effect of blocking the two Ras downstream pathways phosphatidylinositol-3-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal–regulated kinase on melanoma development and regression in the TPRas mouse model. The inhibition of these two signaling cascades by topically applied Ly294002 and U0126 significantly delayed melanoma development and significantly decreased the tumor incidence, particularly when the drugs were applied in combination. Treatment with the inhibitors of established melanomas resulted in complete remission in 33% of mice and partial regression in 46% of mice when drugs were delivered in combination. These responses correlated with increased apoptosis and decreased proliferation both in vitro and in vivo and reduced tumor angiogenesis. In conclusion, this study strongly supports the role of the phosphatidylinositol-3-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal–regulated kinase pathways in the development and maintenance of Ras-dependent melanomas and supports the notion that specific inhibition of these effectors may represent a very promising avenue for the treatment and prevention of the disease. [Mol Cancer Ther 2006;5(12):3071–7]

Hypoxia, melanocytes and melanoma – survival and tumor development in the permissive microenvironment of the skin

The tissue microenvironment plays a critical role in cell survival and growth and can contribute to cell transformation and tumor development. Cellular interactions with the stroma and with other cells provide key signals that control cellular arrest or division, survival or death, and entrance or exit from a quiescent state. Together, these decisions are essential for maintenance of tissue homeostasis. Tissue oxygenation is an important component of the microenvironment that can acutely alter the behavior of a cell through the direct regulation of genes involved in cell survival, apoptosis, glucose metabolism, and angiogenesis. Loss of tissue homeostasis due to, for example, oncogene activation leads to the disruption of these signals and eventually can lead to cell transformation and tumor development. Here we review the role of tissue oxygenation, and in particular physiologic skin hypoxia, on cell survival and senescence and how it contributes to melanocyte transformation and melanoma development.

p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the ‘high‐p53’Mdm4+/− mouse to the well‐established TP‐ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP‐ras0/+ mice on the Mdm4+/− background developed fewer tumors with a delay in the age of onset of melanomas compared to TP‐ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP‐ras0/+: Mdm4+/− mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin‐3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin‐3‐treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti‐melanomagenic activity of p53.

Spontaneous and UV radiation-induced multiple metastatic melanomas in Cdk4R24C/R24C/TPras mice.

Human melanoma susceptibility is often characterized by germ-line inactivating CDKN2A (INK4A/ARF) mutations, or mutations that activate CDK4 by preventing its binding to and inhibition by INK4A. We have previously shown that a single neonatal UV radiation (UVR) dose delivered to mice that carry melanocyte-specific activation of Hras (TPras) increases melanoma penetrance from 0% to 57%. Here, we report that activated Cdk4 cooperates with activated Hras to enhance susceptibility to melanoma in mice. Whereas UVR treatment failed to induce melanomas in Cdk4(R24C/R24C) mice, it greatly increased the penetrance and decreased the age of onset of melanoma development in Cdk4(R24C/R24C)/TPras animals compared with TPras alone. This increased penetrance was dependent on the threshold of Cdk4 activation as Cdk4(R24C/+)/TPras animals did not show an increase in UVR-induced melanoma penetrance compared with TPras alone. In addition, Cdk4(R24C/R24C)/TPras mice invariably developed multiple lesions, which occurred rarely in TPras mice. These results indicate that germ-line defects abrogating the pRb pathway may enhance UVR-induced melanoma. TPras and Cdk4(R24C/R24C)/TPras tumors were comparable histopathologically but the latter were larger and more aggressive and cultured cells derived from such melanomas were also larger and had higher levels of nuclear atypia. Moreover, the melanomas in Cdk4(R24C/R24C)/TPras mice, but not in TPras mice, readily metastasized to regional lymph nodes. Thus, it seems that in the mouse, Hras activation initiates UVR-induced melanoma development whereas the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing more aggressive, metastatic tumors.

TBC1D16 is a Rab4A GTPase activating protein that regulates receptor recycling and EGF receptor signaling

Rab4A is a master regulator of receptor recycling from endocytic compartments to the plasma membrane. The protein TBC1D16 is up-regulated in melanoma, and TBC1D16-overexpressing melanoma cells are dependent on TBC1D16. We show here that TBC1D16 enhances the intrinsic rate of GTP hydrolysis by Rab4A. TBC1D16 is both cytosolic and membrane associated; the membrane-associated pool colocalizes with transferrin and EGF receptors (EGFRs) and early endosome antigen 1, but not with LAMP1 protein. Expression of two TBC1D16 isoforms, but not the inactive R494A mutant, reduces transferrin receptor recycling but has no effect on transferrin receptor internalization. Expression of TBC1D16 alters GFP-Rab4A membrane localization. In HeLa cells, overexpression of TBC1D16 enhances EGF-stimulated EGFR degradation, concomitant with decreased EGFR levels and signaling. Thus, TBC1D16 is a GTPase activating protein for Rab4A that regulates transferrin receptor recycling and EGFR trafficking and signaling.

Hypoxic induction of AKAP12 variant 2 shifts PKA-mediated protein phosphorylation to enhance migration and metastasis of melanoma cells

Significance Scaffold proteins can serve as critical focal points for association of signaling molecules and downstream pathways that regulate tumor growth and invasion. We demonstrate that low oxygen levels, common in solid tumors, can regulate expression of one member of the AKAP scaffold protein family, AKAP12, in melanoma. Genetic inactivation of AKAP12 leads to decreased migration, invasion, and tumor growth in a mouse model of melanoma. Mechanistically, we discovered a switch in protein kinase A (PKA)-regulated phosphorylations under hypoxia that are dependent on AKAP12 and show that PKA is the critical kinase regulating AKAP12-dependent cellular migration. These results provide novel insight into how the tumor microenvironment modulates signal transduction and biological responses through the regulation of a specific variant of the scaffold protein AKAP12. Scaffold proteins are critical hubs within cells that have the ability to modulate upstream signaling molecules and their downstream effectors to fine-tune biological responses. Although they can serve as focal points for association of signaling molecules and downstream pathways that regulate tumorigenesis, little is known about how the tumor microenvironment affects the expression and activity of scaffold proteins. This study demonstrates that hypoxia, a common element of solid tumors harboring low oxygen levels, regulates expression of a specific variant of the scaffold protein AKAP12 (A-kinase anchor protein 12), AKAP12v2, in metastatic melanoma. In turn, through a kinome-wide phosphoproteomic and MS study, we demonstrate that this scaffolding protein regulates a shift in protein kinase A (PKA)-mediated phosphorylation events under hypoxia, causing alterations in tumor cell invasion and migration in vitro, as well as metastasis in an in vivo orthotopic model of melanoma. Mechanistically, the shift in AKAP12-dependent PKA-mediated phosphorylations under hypoxia is due to changes in AKAP12 localization vs. structural differences between its two variants. Importantly, our work defines a mechanism through which a scaffold protein can be regulated by the tumor microenvironment and further explains how a tumor cell can coordinate many critical signaling pathways that are essential for tumor growth through one individual scaffolding protein.

Skin Hypoxia: A Promoting Environmental Factor in Melanomagenesis

Melanomagenesis is a complex phenomenon in which environmental, genetic and host factors play a role. Sun burns in early childhood are a known risk factor in melanoma development. Alteration of prosurvival genes such as Ras and Akt and loss of function of the p16INK4a-CDK4/6-pRb and p14ARF-HDM2-p53 pathways are strongly associated with human melanoma. We have demonstrated that normally occurring skin hypoxia represents a previously unappreciated host promoting factor in melanomagenesis. Melanocytes that express oncogenes such as Akt, and are therefore genetically unstable, show a transform phenotype only in a mild hypoxic environment that resembles the hypoxic status of the skin. Hypoxia, therefore, is not just a prerogative of advanced neoplasia; physiologic tissue hypoxia, through the activity of HIF1α, can function as a promoting factor in tumorigenesis.

Cytotoxic activity of Apomine is due to a novel membrane-mediated cytolytic mechanism independent of apoptosis in the A375 human melanoma cell line

SummaryApomine, a novel bisphosphonate ester, has demonstrated anticancer activity in a variety of cancer cell lines; however, its mechanism of cytotoxicity is not well understood. Previous work has demonstrated that Apomine induces cell death by activation of caspase-3 in several cancer cell types. However, we have demonstrated that Apomine induces cell death in the A375 human melanoma cell line through a novel membrane-mediated mechanism that is independent of caspase-3 activation. This mechanism of membrane lysis may apply to other bisphosphonates and may be an important mechanism for overcoming resistance to apoptosis. Interestingly, Apomine-mediated cell death in the A375 and UACC 3093 human melanoma cell lines is also independent of N-Ras farnesylation, which was a previously described mechanism of action for Apomine in other cancer cell types. These data suggest that Apomine induces cell death through a novel plasma membrane-mediated cytolytic pathway, independent of caspase-3 activation and N-Ras farnesylation.