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

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.

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.

The hypoxic microenvironment of the skin contributes to Akt-mediated melanocyte transformation.

Constitutive activation of Akt characterizes a high percentage of human melanomas and represents a poor prognostic factor of the disease. We show that Akt transforms melanocytes only in a hypoxic environment, which is found in normal skin. The synergy between Akt and hypoxia is HIF1alpha mediated. Inhibition of HIF1alpha decreases Akt transformation capacity in hypoxia and tumor growth in vivo, while overexpression of HIF1alpha allows anchorage-independent growth in normoxia and development of more aggressive tumors. Finally, we show that mTOR activity is necessary to maintain the transformed phenotype by sustaining HIF1alpha activity. Taken together, these findings demonstrate that Akt hyperactivation and HIF1alpha induction by normally occurring hypoxia in the skin significantly contribute to melanoma development.

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.

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.

Targeting integrins and PI3K/Akt-mediated signal transduction pathways enhances radiation-induced anti-angiogenesis.

The integrins and PI3K/Akt are important mediators of the signal transduction pathways involved in tumor angiogenesis and cell survival after exposure to ionizing radiation. Selective targeting of either integrins or PI3K/Akt can radiosensitize tumors. In this study, we tested the hypothesis that the combined inhibition of integrin alphanubeta3 by cRGD and PI3K/Akt by LY294002 would significantly enhance radiation-induced inhibition of angiogenesis by vascular endothelial cells. Treatment with cRGD inhibited the adhesion and tube formation of human umbilical vein endothelial cells (HUVECs). The inhibitory effect was further increased when cRGD and LY294002 were applied simultaneously. Both radiation and cRGD induced Akt phosphorylation, up-regulated COX2 expression, and increased PGE2 production in HUVECs. Treatment with LY294002 effectively inhibited radiation- and cRGD-induced Akt phosphorylation and up-regulation of COX2 and increased apoptosis of HUVECs. The combined use of cRGD and LY294002 enhanced radiation-induced cell killing. The clonogenic survival of HUVECs was decreased from 34% with 2 Gy radiation to 4% with these agents combined. These results demonstrate that combined use of ionizing radiation, cRGD and LY294002 inhibited multiple signaling transduction pathways involved in tumor angiogenesis and enhanced radiation-induced effects on vascular endothelial cells.

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.

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 Deltap85 or kinase-inactive Raf(301) 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.

Effects of perillyl alcohol on melanoma in the TPras mouse model.

This study evaluates the chemopreventive effects of topically applied perillyl alcohol on the development of melanoma in TPras transgenic mice. Our strategy was to target critical pathways in the development of melanoma, in particular, the ras pathway. Ras has been shown in our experimental mouse model, as well as others, to be important in the development and maintenance of melanomas. Perillyl alcohol (POH), a naturally occurring monoterpene, inhibits the isoprenylation of small G protein, including Ras. POH (10 mM) was applied to the shaved dorsal skin of TPras mice starting 1 week before five treatments of dimethylbenz[a]anthracene (50 microg) and was continued for 38 weeks. We observed a delay in the appearance of tumors and a 25-35% reduction in melanoma incidence. POH treatment of melanoma cells in vitro reduced the levels of detectable Ras protein and inhibits the activation of downstream targets, mitogen-activated protein kinases and Akt. POH only minimally induced apoptosis in this system. Pretreatment but not post-treatment of the melanoma cells with POH, however, markedly reduced levels of UV-induced reactive oxygen species. These studies suggest that POH inhibition of the Ras signaling pathway may be an effective target for chemoprevention of melanoma.

Effects of a superpotent melanotropic peptide in combination with solar UV radiation on tanning of the skin in human volunteers.

OBJECTIVE: Three phase 1 clinical trials of a superpotent melanotropic peptide, melanotan-1 (MT-1, or [Nle(4)-D-Phe(7)]alpha-melanocyte-stimulating hormone) were performed to demonstrate safety for MT-1 therapy combined with UV-B light or sunlight. DESIGN: Open-label studies at 2 dose levels of MT-1 combined with small doses of UV-B to the neck or buttock or full sunlight to half of the back. SETTING: Dermatology clinics at the Arizona Health Sciences Center, Tucson. INTERVENTIONS: The first study randomized 4 subjects to MT-1 (0.08 mg/kg per day subcutaneously) and 4 subjects to injections of isotonic sodium chloride (9%) solution for 10 days, followed by neck irradiation with 3 times the minimal erythema dose (MED) of UV-B light. In the next study (n = 12), the MT-1 dosage was increased to 0.16 mg/kg per day for 10 days, with UV-B radiation (0.25-0.75 MED) given to a buttock site for 5 days during (n = 7) or after (n = 5) MT-1 administration. A final study randomized 8 subjects to 3 to 5 days of sunlight to half of the back or to sunlight plus 0.16 mg/kg of MT-1 for 5 days per week for 4 weeks. RESULTS: Tanning in the first study was achieved in 3 of 4 subjects receiving MT-1, and these subjects also had 47% fewer sunburn cells at the irradiated neck site. More skin sites darkened with the higher dose of MT-1 in the second study. In the third study, there was significantly enhanced tanning of the back in the MT-1 group, and this was maintained at least 3 weeks longer than the tanning in the sunlight-only controls, who required 50% more sun-exposure time for equivalent tanning. MAIN OUTCOME MEASURE: There were no pathologic findings at any UV-B or sun-exposed sites in any subject. Toxic effects due to MT-1 were minor, consisting of nausea and transient facial flushing. CONCLUSION: Melanotan-1 can be safely combined with UV-B light or sunlight and appears to act synergistically in the tanning response to light.

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.

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 p16(INK4a)-CDK4/6-pRb and p14(ARF)-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 HIF1alpha, can function as a promoting factor in tumorigenesis.

Melanocyte and keratinocyte carcinogenesis: p53 family protein activities and intersecting mRNA expression profiles.

Melanocytes and keratinocytes were analyzed for potential roles of p53, p73, and p63 tumor suppressor family proteins and of malignancy-specific gene expression changes in the etiology of multi-step cancer. Melanocytes expressed deltaNp73alpha, two p63 isoforms and p53. Although p21 and Noxa mRNA levels increased following DNA damage, p53 family member binding to p21 and Noxa DNA probes was undetectable, suggesting p53 family-independent responses. In contrast, keratinocytes expressed multiple isoforms each of p73 and p63 that were induced to bind p21 and Noxa DNA probes after ionizing (IR) or after ultraviolet B (UVB) irradiation, correlating with p21 and Noxa mRNA induction and with apoptosis. Interestingly, IR-resistant malignant melanocytes and keratinocytes both exhibited Noxa mRNA induction after UVB treatment, correlating with DNA binding of p53 family proteins to the Noxa probe only in keratinocytes. To uncover other malignancy-specific events, we queried mouse initiated keratinocyte clones for early changes that were exacerbated in malignant derivatives and also differentially expressed in human advanced melanoma versus normal melanocytes. Using a new method for ranking and normalization of microarray data for 5000 probe sets, 27 upregulated and 13 downregulated genes satisfied our query. Of these, the majority was associated with late-stage human cancers and six were novel genes. Thus, clonal lineage mouse models representing early through late cancer progression stages may inform the focus on early, potentially causal events from microarray studies of human cancers, facilitating prognosis and molecular therapy.