Gavin P. Robertson

Deregulated Akt3 Activity Promotes Development of Malignant Melanoma

Malignant melanoma is the skin cancer with the most significant impact on man, carrying the highest risk of death from metastasis. Both incidence and mortality rates continue to rise each year, with no effective long-term treatment on the horizon. In part, this reflects lack of identification of critical genes involved and specific therapies targeted to correct these defects. We report that selective activation of the Akt3 protein promotes cell survival and tumor development in 43 to 60% of nonfamilial melanomas. The predominant Akt isoform active in melanomas was identified by showing that small interfering RNA (siRNA) against only Akt3, and not Akt1 or Akt2, lowered the amount of phosphorylated (active) Akt in melanoma cells. The amount of active Akt3 increased progressively during melanoma tumor progression with highest levels present in advanced-stage metastatic melanomas. Mechanisms of Akt3 deregulation occurred through a combination of overexpression of Akt3 accompanying copy number increases of the gene and decreased PTEN protein function occurring through loss or haploinsufficiency of the PTEN gene. Targeted reduction of Akt3 activity with siRNA or by expressing active PTEN protein stimulated apoptotic signaling, which reduced cell survival by increasing apoptosis rates thereby inhibiting melanoma tumor development. Identifying Akt3 as a selective target in melanoma cells provides new therapeutic opportunities for patients in the advanced stages of this disease.

Mutant V599EB-Raf Regulates Growth and Vascular Development of Malignant Melanoma Tumors

Activating mutations of the B-RAF gene are observed in >60% of human melanomas. Approximately 90% of these mutations occur in the activation segment of the kinase domain as a single-base substitution that converts a valine to glutamic acid at codon 599 (V599E) in exon 15. This mutation causes activation of the kinase as well as downstream effectors of the mitogen-activated protein kinase-signaling cascade, leading to melanoma tumor development by an as yet unknown mechanism. In this study, we have identified the role of (V599E)B-Raf in melanoma tumor development by characterizing the mechanism by which this mutant protein promotes melanoma tumorigenesis. Small interfering RNA targeted against B-Raf or a Raf kinase inhibitor (BAY 43-9006) was used to reduce expression and/or activity of (V599E)B-Raf in melanoma tumors. This inhibition led to reduced activity of the mitogen-activated protein kinase-signaling cascade and inhibited tumor development in animals. Targeted reduction of mutant (V599E)B-Raf expression (activity) in melanoma cells before tumor formation inhibited tumorigenesis by reducing the growth potential of melanoma cells. In contrast, reduction of mutant (V599E)B-Raf activity in preexisting tumors prevented further vascular development mediated through decreased vascular endothelial growth factor secretion, subsequently increasing apoptosis in tumors. These effects in combination with reduced proliferative capacity halted growth, but did not shrink the size of preexisting melanoma tumors. Thus, these studies identify the mechanistic underpinnings by which mutant (V599E)B-RAF promotes melanoma development and show the effectiveness of targeting this protein to inhibit melanoma tumor growth.

Calcium Phosphate Nanocomposite Particles for In Vitro Imaging and Encapsulated Chemotherapeutic Drug Delivery to Cancer Cells

Defects change essentially not only the electronic properties but also the chemical properties of graphene, being centers of its chemical activity. Their functionalization is a way to modify the electronic and crystal structure of graphene, which may be important for graphene-based nanoelectronics. Using hydrogen as an example, we have simulated a chemistry of imperfect graphene for a broad class of defects (Stone-Wales (SW) defects, bivacancies, nitrogen substitution impurities, and zigzag edges) by density functional calculations. We have studied also an effect of finite width of graphene nanoribbons on their chemical properties. It is shown that magnetism at graphene edges is fragile, with respect to oxidation, and, therefore, chemical protection of the graphene edges may be required for the application of graphene in spintronics. At the same time, hydrogenation of the SW defects may be a prospective way to create magnetic carbon.Paradigm-shifting modalities to more efficiently deliver drugs to cancerous lesions require the following attributes: nanoscale-size, targetability, and stability under physiological conditions. Often, these nanoscale drug delivery vehicles are limited due to agglomeration, poor solubility, or cytotoxicity. Thus, we have designed a methodology to encapsulate hydrophobic antineoplastic chemotherapeutics within a 20-30 nm diameter, pH-responsive, nonagglomerating, nontoxic calcium phosphate nanoparticle matrix. In the present study, we report on calcium phosphate nanocomposite particles (CPNPs) that encapsulate both fluorophores and chemotherapeutics, are colloidally stable in physiological solution for an extended time at 37 degrees C and can efficaciously deliver hydrophobic antineoplastic agents, such as ceramide, in several cell model systems.

Akt3 and Mutant V600EB-Raf Cooperate to Promote Early Melanoma Development

B-Raf is the most mutated gene in melanoma; however, the mechanism through which it promotes early melanomas remains uncertain. Most nevi contain activated (V600E)B-Raf but few develop into melanoma, and expression in melanocytes is inhibitory with low protein levels present in surviving cells, suggesting unknown cooperative oncogenic events are necessary for melanoma development. Because many melanomas have (V600E)B-Raf and active Akt3, it is possible that these proteins cooperatively facilitate melanocyte transformation. In this study, Akt3 is shown to phosphorylate (V600E)B-Raf to lower its activity as well as that of the downstream mitogen-activated protein kinase (MAPK) pathway to levels promoting early melanoma development. Expression of active Akt3 in early melanoma cells containing (V600E)B-Raf reduced MAPK signaling and promoted anchorage-independent growth. Furthermore, expression of both (V600E)B-Raf and active Akt3 in melanocytes promoted a transformed phenotype. Mechanistically, aberrant Akt3 activity in early melanomas serves to phosphorylate Ser(364) and Ser(428) on (V600E)B-Raf to reduce activity of (V600E)B-Raf to levels that promote rather than inhibit proliferation, which aids melanocytic transformation. Inhibition of (V600E)B-Raf or Akt3 in advanced melanoma cells in which both pathways were active reduced anchorage-independent growth and tumor development in a cooperatively acting manner. Inhibition of Akt3 alone in these cells led to increased MAPK signaling. In summary, these results suggest that activating B-Raf mutations initially promote nevi development, but the resulting high, intense activation of the MAPK pathway inhibits further tumor progression requiring Akt3 activation to bypass this barrier and aid melanoma development.

Transiently Entrapped Circulating Tumor Cells Interact with Neutrophils to Facilitate Lung Metastasis Development

It is unknown why only a minority of circulating tumor cells trapped in lung capillaries form metastases and involvement of immune cells remains uncertain. A novel model has been developed in this study showing that neutrophils regulate lung metastasis development through physical interaction and anchoring of circulating tumor cells to endothelium. Human melanoma cells were i.v. injected into nude mice leading to the entrapment of many cancer cells; however, 24 hours later, very few remained in the lungs. In contrast, injection of human neutrophils an hour after tumor cell injection increased cancer cell retention by approximately 3-fold. Entrapped melanoma cells produced and secreted high levels of a cytokine called interleukin-8 (IL-8), attracting neutrophils and increasing tethering beta(2) integrin expression by 75% to 100%. Intercellular adhesion molecule-1 on melanoma cells and beta(2) integrin on neutrophils interacted, promoting anchoring to vascular endothelium. Decreasing IL-8 secretion from melanoma cells lowered extracellular levels by 20% to 50%, decreased beta(2) integrin on neutrophils by approximately 50%, and reduced neutrophil-mediated extravasation by 25% to 60%, resulting in approximately 50% fewer melanoma cells being tethered to endothelium and retained in lungs. Thus, transendothelial migration and lung metastasis development decreased by approximately 50%, showing that targeting IL-8 in melanoma cells has the potential to decrease metastasis development by disrupting interaction with neutrophils.

Functional and therapeutic significance of Akt deregulation in malignant melanoma.

Identification of specific genes or signaling pathways involved in development of melanoma could lead to new therapies that target and correct these defects. Recent studies have revealed deregulation of the Akt signaling pathway occuring in 43–67% of melanomas. Akt kinase family members, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, share extensive structural similarity and perform common as well as unique functions within cells. The Akt signaling cascade initiates at the cell surface when growth factors or other extracellular stimuli activate phosphoinositide 3-kinase (PI3K). Activated PI3K generates a lipid second messenger, phosphatidylinositol-3,4,5-trisphosphate (PIP3), causing translocation of Akt to the plasma membrane where it becomes phosphorylated and activated. The balance of cellular PIP3 is regulated primarily by a phosphatase called PTEN that reduces PIP3 levels thereby lowering Akt activity. In melanomas, decreased PTEN activity elevates PIP3 levels resulting in Akt activation. Active Akt then phosphorylates downstream cellular proteins that promote melanoma cell proliferation and survival. Recently, Akt3 was discovered to be the predominant isoform activated in sporadic melanomas. Levels of activity increased during melanoma progression with metastatic melanomas having the highest activity. Although mechanisms of Akt3 activation remain to be fully characterized, overexpression of Akt3 and decreased PTEN activity play important roles in this process. Targeted reduction of Akt3 activity decreased survival of melanoma tumor cells leading to inhibition of tumor development, which may be therapeutically effective for shrinking tumors in melanoma patients. This review surveys recent developments in Akt deregulation in melanoma and its potential as a selective therapeutic target in patients in the advanced stages of this disease.

Systemic Delivery of Liposomal Short-Chain Ceramide Limits Solid Tumor Growth in Murine Models of Breast Adenocarcinoma

In vitro tumor cell culture models have illuminated the potential therapeutic utility of elevating the intracellular concentration of the antimitogenic and proapoptotic sphingolipid, ceramide. However, although cell-permeable, short-chain ceramide is an effective apoptotic agent in vitro, its use as an in vivo, systemically delivered therapeutic is limited by its inherent lipid hydrophobicity and physicochemical properties. Here, we report that the systemic i.v. delivery of C6-ceramide (C6) in a pegylated liposomal formulation significantly limited the growth of solid tumors in a syngeneic BALB/c mouse tumor model of breast adenocarcinoma. Over a 3-week treatment period, a well-tolerated dose of 36 mg/kg liposomal-C6 elicited a >6-fold reduction in tumor size compared with empty ghost liposomes. Histologic analyses of solid tumors from liposomal-C6-treated mice showed a marked increase in the presence of apoptotic cells, with a coincident decrease in cellular proliferation and in the development of a microvessel network. Liposomal-C6 accumulated within caveolae and mitochondria, suggesting putative mechanisms by which ceramide induces selective cancer cell cytotoxicity. A pharmacokinetic analysis of systemic liposomal-C6 delivery showed that the pegylated liposomal formulation follows first-order kinetics in the blood and achieves a steady-state concentration in tumor tissue. Confirming the therapeutic utility of i.v. liposomal-C6 administration, we also shown diminution of solid tumor growth in a human xenograft model of breast cancer. Together, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for improved solubility, drug delivery, and antineoplastic efficacy.

Targeting V600EB-Raf and Akt3 Using Nanoliposomal-Small Interfering RNA Inhibits Cutaneous Melanocytic Lesion Development

Most events promoting early melanoma development are yet to be identified, but deregulation of the B-Raf and Akt3 signaling cascades is an important regulator of this process. Approximately 90% of normal moles and approximately 60% of early invasive cutaneous melanomas contain a T1799A B-Raf mutation ((V600E)B-Raf), leading to 10 times higher enzyme activity and constitutive activation of the mitogen-activated protein kinase pathway. Furthermore, approximately 70% of melanomas have elevated Akt3 signaling due to increased gene copy number and PTEN loss. Therefore, targeting (V600E)B-Raf and Akt3 signaling is necessary to prevent or treat cutaneous melanocytic lesions. Agents specifically targeting these proteins are needed, having fewer side effects than those inhibiting both normal and mutant B-Raf protein or targeting all three Akt isoforms. In this study, a unique nanoliposomal-ultrasound-mediated approach has been developed for delivering small interfering RNA (siRNA) specifically targeting (V600E)B-Raf and Akt3 into melanocytic tumors present in skin to retard melanoma development. Novel cationic nanoliposomes stably encapsulate siRNA targeting (V600E)B-Raf or Akt3, providing protection from degradation and facilitating entry into melanoma cells to decrease expression of these proteins. Low-frequency ultrasound using a lightweight four-cymbal transducer array enables penetration of nanoliposomal-siRNA complex throughout the epidermal and dermal layers of laboratory-generated or animal skin. Nanoliposomal-mediated siRNA targeting of (V600E)B-Raf and Akt3 led to a cooperatively acting approximately 65% decrease in early or invasive cutaneous melanoma compared with inhibition of each singly with negligible associated systemic toxicity. Thus, cationic nanoliposomes loaded with siRNA targeting (V600E)B-Raf and Akt3 provide an effective approach for targeted inhibition of early or invasive cutaneous melanomas.

Targeting the MAPK pathway in melanoma: Why some approaches succeed and other fail

The Mitogen Activated Protein Kinase (MAPK) pathway plays a key role in melanoma development making it an important therapeutic target. In normal cells, the tightly regulated pathway relays extracellular signals from cell membrane to nucleus via a cascade of phosphorylation events. In melanomas, dysregulation of the MAPK pathway occurs frequently due to activating mutations in the B-RAF and RAS genes or other genetic or epigenetic modifications, leading to increased signaling activity promoting cell proliferation, invasion, metastasis, migration, survival and angiogenesis. However, identification of ideal pathway member to therapeutically target for maximal clinical benefit to melanoma patients remains a challenge. This review provides an overview of the obstacles faced targeting the MAPK pathway and why certain therapeutic approaches succeed while others fail. The review summarizes the roles played by the proteins, therapeutic potential and the drugs available to target each member of the pathway as well as concerns related to each. Potential for targeting multiple points and inhibiting other pathways along with MAPK inhibition for optimal efficacy are discussed along with explanations for development of drug resistance, which includes discussions related to cross-talk between pathways, RAF kinase isoform switching and phosphatase deregulation. Finally, the use of nanotechnology is reviewed as an approach to target the MAPK pathway using both genetic and pharmacological agents simultaneously targeting multiple points in the pathway or in combination with other cascades.

Targeting Mitogen-Activated Protein Kinase/Extracellular Signal- Regulated Kinase Kinase in the Mutant (V600E)B-Raf Signaling Cascade Effectively Inhibits Melanoma Lung Metastases

Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by (V600E)B-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)-mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant (V600E)B-Raf signaling cascade to inhibit melanoma metastases.