Real-Time Cell Cycle Imaging in a 3D Cell Culture Model of Melanoma, Quantitative Analysis, Optical Clearing, and Mathematical Modeling.

Aberrant cell cycle progression is a hallmark of solid tumors. Therefore, cell cycle analysis is an invaluable technique to study cancer cell biology. However, cell cycle progression has been most commonly assessed by methods that are limited to temporal snapshots or that lack spatial information.

Abrogation of RAB27A expression transiently affects melanoma cell proliferation.

The role of the small GTPase RAB27A as an essential melanosome trafficking regulator in melanocytes is well-accepted. A decade ago, RAB27A was identified as a tumor dependency gene that promotes melanoma cell proliferation. RAB27A has since been linked to another propeller of cancer progression: exosome secretion.

RAB27A promotes melanoma cell invasion and metastasis via regulation of pro-invasive exosomes.

Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival.

HDAC inhibitors restore BRAF-inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma.

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling.

Real-Time Cell Cycle Imaging in a 3D Cell Culture Model of Melanoma.

Aberrant cell cycle progression is a hallmark of solid tumors; therefore, cell cycle analysis is an invaluable technique to study cancer cell biology. However, cell cycle progression has been most commonly assessed by methods that are limited to temporal snapshots or that lack spatial information. Here, we describe a technique that allows spatiotemporal real-time tracking of cell cycle progression of individual cells in a multicellular context.

Cell Cycle Phase-Specific Drug Resistance as an Escape Mechanism of Melanoma Cells.

The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic tumor zones contain clusters of cancer cells that arrest in G1 phase. It is conceivable that neoplastic cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases.

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids.

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid.

Real-time cell cycle imaging during melanoma growth, invasion, and drug response.

Solid cancers are composed of heterogeneous zones containing proliferating and quiescent cells. Despite considerable insight into the molecular mechanisms underlying aberrant cell cycle progression, there is limited understanding of the relationship between the cell cycle on the one side, and melanoma cell motility, invasion, and drug sensitivity on the other side. Utilizing the fluorescent ubiquitination-based cell cycle indicator (FUCCI) to longitudinally monitor proliferation and migration of melanoma cells in 3D culture and in vivo, we found that invading melanoma cells cycle actively, while G1-arrested cells showed decreased invasion.

Targeting glutamine transport to suppress melanoma cell growth.

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression.

Mesenchymal cells hold the key to immune cell recruitment to and migration within melanoma.

Samaniego et al. (this issue) report on distinct tumor-associated mesenchymal cell (MC) populations in human melanomas. FAP(-)CD90(+) peritumoral MCs may be involved in immune cell recruitment from the bloodstream.

Modeling Melanoma In Vitro and In Vivo.

The behavior of melanoma cells has traditionally been studied in vitro in two-dimensional cell culture with cells adhering to plastic dishes. However, in order to mimic the three-dimensional architecture of a melanoma, as well as its interactions with the tumor microenvironment, there has been the need for more physiologically relevant models. This has been achieved by designing 3D in vitro models of melanoma, such as melanoma spheroids embedded in extracellular matrix or organotypic skin reconstructs.

Melanocortin-1 receptor-mediated signalling pathways activated by NDP-MSH and HBD3 ligands.

Binding of melanocortin peptide agonists to the melanocortin-1 receptor of melanocytes results in eumelanin production, whereas binding of the agouti signalling protein inverse agonist results in pheomelanin synthesis. Recently, a novel melanocortin-1 receptor ligand was reported. A β-defensin gene mutation was found to be responsible for black coat colour in domestic dogs.

The recycling endosome protein Rab17 regulates melanocytic filopodia formation and melanosome trafficking.

Rab GTPases including Rab27a, Rab38 and Rab32 function in melanosome maturation or trafficking in melanocytes. A screen to identify additional Rabs involved in these processes revealed the localization of GFP-Rab17 on recycling endosomes (REs) and melanosomes in melanocytic cells. Rab17 mRNA expression is regulated by microphthalmia transcription factor (MITF), a characteristic of known pigmentation genes.

Melanocortin MC₁ receptor in human genetics and model systems.

The melanocortin MC(1) receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC(1) receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage.

The melanocortin-1 receptor gene polymorphism and association with human skin cancer.

The melanocortin-1 receptor (MC1R) is a key gene involved in the regulation of melanin synthesis and encodes a G-protein coupled receptor expressed on the surface of the melanocyte in the skin and hair follicles. MC1R activation after ultraviolet radiation exposure results in the production of the dark eumelanin pigment and the tanning process in humans, providing physical protection against DNA damage. The MC1R gene is highly polymorphic in Caucasian populations with a number of MC1R variant alleles associated with red hair, fair skin, freckling, poor tanning, and increased risk of melanoma and nonmelanoma skin cancer.

PPARgamma agonists attenuate proliferation and modulate Wnt/beta-catenin signalling in melanoma cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcriptional regulators. Accumulating evidence suggests that PPARgamma agonists such as the thiazolidinediones (TZDs) may prove to be useful anti-cancer agents exhibiting anti-proliferative and/or pro-apoptotic affects in a range of cancer cell types including melanoma, however, the mechanisms underlying this effect remain unclear. We have demonstrated the anti-proliferative effects of full and partial PPARgamma modulators in human melanoma cell lines.

Red hair is the null phenotype of MC1R.

The Melanocortin-1 Receptor (MC1R) is a G-protein coupled receptor, which is responsible for production of the darker eumelanin pigment and the tanning response. The MC1R gene has many polymorphisms, some of which have been linked to variation in pigmentation phenotypes within human populations. In particular, the p.

Receptor function, dominant negative activity and phenotype correlations for MC1R variant alleles.

The human melanocortin-1 receptor (MC1R) is a G-protein coupled receptor involved in the regulation of pigmentation. Several MC1R variant alleles are associated with red hair, fair skin and increased skin cancer risk. We have performed a systematic functional analysis of nine common MC1R variants and correlated these results with the strength of the genetic association of each variant allele with pigmentation phenotypes.

Quantitative analysis of MC1R gene expression in human skin cell cultures.

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells.

Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk.

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling.