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. In this study, we used two-dimensional and organotypic melanoma culture models in combination with fluorescent cell cycle indicators to investigate the effects of cell cycle phases on clinically used drugs. We demonstrate that G1-arrested melanoma cells, irrespective of the underlying cause mediating G1 arrest, are resistant to apoptosis induced by the proteasome inhibitor bortezomib or the alkylating agent temozolomide. In contrast, G1-arrested cells were more sensitive to mitogen-activated protein kinase pathway inhibitor-induced cell death. Of clinical relevance, pretreatment of melanoma cells with a mitogen-activated protein kinase pathway inhibitor, which induced G1 arrest, resulted in resistance to temozolomide or bortezomib. On the other hand, pretreatment with temozolomide, which induced G2 arrest, did not result in resistance to mitogen-activated protein kinase pathway inhibitors. In summary, we established a model to study the effects of the cell cycle on drug sensitivity. Cell cycle phase-specific drug resistance is an escape mechanism of melanoma cells that has implications on the choice and timing of drug combination therapies.

A Novel Fully Humanized 3D Skin Equivalent to Model Early Melanoma Invasion.

Metastatic melanoma remains incurable, emphasizing the acute need for improved research models to investigate the underlying biologic mechanisms mediating tumor invasion and metastasis, and to develop more effective targeted therapies to improve clinical outcome. Available animal models of melanoma do not accurately reflect human disease and current in vitro human skin equivalent models incorporating melanoma cells are not fully representative of the human skin microenvironment. We have developed a robust and reproducible, fully humanized three-dimensional (3D) skin equivalent comprising a stratified, terminally differentiated epidermis and a dermal compartment consisting of fibroblast-generated extracellular matrix. Melanoma cells incorporated into the epidermis were able to invade through the basement membrane and into the dermis, mirroring early tumor invasion in vivo. Comparison of our novel 3D melanoma skin equivalent with melanoma in situ and metastatic melanoma indicates that this model accurately recreates features of disease pathology, making it a physiologically representative model of early radial and vertical growth-phase melanoma invasion.

Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.

Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain <10%. Novel treatment strategies are therefore urgently required, particularly for patients bearing BRAF/NRAS wild-type tumors. Targeting autophagy is a means to promote cancer cell death in chemotherapy-resistant tumors, and the aim of this study was to test the hypothesis that cannabinoids promote autophagy-dependent apoptosis in melanoma. Treatment with Δ(9)-Tetrahydrocannabinol (THC) resulted in the activation of autophagy, loss of cell viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.

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. Melanoma cells in a hypoxic environment or treated with mitogen-activated protein kinase pathway inhibitors remained G1-arrested for extended periods of time, with proliferation and invasion resuming after re-exposure to a more favorable environment. We challenge the idea that the invasive and proliferative capacity of melanoma cells are mutually exclusive and further demonstrate that a reversibly G1-arrested subpopulation survives in the presence of targeted therapies.

Targeting the intrinsic apoptosis pathway as a strategy for melanoma therapy.

Melanoma drug resistance is often attributed to abrogation of the intrinsic apoptosis pathway. Targeting regulators of apoptosis is thus considered a promising approach to sensitizing melanomas to treatment. The development of small-molecule inhibitors that mimic natural antagonists of either antiapoptotic members of the BCL-2 family or the inhibitor of apoptosis proteins (IAPs), known as BH3- or SMAC-mimetics, respectively, are helping us to understand the mechanisms behind apoptotic resistance. Studies using BH3-mimetics indicate that the antiapoptotic BCL-2 protein MCL-1 and its antagonist NOXA are particularly important regulators of BCL-2 family signaling, while SMAC-mimetic studies show that both XIAP and the cIAPs must be targeted to effectively induce apoptosis of cancer cells. Although most solid tumors, including melanoma, are insensitive to these mimetic drugs as single agents, combinations with other therapeutics have yielded promising results, and tests combining them with BRAF-inhibitors, which have already revolutionized melanoma treatment, are a clear priority.

Induction of endoplasmic reticulum stress as a strategy for melanoma therapy: is there a future?

Melanoma cells employ several survival strategies, including induction of the unfolded protein response, which mediates resistance to endoplasmic reticulum (ER) stress-induced apoptosis. Activation of oncogenes specifically suppresses ER stress-induced apoptosis, while upregulation of ER chaperone proteins and antiapoptotic BCL-2 family members increases the protein folding capacity of the cell and the threshold for the induction of ER stress-induced apoptosis, respectively. Modulation of unfolded protein response signaling, inhibition of the protein folding machinery and/or active induction of ER stress may thus represent potential strategies for the therapeutic management of melanoma. To this aim, the present article focuses on the current understanding of how melanoma cells avoid or overcome ER stress-induced apoptosis, as well as therapeutic strategies through which to harness ER stress for therapeutic benefit.

Oncogenic BRAF signalling increases Mcl-1 expression in cutaneous metastatic melanoma.

The Bcl-2 family member Mcl-1 is essential for melanoma survival; however, the influence of oncogenic BRAF signalling remains elusive. In this study, Mcl-1 splice variant expression was determined in a panel of melanoma cell lines in relation to BRAF mutational status. Mcl-1L mRNA expression was increased in melanoma cells compared with primary melanocytes with significantly increased mRNA and protein expression observed in BRAF(V600E) mutant melanoma cells. Although no change in Mcl-1S mRNA was observed, Mcl-1S protein expression also increased in BRAF mutant melanoma cells. Additionally, while over-expression of mutant BRAF(V600E) increased both Mcl-1L and Mcl-1S expression, inhibition of hyperactive BRAF signalling resulted in decreased Mcl-1L expression. These studies suggest that the regulation of Mcl-1 expression by BRAF signalling is increased by oncogenic activation of BRAF, revealing a mechanism of apoptotic resistance which may be overcome by the use of more specifically targeted Mcl-1 inhibitors.

Oncogenic B-RAF signaling in melanoma impairs the therapeutic advantage of autophagy inhibition.

PURPOSE: Metastatic melanoma is characterized by extremely poor survival rates and hence novel therapies are urgently required. The ability of many anticancer drugs to activate autophagy, a lysosomal-mediated catabolic process which usually promotes cell survival, suggests targeting the autophagy pathway may be a novel means to augment therapy. EXPERIMENTAL DESIGN: Autophagy and apoptosis were assessed in vitro in human melanoma cell lines in response to clinically achievable concentrations of the endoplasmic reticulum (ER) stress-inducing drugs fenretinide or bortezomib, and in vivo using a s.c. xenograft model. RESULTS: Autophagy was activated in response to fenretinide or bortezomib in B-RAF wild-type cells, shown by increased conversion of LC3 to the autophagic vesicle-associated form (LC3-II) and redistribution to autophagosomes and autolysosomes, increased acidic vesicular organelle formation and autophagic vacuolization. In contrast, autophagy was significantly reduced in B-RAF-mutated melanoma cells, an effect attributed partly to oncogenic B-RAF. Rapamycin treatment was unable to stimulate LC3-II accumulation or redistribution in the presence of mutated B-RAF, indicative of de-regulated mTORC1-dependent autophagy. Knockdown of Beclin-1 or ATG7 sensitized B-RAF wild-type cells to fenretinide- or bortezomib-induced cell death, demonstrating a pro-survival function of autophagy. In addition, autophagy was partially reactivated in B-RAF-mutated cells treated with the BH3 mimetic ABT737 in combination with fenretinide or bortezomib, suggesting autophagy resistance is partly mediated by abrogated Beclin-1 function. CONCLUSIONS: Our findings suggest inhibition of autophagy in combination with ER stress-inducing agents may represent a means by which to harness autophagy for the therapeutic benefit of B-RAF wild-type melanoma.

Targeting GRP78 to enhance melanoma cell death.

Targeting endoplasmic reticulum stress-induced apoptosis may offer an alternative therapeutic strategy for metastatic melanoma. Fenretinide and bortezomib induce apoptosis of melanoma cells but their efficacy may be hindered by the unfolded protein response, which promotes survival by ameliorating endoplasmic reticulum stress. The aim of this study was to test the hypothesis that inhibition of GRP78, a vital unfolded protein response mediator, increases cell death in combination with endoplasmic reticulum stress-inducing agents. Down-regulation of GRP78 by small-interfering RNA increased fenretinide- or bortezomib-induced apoptosis. Treatment of cells with a GRP78-specific subtilase toxin produced a synergistic enhancement with fenretinide or bortezomib. These data suggest that combining endoplasmic reticulum stress-inducing agents with strategies to down-regulate GRP78, or other components of the unfolded protein response, may represent a novel therapeutic approach for metastatic melanoma.

Targeting X-linked inhibitor of apoptosis protein to increase the efficacy of endoplasmic reticulum stress-induced apoptosis for melanoma therapy.

Melanoma remains notoriously resistant to current chemotherapeutics, leaving an acute need for novel therapeutic approaches. The aim of this study was to determine the prognostic and therapeutic significance of X-linked inhibitor of apoptosis protein (XIAP) in melanoma through correlation of XIAP expression with disease stage, RAS/RAF mutational status, clinical outcome, and susceptibility to endoplasmic reticulum (ER) stress-induced cell death. XIAP expression and N-RAS/B-RAF mutational status were retrospectively determined in a cohort of 55 primary cutaneous melanocytic lesions selected and grouped according to the American Joint Committee on Cancer staging system. Short hairpin RNA interference of XIAP was used to analyze the effect of XIAP expression on ER stress-induced apoptosis in response to fenretinide or bortezomib in vitro. The results showed that XIAP positivity increased with progressive disease stage, although there was no significant correlation between XIAP positivity and combined N-RAS/B-RAF mutational status or clinical outcome. However, XIAP knockdown significantly increased ER stress-induced apoptosis of melanoma cells in a caspase-dependant manner. The correlation of XIAP expression with disease stage, as well as data showing that XIAP knockdown significantly increases fenretinide and bortezomib-induced apoptosis of metastatic melanoma cells, suggests that XIAP may prove to be an effective therapeutic target for melanoma therapy.

Combining the endoplasmic reticulum stress-inducing agents bortezomib and fenretinide as a novel therapeutic strategy for metastatic melanoma.

PURPOSE: Single-agent chemotherapy is largely the treatment of choice for systemic therapy of metastatic melanoma, but survival rates are low, and novel adjuvant and systemic therapies are urgently required. Endoplasmic reticulum (ER) stress is a potential therapeutic target, and two relatively new drugs, fenretinide and bortezomib (Velcade), each acting via different cellular mechanisms, induce ER stress leading to apoptosis in melanoma cells. The aim of this study was to test the hypothesis that apoptosis of melanoma cells may be increased by combining clinically achievable concentrations of fenretinide and bortezomib. EXPERIMENTAL DESIGN: Three human melanoma cell lines were used to assess changes in viability and the induction of apoptosis in response to fenretinide, bortezomib, or both drugs together. A s.c. xenograft model was used to test responses in vivo. RESULTS: Fenretinide and bortezomib synergistically decreased viability and increased apoptosis in all three melanoma lines at clinically achievable concentrations. This was also reflected by increased expression of GADD153, a marker of ER stress-induced apoptosis. In vivo, fenretinide in combination with bortezomib gave a marked reduction in xenograft tumor volume and an increase in apoptosis compared with fenretinide or bortezomib alone. The cell cycle stage of tumor cells in vivo were similar to that predicted from the effects of each drug or the combination in vitro. CONCLUSIONS: These results suggest that fenretinide and bortezomib, both of which are available in clinical formulation, warrant clinical evaluation as a combination therapy for metastatic melanoma.