Mariusz L. Hartman

MITF in melanoma: mechanisms behind its expression and activity

MITF (microphthalmia-associated transcription factor) represents a melanocytic lineage-specific transcription factor whose role is profoundly extended in malignant melanoma. Over the last few years, the function of MITF has been tightly connected to plasticity of melanoma cells. MITF participates in executing diverse melanoma phenotypes defined by distinct gene expression profiles. Mutation-dependent alterations in MITF expression and activity have been found in a relatively small subset of melanomas. MITF activity is rather modulated by its upstream activators and suppressors operating on transcriptional, post-transcriptional and post-translational levels. These regulatory mechanisms also include epigenetic and microenvironmental signals. Several transcription factors and signaling pathways involved in the regulation of MITF expression and/or activity such as the Wnt/β-catenin pathway are broadly utilized by various types of tumors, whereas others, e.g., BRAFV600E/ERK1/2 are more specific for melanoma. Furthermore, the MITF activity can be affected by the availability of transcriptional co-partners that are often redirected by MITF from their own canonical signaling pathways. In this review, we discuss the complexity of a multilevel regulation of MITF expression and activity that underlies distinct context-related phenotypes of melanoma and might explain diverse responses of melanoma patients to currently used therapeutics.

Anti-apoptotic proteins on guard of melanoma cell survival

Apoptosis plays a pivotal role in sustaining proper tissue development and homeostasis. Evading apoptosis by cancer cells is a part of their adaption to microenvironment and therapies. Cellular integrity is predominantly maintained by pro-survival members of Bcl-2 family and IAPs. Melanoma cells are characterized by a labile and stage-dependent phenotype. Pro-survival molecules can protect melanoma cells from apoptosis and mediate other processes, thus enhancing aggressive phenotype. The essential role of Bcl-2, Mcl-1, Bcl-X(L), livin, survivin and XIAP was implicated for melanoma, often in a tumor stage-dependent fashion. In this review, the current knowledge of pro-survival machinery in melanoma is discussed.

Pro-Survival Role of MITF in Melanoma

Melanoma is a therapy-resistant skin cancer due to numerous mechanisms supporting cell survival. Although components of melanoma cytoprotective mechanisms are overexpressed in many types of tumors, some of their regulators are characteristic for melanoma. Several genes mediating pro-survival functions have been identified as direct targets of microphthalmia-associated transcription factor (MITF), a melanocyte-specific modulator also recognized as a lineage addiction oncogene in melanoma. BRAF(V600E) and other proteins deregulated in melanoma influence MITF expression and activity, or they are the partners of MITF in melanoma response to radiotherapy and chemotherapeutics. In this review, the pro-survival activity of MITF is discussed.

Natural Compounds' Activity against Cancer Stem-Like or Fast-Cycling Melanoma Cells

Background Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. Methods We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. Findings Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC. Conclusion Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.

Parthenolide enhances dacarbazine activity against melanoma cells

Dacarbazine induces a clinical response only in 15% of melanoma patients. New treatment strategies may involve combinations of drugs with different modes of action to target the tumor heterogeneity. We aimed to determine whether the combined treatment of heterogeneous melanoma cell populations in vitro with the alkylating agent dacarbazine and the nuclear factor-&kgr;B inhibitor parthenolide could be more effective than either drug alone. A panel of melanoma cell lines, including highly heterogeneous populations derived from surgical specimens, was treated with dacarbazine and parthenolide. The effect of drugs on the viable cell number was examined using an acid phosphatase activity assay, and the combination effect was determined by median-effect analysis. Cell death and cell-cycle arrest were assessed by flow cytometry. Gene expression was measured by real-time PCR and changes in the protein levels were evaluated by western blotting. Secretion of vascular endothelial growth factor and interleukin-8 was determined using an enzyme-linked immunosorbent assay. The self-renewing capacity was assessed using a clonogenic assay. Dacarbazine was less effective in heterogeneous melanoma populations than in the A375 cell line. Parthenolide and dacarbazine synergistically reduced the viable cell numbers. Both drugs induced cell-cycle arrest and apoptotic cell death. Importantly, parthenolide abrogated the baseline and dacarbazine-induced vascular endothelial growth factor secretion from melanoma cells in heterogeneous populations, whereas interleukin-8 secretion was not significantly affected by either drug. Parthenolide eradicated melanoma cells with self-renewing capacity also in cultures simultaneously treated with dacarbazine. The combination of parthenolide and dacarbazine might be considered as a new therapeutic modality against metastatic melanoma.

Pro-apoptotic activity of BH3-only proteins and BH3 mimetics: from theory to potential cancer therapy.

The evasion of cancer cells from the induction of cell death pathways results in the resistance of tumor to current treatment modalities. Therefore, the resistance to cell death, one of the hallmarks of cancer, is a major target in the development of new approaches to selectively affect cancer cells. The complex interplay between individual members of Bcl-2 family regulates both cell survival and the mitochondrial pathway of apoptosis by maintaining mitochondrial membrane integrity (anti-apoptotic Bcl-2 subfamily) and by triggering its disruption in response to stress stimuli (Bax-like subfamily). BH3-only proteins, another Bcl-2 subfamily, act either by direct stimulation of pro-apoptotic proteins of the Bax subfamily or by interfering with anti-apoptotic proteins of the Bcl-2 subfamily. Thus, pro-apoptotic BH3 mimetics, thought to function as BH3-only proteins, are expected to improve the effectiveness of cancer treatment. BH3 mimetics could be either natural or synthetic, peptidic or only based on a helical peptide-like scaffold. Experimental and clinical evidence indicates that BH3 mimetics may not be sufficient to cure cancer patients when used as a single agent. BH3 profiling of cancer cells was introduced to better predict the in vivo responsiveness of tumor to BH3 mimetics combined with conventional therapies. In summary, targeting the Bcl-2 proteins is a promising tool with potential to generate new treatment modalities and to complement existing anti-cancer therapies. This review presents the current knowledge on BH3-only proteins and the spectrum of strategies employing BH3 mimetics in preclinical and clinical studies that aim at tumor targeting.

Gene Expression Profiling Identifies Microphthalmia-Associated Transcription Factor (MITF) and Dickkopf-1 (DKK1) as Regulators of Microenvironment-Driven Alterations in Melanoma Phenotype

Background The diversity of functional phenotypes observed within a tumor does not exclusively result from intratumoral genetic heterogeneity but also from the response of cancer cells to the microenvironment. We have previously demonstrated that the morphological and functional phenotypes of melanoma can be dynamically altered upon external stimuli. Findings In the present study, transcriptome profiles were generated to explore the molecules governing phenotypes of melanospheres grown in the bFGF(+)EGF(+) serum-free cultures and monolayers maintained in the serum-containing medium. Higher expression levels of MITF-dependent genes that are responsible for differentiation, e.g., TYR and MLANA, and stemness-related genes, e.g., ALDH1A1, were detected in melanospheres. These results were supported by the observation that the melanospheres contained more pigmented cells and cells exerting the self-renewal capacity than the monolayers. In addition, the expression of the anti-apoptotic, MITF-dependent genes e.g., BCL2A1 was also higher in the melanospheres. The enhanced activity of MITF in melanospheres, as illustrated by the increased expression of 74 MITF-dependent genes, identified MITF as a central transcriptional regulator in melanospheres. Importantly, several genes including MITF-dependent ones were expressed in melanospheres and original tumors at similar levels. The reduced MITF level in monolayers might be partially explained by suppression of the Wnt/β-catenin pathway, and DKK1, a secreted inhibitor of this pathway, was highly up-regulated in monolayers in comparison to melanospheres and original tumors. Furthermore, the silencing of DKK1 in monolayers increased the percentage of cells with self-renewing capacity. Conclusions Our study indicates that melanospheres can be used to unravel the molecular pathways that sustain intratumoral phenotypic heterogeneity. Melanospheres directly derived from tumor specimens more accurately mirrored the morphology and gene expression profiles of the original tumors compared to monolayers. Therefore, melanospheres represent a relevant preclinical tool to study new anticancer treatment strategies.

Phenotypic diversity of patient-derived melanoma populations in stem cell medium.

Melanomas are highly heterogeneous tumors and there is no treatment effective at achieving long-term remission for metastatic melanoma patients. Thus, an appropriate model system for studying melanoma biology and response to drugs is necessary. It has been shown that composition of the medium is a critical factor in preserving the complexity of the tumor in in vitro settings, and melanospheres maintained in stem cell medium are a good model in this respect. In the present study, we observed that not all nodular melanoma patient-derived cell populations grown in stem cell medium were capable of forming melanospheres, and cell aggregates and anchorage-independent single-cell cultures emerged instead. Self-renewing capacity and unlimited growth potential indicated the presence of cells with stem-like properties in all patient-derived populations but immunophenotype and MITF expression exhibited variability. Enhanced MITF expression and activity was observed in melanospheres in comparison with cell aggregates and single-cell culture, and hypoxic-like conditions that increased the ability of single-cell population to form melanospheres enhanced MITF expression and cell pigmentation as well. Thus, MITF seems to be a critical transcription factor for formation of both patient-derived and hypoxia-induced melanospheres. After 2 years of continuous culturing, melanospheres progressively underwent transition into cell aggregates that was accompanied by changes in expression of several MITF-dependent genes associated with melanogenesis and survival and alterations in the composition of subpopulations but not in the frequency of ABCB5-positive cells. Several biological properties of parent tumor are well preserved in patient-derived melanospheres, but during prolonged culturing the heterogeneity is substantially lost when the melanospheres are substituted by cell aggregates. This should be considered when cell aggregates instead of melanospheres are used in the study of melanoma biology and cell response to drugs.

Parthenolide induces MITF-M downregulation and senescence in patient-derived MITF-M high melanoma cell populations

The activity of the M isoform of microphthalmia-associated transcription factor (MITF-M) has been attributed to regulation of differentiation, proliferation, survival and senescence of melanoma cells. MITF expression was shown to be antagonized by the activation of transcription factor NF-κB. Parthenolide, an inhibitor of NF-κB, has not been yet reported to affect MITF-M expression. Our results obtained in patient-derived melanoma cell populations indicate that parthenolide efficiently decreases the MITF-M level. This is neither dependent on p65/NF-κB signaling nor RAF/MEK/ERK pathway activity as inhibition of MEK by GSK1120212 (trametinib) and induction of ERK1/2 activity by parthenolide itself do not interfere with parthenolide-triggered depletion of MITF-M in both wild-type BRAF and BRAFV600E melanoma populations. Parthenolide activity is not prevented by inhibitors of caspases, proteasomal and lysosomal pathways. As parthenolide reduces MITF-M transcript level and HDAC1 protein level, parthenolide-activated depletion of MITF-M protein may be considered as a result of transcriptional regulation, however, the influence of parthenolide on other elements of a dynamic control over MITF-M cannot be ruled out. Parthenolide induces diverse effects in melanoma cells, from death to senescence. The mode of the response to parthenolide is bound to the molecular characteristics of melanoma cells, particularly to the basal MITF-M expression level but other cell-autonomous differences such as NF-κB activity and MCL-1 level might also contribute. Our data suggest that parthenolide can be developed as a drug used in combination therapy against melanoma when simultaneous inhibition of MITF-M, NF-κB and HDAC1 is needed.

Vemurafenib and trametinib reduce expression of CTGF and IL-8 in V600E BRAF melanoma cells

Clinical evidence has revealed that while RAS/RAF/MEK/ERK pathway is a crucial component of melanomagenesis, other signaling pathways can also contribute to the malignant growth and development of resistance to targeted therapies. We explored the response of V600EBRAF melanoma cells derived from surgical specimens and grown in stem cell medium to vemurafenib and trametinib, drugs targeting the activity of V600EBRAF and MEK1/2, respectively. Cell growth and apoptosis were monitored by real-time imaging system, immunophenotype and cell cycle by flow cytometry, gene expression by quantitative real-time PCR, immunoblotting and enzyme-linked immunosorbent assay. The V600EBRAF melanoma cell populations were diverse. Differences in morphology, pigmentation, cell cycle profiles, and immunophenotype were observed. At the molecular level, melanoma cells differed in the phosphorylation of ERK1/2, NF-κB, and β-catenin, and expression of several relevant genes, including MITF-M, DKK1, CCND1, BRAF, CXCL8, and CTGF. Despite having different characteristics, melanoma cells responded similarly to vemurafenib and trametinib. Both drugs reduced ERK1/2 phosphorylation and percentages of cells expressing Ki-67 at high level, inhibited expression of CCND1 and induced cell cycle arrest in the Go/G1 phase. These expected cytostatic effects were accompanied by increased CD271 expression, a marker of stem-like cells. NF-κB activity was reduced by both drugs, however, not completely abolished, whereas the level of active β-catenin was increased by drugs in three out of six cell populations. Interestingly, expression of IL-8 and CTGF was significantly reduced by treatment with vemurafenib and trametinib. Simultaneous inhibition of NF-κB activity and induction of ERK1/2 phosphorylation revealed that CTGF expression depends on ERK1/2 activity but not on NF-κB activity. Both, the positive effects of treatment with vemurafenib and trametinib such as the newly identified CTGF suppression and undesired effects such as increased CD271 expression suggesting selection of melanoma stem-like cells should be considered in the development of combination treatment for melanoma patients.