Pro-Apoptotic Activity of MCL-1 Inhibitor in Trametinib-Resistant Melanoma Cells Depends on Their Phenotypes and Is Modulated by Reversible Alterations Induced by Trametinib Withdrawal.

BACKGROUND: Although BRAFV600/MEK inhibitors improved the treatment of melanoma patients, resistance is acquired almost inevitably. METHODS: Trametinib withdrawal/rechallenge and MCL-1 inhibition in trametinib-resistance models displaying distinct p-ERK1/2 levels were investigated. RESULTS: Trametinib withdrawal/rechallenge caused reversible changes in ERK1/2 activity impacting the balance between pro-survival and pro-apoptotic proteins. Reversible alterations were found in MCL-1 levels and MCL-1 inhibitors, BIM and NOXA. Taking advantage of melanoma cell dependency on MCL-1 for survival, we used S63845. While it was designed to inhibit MCL-1 activity, we showed that it also significantly reduced NOXA levels. S63845-induced apoptosis was detected as the enhancement of Annexin V-positivity, caspase-3/7 activation and histone H2AX phosphorylation. Percentages of Annexin V-positive cells were increased most efficiently in trametinib-resistant melanoma cells displaying the p-ERK1/2low/MCL-1low/BIMhigh/NOXAlow phenotype with EC50 values at concentrations as low as 0.1 µM. Higher ERK1/2 activity associated with increased MCL-1 level and reduced BIM level limited pro-apoptotic activity of S63845 further influenced by a NOXA level. CONCLUSIONS: Our study supports the notion that the efficiency of an agent designed to target a single protein can largely depend on the phenotype of cancer cells. Thus, it is important to define appropriate phenotype determinants to stratify the patients for the novel therapy.

Trametinib-Resistant Melanoma Cells Displaying MITFhigh/NGFRlow/IL-8low Phenotype Are Highly Responsive to Alternating Periods of Drug Withdrawal and Drug Rechallenge.

Despite significant advances in targeted therapies against the hyperactivated BRAFV600/MEK pathway for patients with unresectable metastatic melanoma, acquired resistance remains an unsolved clinical problem. In this study, we focused on melanoma cells resistant to trametinib, an agent broadly used in combination therapies. Molecular and cellular changes were assessed during alternating periods of trametinib withdrawal and rechallenge in trametinib-resistant cell lines displaying either a differentiation phenotype (MITFhigh/NGFRlow) or neural crest stem-like dedifferentiation phenotype (NGFRhigh/MITFlow). Neither drug withdrawal nor drug rechallenge induced cell death, and instead of loss of fitness, trametinib-resistant melanoma cells adapted to altered conditions by phenotype switching. In resistant cells displaying a differentiation phenotype, trametinib withdrawal markedly decreased MITF level and activity, which was associated with reduced cell proliferation capacity, and induced stemness assessed as NGFR-positive cells and senescence features, including IL-8 expression and secretion. All these changes could be reversed by trametinib re-exposure, which emphasizes melanoma cell plasticity. Trametinib-resistant cells displaying a dedifferentiation phenotype were less responsive presumably due to the already low level of MITF, a master regulator of the melanoma phenotype. Considering new directions of the development of anti-melanoma treatment, our study suggests that the phenotype of melanomas resistant to targeted therapy might be a crucial determinant of the selection of second-line therapy for melanoma patients.

BCL-G: 20 years of research on a non-typical protein from the BCL-2 family.

Proteins from the BCL-2 family control cell survival and apoptosis in health and disease, and regulate apoptosis-unrelated cellular processes. BCL-Gonad (BCL-G, also known as BCL2-like 14) is a non-typical protein of the family as its long isoform (BCL-GL) consists of BH2 and BH3 domains without the BH1 motif. BCL-G is predominantly expressed in normal testes and different organs of the gastrointestinal tract. The complexity of regulatory mechanisms of BCL-G expression and post-translational modifications suggests that BCL-G may play distinct roles in different types of cells and disorders. While several genetic alterations of BCL2L14 have been reported, gene deletions and amplifications prevail, which is also confirmed by the analysis of sequencing data for different types of cancer. Although the studies validating the phenotypic consequences of genetic manipulations of BCL-G are limited, the role of BCL-G in apoptosis has been undermined. Recent studies using gene-perturbation approaches have revealed apoptosis-unrelated functions of BCL-G in intracellular trafficking, immunomodulation, and regulation of the mucin scaffolding network. These studies were, however, limited mainly to the role of BCL-G in the gastrointestinal tract. Therefore, further efforts using state-of-the-art methods and various types of cells are required to find out more about BCL-G activities. Deciphering the isoform-specific functions of BCL-G and the BCL-G interactome may result in the designing of novel therapeutic approaches, in which BCL-G activity will be either imitated using small-molecule BH3 mimetics or inhibited to counteract BCL-G upregulation. This review summarizes two decades of research on BCL-G.

BH3 mimetics potentiate pro-apoptotic activity of encorafenib in BRAFV600E melanoma cells.

BRAFV600- and MEK1/2-targeting therapies rarely produce durable response in melanoma patients. We investigated five BRAFV600E melanoma cell lines derived from drug-naïve tumor specimens to assess cell death response to encorafenib (Braftovi), a recently FDA-approved BRAFV600 inhibitor. Drug-naïve cell lines (i) did not harbor damaging alterations in genes encoding core apoptotic machinery, but they differed in (ii) mitochondrial priming as demonstrated by whole-cell BH3 profiling, and (iii) levels of selected anti-apoptotic proteins. Encorafenib modulated the balance between apoptosis-regulating proteins as it upregulated BIM and BMF, and attenuated NOXA, but did not affect the levels of pro-survival proteins except for MCL-1 and BCL-XL in selected cell lines. Induction of apoptosis could be predicted using Dynamic BH3 profiling. The extent of apoptosis was dependent on both (i) cell-intrinsic proximity to the apoptotic threshold (initial mitochondrial priming) and (ii) the abundance of encorafenib-induced BIM (iBIM; drug-induced change in priming). While co-inhibition of MCL-1 and BCL-XL/BCL-2 was indispensable for apoptosis in drug-naïve cells, encorafenib altered cell dependence to MCL-1, and reliance on BCL-XL/BCL-2 was additionally found in cell lines that were highly primed to apoptosis by encorafenib. This translated into robust apoptosis when encorafenib was combined with selective BH3 mimetics. Our study provides a mechanistic insight into the role of proteins from the BCL-2 family in melanoma cell response to targeted therapy, and presents preclinical evidence that (i) MCL-1 is a druggable target to potentiate encorafenib activity, whereas (ii) pharmacological inhibition of BCL-XL/BCL-2 might be relevant but only for a narrow group of encorafenib-treated patients.

17-Aminogeldanamycin Inhibits Constitutive Nuclear Factor-Kappa B (NF-κB) Activity in Patient-Derived Melanoma Cell Lines.

Melanoma remains incurable skin cancer, and targeting heat shock protein 90 (HSP90) is a promising therapeutic approach. In this study, we investigate the effect of 17-aminogeldanamycin, a potent HSP90 inhibitor, on nuclear factor-kappa B (NF-κB) activity in BRAFV600E and NRASQ61R patient-derived melanoma cell lines. We performed time-lapse microscopy and flow cytometry to monitor changes in cell confluence and viability. The NF-κB activity was determined by immunodetection of phospho-p65 and assessment of expression of NF-κB-dependent genes by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Constitutive activity of p65/NF-κB was evident in all melanoma cell lines. Differences in its level might be associated with genetic alterations in CHUK, IL1B, MAP3K14, NFKBIE, RIPK1, and TLR4, while differences in transcript levels of NF-κB-inducible genes revealed by PCR array might result from the contribution of other regulatory mechanisms. 17-Aminogeldanamycin markedly diminished the level of phospho-p65, but the total p65 protein level was unaltered, indicating that 17-aminogeldanamycin inhibited activation of p65/NF-κB. This conclusion was supported by significantly reduced expression of selected NF-κB-dependent genes: cyclin D1 (CCND1), C-X-C motif chemokine ligand 8 (CXCL8), and vascular endothelial growth factor (VEGF), as shown at transcript and protein levels, as well as secretion of IL-8 and VEGF. Our study indicates that 17-aminogeldanamycin can be used for efficient inhibition of NF-κB activity and the simultaneous diminution of IL-8 and VEGF levels in the extracellular milieu of melanoma.

BCL-w: apoptotic and non-apoptotic role in health and disease.

The BCL-2 family of proteins integrates signals that trigger either cell survival or apoptosis. The balance between pro-survival and pro-apoptotic proteins is important for tissue development and homeostasis, while impaired apoptosis contributes to several pathologies and can be a barrier against effective treatment. BCL-w is an anti-apoptotic protein that shares a sequence similarity with BCL-XL, and exhibits a high conformational flexibility. BCL-w level is controlled by a number of signaling pathways, and the repertoire of transcriptional regulators largely depends on the cellular and developmental context. As only a few disease-relevant genetic alterations of BCL2L2 have been identified, increased levels of BCL-w might be a consequence of abnormal activation of signaling cascades involved in the regulation of BCL-w expression. In addition, BCL-w transcript is a target of a plethora of miRNAs. Besides its originally recognized pro-survival function during spermatogenesis, BCL-w has been envisaged in different types of normal and diseased cells as an anti-apoptotic protein. BCL-w contributes to survival of senescent and drug-resistant cells. Its non-apoptotic role in the promotion of cell migration and invasion has also been elucidated. Growing evidence indicates that a high BCL-w level can be therapeutically relevant in neurodegenerative disorders, neuron dysfunctions and after small intestinal resection, whereas BCL-w inhibition can be beneficial for cancer patients. Although several drugs and natural compounds can bi-directionally affect BCL-w level, agents that selectively target BCL-w are not yet available. This review discusses current knowledge on the role of BCL-w in health, non-cancerous diseases and cancer.

Non-Apoptotic Cell Death Signaling Pathways in Melanoma.

Resisting cell death is a hallmark of cancer. Disturbances in the execution of cell death programs promote carcinogenesis and survival of cancer cells under unfavorable conditions, including exposition to anti-cancer therapies. Specific modalities of regulated cell death (RCD) have been classified based on different criteria, including morphological features, biochemical alterations and immunological consequences. Although melanoma cells are broadly equipped with the anti-apoptotic machinery and recurrent genetic alterations in the components of the RAS/RAF/MEK/ERK signaling markedly contribute to the pro-survival phenotype of melanoma, the roles of autophagy-dependent cell death, necroptosis, ferroptosis, pyroptosis, and parthanatos have recently gained great interest. These signaling cascades are involved in melanoma cell response and resistance to the therapeutics used in the clinic, including inhibitors of BRAFmut and MEK1/2, and immunotherapy. In addition, the relationships between sensitivity to non-apoptotic cell death routes and specific cell phenotypes have been demonstrated, suggesting that plasticity of melanoma cells can be exploited to modulate response of these cells to different cell death stimuli. In this review, the current knowledge on the non-apoptotic cell death signaling pathways in melanoma cell biology and response to anti-cancer drugs has been discussed.

Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity.

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines. There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.

TYRP1 mRNA level is stable and MITF-M-independent in drug-naïve, vemurafenib- and trametinib-resistant BRAFV600E melanoma cells.

TYRP1 mRNA is of interest due to its potential non-coding role as a sponge sequestering tumor-suppressive miRs in melanoma. To our knowledge, there is no report on changes in TYRP1 expression in melanomas after development of resistance to targeted therapies. We used patient-derived drug-naïve RASQ61R and BRAFV600E melanoma cell lines. In BRAFV600E melanoma cells, resistance to vemurafenib and trametinib was developed. A time-lapse fluorescence microscope was used to rate proliferation, qRT-PCR and Western blotting were used to assess TYRP1 expression and MITF-M level and activity. A high TYRP1 protein level in RASQ61R cells corresponded with high TYRP1 mRNA level, whereas undetectable TYRP1 protein in BRAFV600E cells was accompanied by medium mRNA level, also in cells carrying NF1R135W variant in addition. TYRP1 expression was MITF-M-independent, since similar transcript status was found in MITF-Mhigh and MITF-Mlow cells. For the first time, we showed that TYRP1 expression remained unaltered in melanoma cells that became resistant to vemurafenib or trametinib, including those cells losing MITF-M. Also drug discontinuation in resistant cells did not substantially affect TYRP1 expression. To verify in vitro results, publicly available microarray data were analyzed. TYRP1 transcript levels stay unaltered in the majority of paired melanoma samples from patients before treatment and after relapse caused by resistance to targeted therapies. As TYRP1 mRNA level remains unaltered in melanoma cells during development of resistance to vemurafenib or trametinib, therapies developed to terminate a sponge activity of TYRP1 transcript may be extended to patients that relapse with resistant disease.

Inhibitors of HSP90 in melanoma.

HSP90 (heat shock protein 90) is an ATP-dependent molecular chaperone involved in a proper folding and maturation of hundreds of proteins. HSP90 is abundantly expressed in cancer, including melanoma. HSP90 client proteins are the key oncoproteins of several signaling pathways controlling melanoma development, progression and response to therapy. A number of natural and synthetic compounds of different chemical structures and binding sites within HSP90 have been identified as selective HSP90 inhibitors. The majority of HSP90-targeting agents affect N-terminal ATPase activity of HSP90. In contrast to N-terminal inhibitors, agents interacting with the middle and C-terminal domains of HSP90 do not induce HSP70-dependent cytoprotective response. Several inhibitors of HSP90 were tested against melanoma in pre-clinical studies and clinical trials, providing evidence that these agents can be considered either as single or complementary therapeutic strategy. This review summarizes current knowledge on the role of HSP90 protein in cancer with focus on melanoma, and provides an overview of structurally different HSP90 inhibitors that are considered as potential therapeutics for melanoma treatment.

Physiologically Relevant Oxygen Concentration (6% O2) as an Important Component of the Microenvironment Impacting Melanoma Phenotype and Melanoma Response to Targeted Therapeutics In Vitro.

Cancer cell phenotype largely depends on oxygen availability. The atmospheric oxygen concentration (21%) used in in vitro studies is much higher than in any human tissue. Using well-characterized patient-derived melanoma cell lines, we compared: (i) activities of several signaling pathways, and (ii) the effects of vemurafenib and trametinib in hyperoxia (21% O2), normoxia (6% O2) and hypoxia (1% O2). A high plasticity of melanoma cells in response to changes in oxygen supplementation and drug treatment was observed, and the transcriptional reprograming and phenotypic changes varied between cell lines. Normoxia enhanced the expression of vascular endothelial growth factor (VEGF), glucose metabolism/transport-related genes, and changed percentages of NGFR- and MITF-positive cells in cell line-dependent manner. Increased protein stability might be responsible for high PGC1α level in MITFlow melanoma cells. Vemurafenib and trametinib while targeting the activity of MAPK/ERK pathway irrespective of oxygen concentration, were less effective in normoxia than hyperoxia in reducing levels of VEGF, PGC1α, SLC7A11 and Ki-67-positive cells in cell line-dependent manner. In conclusion, in vitro studies performed in atmospheric oxygen concentration provide different information on melanoma cell phenotype and response to drugs than performed in normoxia, which might partially explain the discrepancies between results obtained in vitro and in clinical settings.

Plasticity of Drug-Naïve and Vemurafenib- or Trametinib-Resistant Melanoma Cells in Execution of Differentiation/Pigmentation Program.

Melanoma plasticity creates a plethora of opportunities for cancer cells to escape treatment. Thus, therapies must target all cancer cell subpopulations bearing the potential to contribute to disease. The role of the differentiation/pigmentation program in intrinsic and acquired drug resistance is largely uncharacterized. MITF level and expression of MITF-dependent pigmentation-related genes, MLANA, PMEL, TYR, and DCT, in drug-naïve and vemurafenib- or trametinib-treated patient-derived melanoma cell lines and their drug-resistant counterparts were analysed and referred to genomic alterations. Variability in execution of pigmentation/differentiation program was detected in patient-derived melanoma cell lines. Acute treatment with vemurafenib or trametinib enhanced expression of pigmentation-related genes in MITF-Mhigh melanoma cells, partially as the consequence of transcriptional reprograming. During development of resistance, changes in pigmentation program were not unidirectional, but also not universal as expression of different pigmentation-related genes was diversely affected. In selected resistant cell lines, differentiation/pigmentation was promoted and might be considered as one of drug-tolerant phenotypes. In other resistant lines, dedifferentiation was induced. Upon drug withdrawal ("drug holiday"), the dedifferentiation process in resistant cells either was enhanced but reversed by drug reexposure suggesting involvement of epigenetic mechanisms or was irreversible. The irreversible dedifferentiation might be connected with homozygous loss-of-function mutation in MC1R, as MC1RR151C +/+ variant was found exclusively in drug-naïve MITF-Mlow dedifferentiated cells and drug-resistant cells derived from MITFhigh/MC1RWT cells undergoing irreversible dedifferentiation. MC1RR151C +/+ variant might be further investigated as a parameter potentially impacting melanoma patient stratification and aiding in treatment decision.

17-Aminogeldanamycin selectively diminishes IRE1α-XBP1s pathway activity and cooperatively induces apoptosis with MEK1/2 and BRAFV600E inhibitors in melanoma cells of different genetic subtypes.

Outcomes of melanoma patient treatment remain unsatisfactory despite accessibility of oncoprotein-targeting drugs and immunotherapy. Here, we reported that 17-aminogeldanamycin more potently activated caspase-3/7 in BRAFV600E melanoma cells than geldanamycin, another inhibitor of heat shock protein 90 (HSP90). 17-aminogeldanamycin alleviated self-triggered compensatory increase in HSP70 mRNA level and induced endoplasmic reticulum (ER) stress, which was followed by selective diminution of cytoprotective IRE1α-XBP1s pathway activity of unfolded protein response (UPR), inhibition of ERK1/2 activity and induction of apoptosis. Concomitantly, ATF6/p50 level and expression of PERK-dependent genes, CHOP and BIM, remained unaltered. This might result from an inframe deletion in EIF2AK3 leading to a PERKL21del variant revealed by whole-exome sequencing in melanoma cell lines. 17-aminogeldanamycin exhibited similar activity in NRASQ61R melanoma cells that harbored a heterozygous inactivating variant of NAD(P)H:quinone oxidoreductase 1 (NQO1P187S). In addition, 17-aminogeldanamycin acted cooperatively with trametinib (an inhibitor of MEK1/2) and vemurafenib (an inhibitor of BRAFV600E) in induction of apoptosis in melanoma cell lines as evidenced by in-cell caspase-3/7 activation and PARP cleavage that occurred earlier compared with either drug used alone. As trametinib and vemurafenib did not significantly affect HSP70 and GRP78 transcript levels, cooperation of MEK/BRAFV600E inhibitors and 17-aminogeldanamycin might result from a concurrent inhibition of the RAS/RAF/MEK/ERK cascade and IRE1α-dependent signaling, and cell-intrinsic ER homeostasis can determine the extent of the drug cooperation. Our study indicates that 17-aminogeldanamycin takes several advantages compared with other HSP90-targeting compounds, and can complement activity of BRAF/MEK inhibitors in melanoma cells of different genetic subtypes.

Whole-exome sequencing reveals novel genetic variants associated with diverse phenotypes of melanoma cells.

We have extensively studied the phenotypic heterogeneity of patient-derived melanoma cells. Here, whole-exome sequencing revealed novel variants of genes associated with the MAPK, NOTCH, Hippo, cell-cycle, senescence, and ubiquitin-dependent pathways, which could contribute to the observed phenotypic diversity between cell lines. Focusing on mutations in the MAPK pathway-associated genes, we found BRAF (BRAFV600E ) and RAS subtypes, including NRASQ61R and the rare HRASQ61R variant, and additional alterations potentially leading to different ERK1/2 activity. Both RASQ61R cell lines harbored a MEK1P124S variant and exerted a low level of phospho-MEK1/2. Activity of the MAPK pathway was further attenuated in NRASQ61R /MEKP124S cells by trametinib, and this effect was also shown in HRASQ61R /MEKP124S melanoma cells. The observed variability in doubling time might be a consequence of diverse MAPK and PI3K/AKT pathway activities, but not exclusively, as a senescence program was also executed to different extent in distinct melanoma cell lines. Low percentages of senescent cells might result from mutations in CDKN2A, E2F3, and EZH2, and a high c-MYC expression. Vemurafenib and trametinib induced senescence concomitantly with c-MYC downregulation and irrespectively of CDKN2A mutation, but the EZH2S412C variant might limit senescence induction. Damaging alterations in Hippo pathway-associated genes were accompanied with variability in the phosphorylation level of YAP1/TAZ and CTGF expression. Our study also suggests opposite activity of NOTCH2F1209V and NOTCH2N2002S variants. Additionally, we found a novel FBXW7V418M variant that retained its function in melanoma cells. The obtained molecular data might be further exploited in genotype-phenotype relationship studies and in identifying novel biomarkers and therapies for melanomas.

Exogenous growth factors bFGF, EGF and HGF do not influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro.

It has been shown that the response of V600EBRAF melanoma cells to targeted therapeutics is affected by growth factors. We have investigated the influence of three different growth factors, bFGF, EGF and HGF used either alone or in combination, on the response of V600EBRAF melanoma cell populations established from surgical specimens to vemurafenib and trametinib, targeting V600EBRAF and MEK1/2, respectively. We report that proliferation and phenotype of V600EBRAF melanoma cell populations were not detectably influenced by exogenous growth factors. Neither cell distribution in cell cycle and CCND1 expression nor activity of signaling pathways crucial for melanoma development and maintenance, including the RAF/MEK/ERK pathway, WNT/ß-catenin pathway and NF-κB signaling, were affected by the presence of different growth factors. We furthermore show that vemurafenib and trametinib abrogated the activity of ERK1/2, arrested cells in G0/G1 cell cycle phase, triggered apoptosis, induced changes in the expression of CXCL8, CCND1 and CTGF and the frequency of Ki-67high and CD271high cells. These effects were, however, similar in the presence of different growth factors. Interestingly, comparable results were also obtained for melanoma cells grown without exogenous growth factors bFGF, EGF and HGF for a period as long as 4 months prior the drug treatment. We conclude that the composition or lack of exogenous growth factors bFGF, EGF and HGF do not markedly influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro. Our results question the necessity of these growth factors in the medium that is used for culturing V600EBRAF melanoma cells.

Vemurafenib and trametinib reduce expression of CTGF and IL-8 in V600EBRAF 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 ß-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 ß-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.

[BRAF mutation in progression and therapy of melanoma, papillary thyroid carcinoma and colorectal adenocarcinoma]. / Znaczenie mutacji BRAF dla progresji i terapii czerniaka, raka brodawkowatego tarczycy i raka jelita grubego.

BRAF is mutated at a high frequency in various malignancies, including melanoma, papillary thyroid carcinoma and colorectal adenocarcinoma. BRAF is an element of the RAS/RAF/MEK/ERK (MAPK) pathway, which when constitutively active can lead to increased proliferation rate, enhanced survival, invasion and metastasis. The development of small molecule inhibitors of mutant BRAF kinase has changed the care of patients, especially with melanoma. Despite the success in treating melanoma with inhibitors of mutant BRAF and other elements of RAS/RAF/MEK/ERK (MAPK) pathway, resistance limits the long-term responsiveness to these drugs. The resistance mechanisms to MAPK pathway inhibition are complex, occur at genomic and phenotypic levels, and frequently the same patient can simultaneously develop diverse mechanisms of resistance in different progressive metastases or even in the same lesion. In the current review, we summarize recent research on mutations in BRAF and their importance for the development of tumor. This review will also give an overview on the current knowledge concerning therapies for patients harboring mutation in BRAF and discusses the diverse mechanisms of resistance developed in response to these targeted therapies.

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 BRAF(V600E) 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.

MCL-1, BCL-XL and MITF Are Diversely Employed in Adaptive Response of Melanoma Cells to Changes in Microenvironment.

Melanoma cells can switch their phenotypes in response to microenvironmental insults. Heterogeneous melanoma populations characterized by long-term growth and a high self-renewal capacity can be obtained in vitro in EGF(+)bFGF(+) medium whilst invasive potential of melanoma cells is increased in serum-containing cultures. In the present study, we have shown that originally these patient-derived melanoma populations exhibit variable expression of pro-survival genes from the BCL-2 family and inhibitors of apoptosis (IAPs), and differ in the baseline MCL-1 transcript stability as well. While being transferred to serum-containing medium, melanoma cells are well protected from death. Immediate adaptive response of melanoma cells selectively involves a temporary MCL-1 increase, both at mRNA and protein levels, and BCL-XL can complement MCL-1, especially in MITFlow populations. Thus, the extent of MCL-1 and BCL-XL contributions seems to be cell context-dependent. An increase in MCL-1 level results from a transiently enhanced stability of its transcript, but not from altered protein turnover. Inhibition of MCL-1 preceding transfer to serum-containing medium caused the induction of cell death in a subset of melanoma cells, which confirms the involvement of MCL-1 in melanoma cell survival during the rapid alteration of growth conditions. Additionally, immediate response to serum involves the transient increase in MITF expression and inhibition of ERK-1/2 activity. Uncovering the mechanisms of adaptive response to rapid changes in microenvironment may extend our knowledge on melanoma biology, especially at the stage of dissemination.

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.