Structural and Biofunctional Insights into the Cyclo(Pro-Pro-Phe-Phe-) Scaffold from Experimental and In Silico Studies: Melanoma and Beyond.

Short peptides have great potential as safe and effective anticancer drug leads. Herein, the influence of short cyclic peptides containing the Pro-Pro-Phe-Phe sequence on patient-derived melanoma cells was investigated. Cyclic peptides such as cyclo(Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe-), called CLA, and cyclo(Pro-homoPro-ß3homoPhe-Phe-), called P11, exert the cytotoxic and the cytostatic effects in melanoma cells, respectively. CLA was the most active peptide as it reduced the viability of melanoma cells to 50% of control at about 10 µM, whereas P11 at about 40 µM after 48 h incubation. Interestingly, a linear derivative of P11 did not induce any effect in melanoma cells confirming previous studies showing that cyclic peptides exert better biological activity compared to their linear counterparts. According to in silico predictions, cyclic tetrapeptides show a better pharmacokinetic and toxic profile to humans than CLA. Notably, the spatial structure of those peptides containing synthetic amino acids has not been explored yet. In the Cambridge Structural Database, there is only one such cyclic tetrapeptide, cyclo((R)-ß2homoPhe-D-Pro-Lys-Phe-), while in the Protein Data Bank-none. Therefore, we report the first crystal structure of cyclo(Pro-Pro-ß3homoPhe-Phe-), denoted as 4B8M, a close analog of P11, which is crucial for drug discovery. Comparative molecular and supramolecular analysis of both structures was performed. The DFT findings revealed that 4B8M is well interpreted in the water solution. The results of complex Hirshfeld surface investigations on the cooperativity of interatomic contacts in terms of electrostatic and energetic features are provided. In short, the enrichment ratio revealed O…H/H…O and C…H/H…C as privileged intercontacts in the crystals in relation to basic and large supramolecular H-bonding synthon patterns. Furthermore, the ability of self-assemble 4B8M leading to a nanotubular structure is also discussed.

WNT Signaling in Melanoma.

WNT-signaling controls important cellular processes throughout embryonic development and adult life, so any deregulation of this signaling can result in a wide range of pathologies, including cancer. WNT-signaling is classified into two categories: ß-catenin-dependent signaling (canonical pathway) and ß-catenin-independent signaling (non-canonical pathway), the latter can be further divided into WNT/planar cell polarity (PCP) and calcium pathways. WNT ligands are considered as unique directional growth factors that contribute to both cell proliferation and polarity. Origin of cancer can be diverse and therefore tissue-specific differences can be found in WNT-signaling between cancers, including specific mutations contributing to cancer development. This review focuses on the role of the WNT-signaling pathway in melanoma. The current view on the role of WNT-signaling in cancer immunity as well as a short summary of WNT pathway-related drugs under investigation are also provided.

Histone Deacetylases (HDAC) Inhibitor-Valproic Acid Sensitizes Human Melanoma Cells to Dacarbazine and PARP Inhibitor.

The inhibition of histone deacetylases (HDACs) holds promise as a potential anti-cancer therapy as histone and non-histone protein acetylation is frequently disrupted in cancer, leading to cancer initiation and progression. Additionally, the use of a histone deacetylase inhibitor (HDACi) such as the class I HDAC inhibitor-valproic acid (VPA) has been shown to enhance the effectiveness of DNA-damaging factors, such as cisplatin or radiation. In this study, we found that the use of VPA in combination with talazoparib (BMN-673-PARP1 inhibitor-PARPi) and/or Dacarbazine (DTIC-alkylating agent) resulted in an increased rate of DNA double strand breaks (DSBs) and reduced survival (while not affecting primary melanocytes) and the proliferation of melanoma cells. Furthermore, the pharmacological inhibition of class I HDACs sensitizes melanoma cells to apoptosis following exposure to DTIC and BMN-673. In addition, the inhibition of HDACs causes the sensitization of melanoma cells to DTIV and BMN-673 in melanoma xenografts in vivo. At the mRNA and protein level, the histone deacetylase inhibitor downregulated RAD51 and FANCD2. This study aims to demonstrate that combining an HDACi, alkylating agent and PARPi could potentially enhance the treatment of melanoma, which is commonly recognized as being among the most aggressive malignant tumors. The findings presented here point to a scenario in which HDACs, via enhancing the HR-dependent repair of DSBs created during the processing of DNA lesions, are essential nodes in the resistance of malignant melanoma cells to methylating agent-based therapies.

The influence of follistatin on mechanical properties of bone tissue in growing mice with overexpression of follistatin.

Mechanical competence of bones is mainly associated with tissue quality that depends on proper bone metabolism processes. An imbalance in the regulation of bone metabolism leads to pathological changes in bone tissue leading to susceptibility to bone fractures and bone deterioration processes. Bone metabolism is regulated to a large extent by the members of the transforming growth factor-ß superfamily, i.e., activins and bone morphogenetic proteins. However, their function is regulated by a single-chain protein called follistatin (FS). The aim of this study was to test the hypothesis that overexpression of FS in growing mice results in impairments in bone morphology and mechanical properties. Moreover, we wanted to investigate how geometrical, structural and material properties of bone tissue change with age. The experiment was performed on growing C57BL/6 TgNK14-mFst/6J mice, overexpressing FS (F mice) versus C57BL/6J mice used as controls (C mice). To establish how overexpression of FS influences bone tissue quality, we studied mice femurs to determine geometrical, structural and material properties of the skeleton. To determine mechanical resistance of bone tissue, femurs were loaded to failure in a three-point bending test. Obtained results indicated that overexpression of FS negatively influences bone metabolism. It was found that mutation results with a significant decrease of all measured biomechanical strength variables in F mice in comparison to C mice. Overexpression of FS leads to decreased quality of skeleton, increasing susceptibility to bone fractures.

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.

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.

Role of miRNAs in Melanoma Metastasis.

Tumour metastasis is a multistep process. Melanoma is a highly aggressive cancer and metastasis accounts for the majority of patient deaths. microRNAs (miRNAs) are non-coding RNAs that affect the expression of their target genes. When aberrantly expressed they contribute to the development of melanoma. While miRNAs can act locally in the cell where they are synthesized, they can also influence the phenotype of neighboring melanoma cells or execute their function in the direct tumour microenvironment by modulating ECM (extracellular matrix) and the activity of fibroblasts, endothelial cells, and immune cells. miRNAs are involved in all stages of melanoma metastasis, including intravasation into the lumina of vessels, survival during circulation in cardiovascular or lymphatic systems, extravasation, and formation of the pre-metastatic niche in distant organs. miRNAs contribute to metabolic alterations that provide a selective advantage during melanoma progression. They play an important role in the development of drug resistance, including resistance to targeted therapies and immunotherapies. Distinct profiles of miRNA expression are detected at each step of melanoma development. Since miRNAs can be detected in liquid biopsies, they are considered biomarkers of early disease stages or response to treatment. This review summarizes recent findings regarding the role of miRNAs in melanoma metastasis.

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.

Modulation of WNT/ß-catenin pathway in melanoma by biologically active components derived from plants.

Metastatic melanoma is an aggressive cancer, often resistant to treatment. Therefore, it is essential to determine the molecular mechanisms leading to melanoma or underlying resistance to therapy, and the response to targeted inhibition of the RAS/BRAF/MEK/ERK pathway was a good lesson in this respect. Aberrant WNT/ß-catenin pathway is observed in melanoma, and the modulators of this signaling cascade have been under investigation in the context of therapy as well as chemoprevention. Several natural compounds were recognized as being capable of targeting elements of the WNT/ß-catenin pathway in various cancers, however, only a few of them can modulate this pathway in melanoma. This review examines recent research on the role of the WNT/ß-catenin pathway in tumor development and maintenance, as well as summarizes the current knowledge concerning the modulation of this pathway in melanoma by active compounds of natural origin.

Pentoxifylline Inhibits WNT Signalling in ß-Cateninhigh Patient-Derived Melanoma Cell Populations.

BACKGROUND: The heterogeneity of melanoma needs to be addressed and combination therapies seem to be necessary to overcome intrinsic and acquired resistance to newly developed immunotherapies and targeted therapies. Although the role of WNT/ß-catenin pathway in melanoma was early demonstrated, its contribution to the lack of the melanoma patient response to treatment was only recently recognized. Using patient-derived melanoma cell populations, we investigated the influence of pentoxifylline on melanoma cells with either high or low expression of ß-catenin. FINDINGS: Our results indicate that pentoxifylline inhibits the activity of the canonical WNT pathway in melanoma cell populations with high basal activity of this signalling. This is supported by lowered overall activity of transcription factors TCF/LEF and reduced nuclear localisation of active ß-catenin. Moreover, treatment of ß-cateninhigh melanoma cell populations with pentoxifylline induces downregulation of genes that are targets of the WNT/ß-catenin pathway including connective tissue growth factor (CTGF) and microphthalmia-associated transcription factor (MITF-M), a melanocyte- and melanoma cell-specific regulator. CONCLUSIONS: These results suggest that pentoxifylline, a drug approved by the FDA in the treatment of peripheral arterial disease, might be tested in a subset of melanoma patients with elevated activity of ß-catenin. This pharmaceutical might be tested as an adjuvant drug in combination therapies when the response to immunotherapy is prevented by high activity of the WNT/ß-catenin pathway.

PARP1 inhibitor olaparib (Lynparza) exerts synthetic lethal effect against ligase 4-deficient melanomas.

Cancer including melanoma may be ''addicted" to double strand break (DSB) repair and targeting this process could sensitize them to the lethal effect of DNA damage. PARP1 exerts an important impact on DSB repair as it binds to both single- and double- strand breaks. PARP1 inhibitors might be highly effective drugs triggering synthetic lethality in patients whose tumors have germline or somatic defects in DNA repair genes. We hypothesized that PARP1-dependent synthetic lethality could be induced in melanoma cells displaying downregulation of DSB repair genes. We observed that PARP1 inhibitor olaparib sensitized melanomas with reduced expression of DNA ligase 4 (LIG4) to an alkylatimg agent dacarbazine (DTIC) treatment in vitro, while normal melanocytes remained intact. PARP1 inhibition caused accumulation of DSBs, which was associated with apoptosis in LIG4 deficient melanoma cells. Our hypothesis that olaparib is synthetic lethal with LIG4 deficiency in melanoma cells was supported by selective anti-tumor effects of olaparib used either alone or in combination with dacarbazine (DTIC) in LIG4 deficient, but not LIG4 proficient cells. In addition, olaparib combined with DTIC inhibited the growth of LIG4 deficient human melanoma xenografts. This work for the first time demonstrates the effectiveness of a combination of PARP1 inhibitor olaparib and alkylating agent DTIC for treating LIG4 deficient melanomas. In addition, analysis of the TCGA and transcriptome microarray databases revealed numerous individual melanoma samples potentially displaying specific defects in DSB repair pathways, which may predispose them to synthetic lethality triggered by PARP1 inhibitor combined with a cytotoxic drug.

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.

MiRNA in melanoma-derived exosomes.

Proteins, RNAs and viruses can be spread through exosomes, therefore transport utilizing these nanovesicles is of the great interest. MiRNAs are common exosomal constituents capable of influencing expression of a variety of target genes. MiRNA signatures of exosomes are unique in cancer patients and differ from those in normal controls. The knowledge about miRNA profiles of tumor-derived exosomes may contribute to better diagnosis, determination of tumor progression and response to treatment, as well as to the development of targeted therapies. We summarize the current knowledge with regard to miRNAs that are found in exosomes derived from tumors, particularly from melanoma.

Angiotensin modulates human mammary epithelial cell motility.

INTRODUCTION: Angiotensin II is an effector peptide showing multiple physiological effects, such as regulation of vascular tone, tissue growth and remodelling. Postlactational involution of mammary gland involves changes such as high matrix metalloproteinase activity and release of bioactive fragments of fibronectin and laminin, which may be directly regulated by angiotensin II. The aim of the present study was to evaluate the influence of angiotensin II on proliferation, viability and motility of normal human mammary epithelial cells (184A1 cell line) and to determine the role of angiotensin II receptors in these processes. MATERIALS AND METHODS: Real-time reverse transcription-PCR, western blot and gelatin zymography were used to study the effect of angiotensin II on the expression of angiotensin receptors and matrix metalloproteinases in 184A1 cells. WST-1, AlamarBlue and BrdU assays were used as indicators of cell viability and proliferation after angiotensin II stimulation. Boyden chamber assays and monolayer wound migration assay were used to evaluate in vitro the changes in cell adhesion, migration and invasion. RESULTS: Angiotensin II increased motility of the 184A1 cells and the ability of wound closure. Modifications in cell-substrate adhesion systems and increased secretion and activity of matrix metalloproteinases were also observed. The effect of angiotensin II was abolished by blocking angiotensin type 1 receptor with specific inhibitors candesartan and losartan. CONCLUSIONS: The results indicate that angiotensin II modulates cell behaviour via AT1-R and stimulates secretion of MMP-2 by human mammary epithelial cells.

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/ß-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.