Melanoma Cell Lines Research Articles

Tumor reactive T cells from the cerebrospinal fluid of patients with leptomeningeal disease from melanoma.

2027 Background: Leptomeningeal disease (LMD) is a devastating complication from cancer, with a median survival of 6-8 weeks. The incidence of LMD is highest in melanoma (5-25%) carrying the worst prognosis. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) showed complete and durable responses in patients with advanced metastatic melanoma. CSF-derived T cell populations in LMD shift to more exhausted phenotypes compared to extracranial and brain metastases. Ex vivo large T cell expansion can overcome this shortfall, with adoptive T cell transfer representing a novel therapeutic approach to treat melanoma LMD (M-LMD). The goal of this study is to optimize rapid CSF-derived tumor reactive T cell expansion in M-LMD, and evaluate the immune microenvironment in CSF compared to peripheral immunity and anti-tumoral response. Methods: CSF was collected from M-LMD patients (n=6) via lumbar puncture or Ommaya. Cells were plated following established TIL culture protocols to determine optimal expansion conditions i.e.: 1) 6000 IU/mL IL-2, 2) IL-2+OKT3, 3) IL-2+anti-4-1BB agonistic antibody, 4) IL-2+IL-7+IL-15+IL-21, 5) IL-2+anti-CD3/CD28 human dynabeads, & 6) IL-2+anti-CD3/CD28/CD137 human dynabeads. After 4 weeks, T cells were propagated via rapid expansion protocol (REP) and phenotyped for CD4+ and CD8+ T cells with flow cytometry. T cells were co-cultured for 18-24 hours with HLA-matched melanoma cell lines to assess functionality, and IFN-y levels evaluated in supernatant. Ongoing studies are evaluating other biomarkers i.e., chemokines via Luminex. To examine the expanded TCR clonotypes, TCR beta seq was done in CSF-derived T cells at different times point of expansion and peripheral T cells. Results: Collected CSF yielded an average 8.12e4 viable cells for expansion (range 5e2-2.43e6). After initial culture in IL-2, 61.9% of samples showed increased cell yield with an average 101.68-fold expansion (range 2.35-1054). PreREP T cells were predominantly CD4+ (45.7%). REP was completed with a 100% success rate (mean 187.47-fold expansion). Post-REP flow cytometry analysis revealed similar results with further expansion of T cells. Additional culture supplements (Methods #2-6) produced similar expansion with a reduced T cell input requirement and demonstrated the potential to enrich for CD8+ T cells. M-LMD T cells were highly functional, producing substantial levels of IFN-y in response to HLA-matched melanoma cell lines. Anti-CD3/CD28 produced a larger expansion of T cells with higher levels of IFN-y. Preliminary TCR beta sequencing data revealed a distinct clonal distribution between peripheral and CSF-derived T cells. Conclusions: Therapies for LMD patients are desperately needed. Results demonstrate successful expansion of T cells ex vivo from CSF in M-LMD. Results raise potential to use autologous CSF-derived T cells as therapeutic strategy for patients with LMD.

The novel prognostic marker SPOCK2 regulates tumour progression in melanoma.

Secreted protein acidic and cysteine rich/osteonectin, cwcv and kazal-like domain proteoglycan 2 (SPOCK2) is a protein that regulates cell differentiation and growth. Recent studies have reported that SPOCK2 plays important roles in the progression of various human cancers; however, the role of SPOCK2 in melanoma remains unknown. Therefore, this study investigated the roles of SPOCK2 and the related mechanisms in melanoma progression. To evaluate the clinical significance of SPOCK2 expression in patients with melanoma, we analysed the association between SPOCK2 expression and its prognostic value for patients with melanoma using systematic multiomic analysis. Subsequently, to investigate the roles of Spock2 in melanoma progression invitro and invivo, we knocked down Spock2 in the B16F10 melanoma cell line. High SPOCK2 levels were positively associated with good prognosis and long survival rate of patients with melanoma. Spock2 knockdown promoted melanoma cell proliferation by inducing the cell cycle and inhibiting apoptosis. Moreover, Spock2 downregulation significantly increased cell migration and invasion by upregulating MMP2 and MT1-MMP. The increased cell proliferation and migration were inhibited by MAPK inhibitor, and ERK phosphorylation was considerably enhanced in Spock2 knockdown cells. Therefore, Spock2 could function as a tumour suppressor gene to regulate melanoma progression by regulating the MAPK/ERK signalling pathway. Additionally, Spock2 knockdown cell injection induced considerable tumour growth and lung metastasis in C57BL6 mice compared to that in the control group. Our findings suggest that SPOCK2 plays crucial roles in malignant progression of melanoma and functions as a novel therapeutic target of melanoma.

GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples.

Although not completely understood, the role of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and epithelial skin tumors has been reported before. In this study, we confirmed in various melanoma cell line models that keratin 16 (KRT16) and S100 Calcium-Binding Protein A7 (S100A7) are transcriptional targets of GLI Family Zinc Finger (GLI) proteins. Besides their important role in protecting and maintaining the epidermal barrier, keratins are somehow tightly connected with the S100 family of proteins. We found that stronger expression of KRT16 indeed corresponds to stronger expression of S100A7 in our clinical melanoma samples. We also report a trend regarding staining of GLI1, which corresponds to stronger staining of GLI3, KRT16, and S100A7 proteins. The most interesting of our findings is that all the proteins are detected specifically in the epidermis overlying the tumor, but rarely in the tumor itself. The examined proteins were also not detected in the healthy epidermis at the edges of the sample, suggesting that the staining is specific to the epidermis overlaying the tumor mass. Of all proteins, only S100A7 demonstrated a statistically significant trend regarding tumor staging and staining intensity. Results from our clinical samples prove that immune infiltration is an important feature of melanoma. Pigmentophages and tumor-infiltrating lymphocytes (TIL) demonstrate a significant association with tumor stage, while mononuclear cells are equally present in all stages. For S100A7, we found an association between the number of TILs and staining intensity. Considering these new findings presented in our study, we suggest a more detailed examination of the possible role of the S100A7 protein as a biomarker in melanoma.

Identification of BRAF Inhibitor Resistance-associated lncRNAs Using Genome-scale CRISPR-Cas9 Transcriptional Activation Screening.

Approximately 50% of melanomas harbor the BRAF V600E mutation and targeted therapies using BRAF inhibitors improve patient outcomes. Nonetheless, resistance to BRAF inhibitors develops rapidly and remains a challenge in melanoma treatment. In this study, we attempted to isolate long noncoding RNAs (lncRNAs) involved in BRAF inhibitor resistance using a comprehensive screening method. We used a CRISPR-Cas9 synergistic activation mediator (SAM) protein complex in a genome-scale transcriptional activation assay to screen for candidate lncRNA genes related to BRAF inhibitor resistance. Correlation analysis was performed between expression levels of isolated lncRNA genes and IC50 of dabrafenib in a BRAF-mutated melanoma cell line. Next, online databases were used to construct the lncRNA-miRNA-mRNA regulatory network. Finally, we evaluated the significance of the expression levels of these lncRNAs and mRNAs as biomarkers using clinical specimens. We isolated three BRAF inhibitor resistance-associated lncRNA genes, namely SNHG16, NDUFV2-AS1, and LINC01502. We constructed a lncRNA-miRNA-mRNA network of 13 nodes consisting of three lncRNAs, six miRNAs, and four mRNAs. The lncRNAs and target mRNAs from each regulatory axis significantly and positively correlated with each other. Finally, Kaplan-Meier analysis showed that higher expression levels of MITF, which was up-regulated by LINC01502, were significantly associated with worse prognosis in BRAF V600E-mutated melanoma. The identification of these BRAF inhibitor resistance-associated lncRNA genes at the genomic scale and the establishment of the lncRNA-miRNA-mRNA regulatory network provides new insights into the underlying mechanisms of BRAF inhibitor resistance in melanoma.

MIIP downregulates PD-L1 expression through HDAC6 in cutaneous melanoma

PurposeImmune checkpoint inhibitors have improved the objective response rate and survival of melanoma patients. However, there are still many melanoma patients suffering from disease progression due to primary or secondary immune checkpoint inhibitor resistance, as is observed in the failure of anti-PD1/PD-L1 therapy. While the expression of valuable markers, such as TMB, MSI, and PD-L1, could serve as effective predictors of anti-checkpoint inhibitor therapies, tumor cell PD-L1 expression and its regulating mechanism would significantly affect the anti-PD-1 immunotherapy response and efficacy. Therefore, it is urgent to determine the function of PD-1/PD-L1 expression in melanoma and its associated pathways to enhance the efficacy of anti-PD-1 therapies.MethodsA cohort of 133 patients with histologically confirmed melanoma from Tianjin Medical University Cancer Institute & Hospital were included in this study. We performed immunohistochemical staining to detect the expression of Migration and invasion inhibitory protein (MIIP), HDAC6 and PD-L1. Kaplan–Meier and log-rank test were used for survival analysis. As for vitro, Western blot was used in melanoma cell lines to verify the signaling pathway that MIIP regulates PD-L1 expression.ResultsMIIP expression was decreased in melanoma and that the negative expression of MIIP was correlated with worse overall survival. The positive expression of HDAC6, a molecule that is downstream of MIIP, had a positive trend with decreased overall survival. At the same time, the positive expression of PD-L1, a crucial costimulatory molecule, was associated with decreased overall survival. Furthermore, there was a positive association between HDAC6 and PD-L1 protein expression (p < 0.01), and this correlation is more prominent in cutaneous melanoma than acral melanoma. In cutaneous melanoma cell lines, we found that increasing MIIP led to decreased HDAC6, pSTAT3, and PD-L1 expression. Knocking down MIIP led to increased HDAC6, pSTAT3, and PD-L1 expression. Combining the published results, showing that HDAC6 can regulate PD-L1 expression through STAT3, our present data suggest that MIIP inhibits the expression of PD-L1 by downregulating HDAC6 in melanoma. Most importantly, methods for targeting MIIP-HDAC6-PD-L1 pathways, such as treatment with HDAC6 inhibitors, might indicate a new therapeutic approach for enhancing immune checkpoint inhibitor therapies in melanoma.ConclusionsOur findings highlight the immunomodulatory effects of MIIP in the inhibition of PD-L1 expression by downregulating HDAC6 in melanoma. Methods for targeting MIIP-HDAC6-PD-L1 pathways might be new therapeutic approaches for enhancing immune checkpoint inhibitor therapies in melanoma.

Effect of a novel siRNA delivery system, siRNA ternary complex, on melanoma lung metastasis

In this study, we determined effects of an anionic siRNA delivery vector, siRNA ternary complex, which is constructed with biodegradable dendrigraft poly-L-lysine (DGL) and γ-polyglutamic acid (γ-PGA) on the melanoma cells and melanoma lung metastasis. The siRNA ternary complex showed high cellular uptake and silencing effect in melanoma cell line B16-F10/Luc cells. After intravenous administration of the siRNA ternary complex, high silencing effect was also observed in the lung of B16-F10/Luc melanoma metastasis model mice. Therefore, we applied vascular endothelial growth factor (VEGF)-siRNA on the siRNA ternary complex and determined the effect on the melanoma lung metastasis. The siRNA ternary complex containing VEGF-siRNA reduced VEGF protein levels significantly in in vitro and in vivo, and the complex successfully inhibited melanoma lung metastasis. This biodegradable and effective siRNA delivery vector, siRNA ternary complex, could be available for clinical trials.

Sex-dependent interaction of PTGS2 with miR-146a as risk factor for melanoma and the impact of sex hormones in gene expression in skin cells.

Gender disparity in melanoma is a complex issue where sex hormones could be engaged. Differences in genetic variations are important in understanding the mechanisms of sex disparity in melanoma. Post-transcriptional regulation of prostaglandin-endoperoxide synthase (PTGS2) mRNA occurs through a complex interplay of specific trans-acting RNA-binding proteins and microRNAs. MiR-146a is a key player in melanoma, modulating immune responses and tumor microenvironment (TME). Polymorphisms in PTGS2 gene rs20415G<C and miR-146a gene rs2910164G>C have been associated with an increased risk of melanoma. Epistasis between polymorphisms rs20415G<C and rs2910164G>C was investigated by genotyping 453 melanoma patients and 382 control individuals. The effects of testosterone and 17β-estradiol were analyzed in keratinocytes and two melanoma cell lines. The rs2910164GG showed a higher risk in the presence of the genotype rs20417CC in the male population. Testosterone and 17β-estradiol act differently on PTGS2 and miR-146a expression, depending on the cell type. Testosterone augments PTGS2 gene expression in keratinocytes and miR-146a in melanoma cells. While 17β-estradiol only increases miR-146a expression in HaCaT cells. The present study indicates a sex-specific relation between miR-146a and PTGS2 polymorphisms with melanoma cancer risk. Testosterone and 17β-estradiol act differently on the expression of PTGS2 and miR-146a depending on the skin cell type.

Loss of p14 diminishes immunogenicity in melanoma via non-canonical Wnt signaling by reducing the peptide surface density.

Immunotherapy has achieved tremendous success in melanoma. However, only around 50% of advanced melanoma patients benefit from immunotherapy. Cyclin-dependent kinase inhibitor 2A (CDKN2A), encoding the two tumor-suppressor proteins p14ARF and p16INK4a, belongs to the most frequently inactivated gene loci in melanoma and leads to decreased Tcell infiltration. While the role of p16INK4a has been extensively investigated, knowledge about p14ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished their recognition and killing by melanoma differentiation antigen (MDA)-specific Tcells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. Immunopeptidomic analyses revealed that antigen presentation via human leukocyte antigen class I (HLA-I) molecules was enhanced upon p14ARF downregulation in general, but absolute and relative expression of cognate peptides was decreased. However, this phenotype is associated with a favorable outcome for melanoma patients. Limiting Wnt5a signaling reverted this phenotype, suggesting an involvement of non-canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA-specific Tcell responses by decreasing both absolute and relative MDA-peptide presentation in melanoma.

Curcumin modulates purinergic signaling and inflammatory response in cutaneous metastatic melanoma cells.

Cutaneous melanoma (CM) poses a therapeutic challenge due to its aggressive nature and often limited response to conventional treatments. Exploring novel therapeutic targets is essential, and natural compounds have emerged as potential candidates. This study aimed to elucidate the impact of curcumin, a natural compound known for its anti-inflammatory, antioxidant, and anti-tumor properties, on metastatic melanoma cells, focusing on the purinergic system and immune responses. Human melanoma cell line SK-Mel-28 were exposed to different curcumin concentrations for either 6 or 24h, after which we assessed components related to the purinergic system and the inflammatory cascade. Using RT-qPCR, we assessed the gene expression of CD39 and CD73 ectonucleotidases, as well as adenosine deaminase (ADA). Curcumin effectively downregulated CD39, CD73, and ADA gene expression. Flow cytometry analysis revealed that curcumin significantly reduced CD39 and CD73 protein expression at specific concentrations. Moreover, the A2A receptor's protein expression decreased across all concentrations. Enzymatic activity assays demonstrated that curcumin modulated CD39, CD73, and ADA activities, with effects dependent on concentration and duration of treatment. Extracellular ATP levels increased after 24h of curcumin treatment, emphasizing its role in modulating hydrolytic activity. Curcumin also displayed anti-inflammatory properties by reducing NLRP3 gene expression and impacting the levels of key inflammatory cytokines. In conclusion, this study unveils the potential of curcumin as a promising adjuvant in CM treatment. Curcumin modulates the expression and activity of crucial components of the purinergic system and exhibits anti-inflammatory effects, indicating its potential therapeutic role in combating CM. These findings underscore curcumin's promise and warrant further investigation in preclinical and clinical settings for melanoma management.

Singlet oxygen production of Zn-Ag-In-S quantum dots for photodynamic treatment of cancer cells and bacteria.

Zn-Ag-In-S (ZAIS) quantum dots (QDs) were synthesized with various Ag-to-In ratios and used as novel photosensitizers for photodynamic therapy (PDT) on cancer cell inhibition and bacterial sterilization, and their structural, optical, and photodynamic properties were investigated. The alloyed QDs displayed a photoluminescence quantum yield of 72% with a long fluorescence lifetime of 5.3μs when the Ag-to-In ratio was 1:3, suggesting a good opportunity as a dual functional platform for fluorescence imaging and PDT. The ZAIS QDs were then coated with amphiphilic brush copolymer poly(maleic anhydride-alt-1-octadecene) (PMAO) before application. The 1O2 quantum yield of the ZAIS/PMAO was measured to be 8%, which was higher than previously reported CdSe QDs and comparable to some organic photosensitizers. Moreover, the ZAIS QDs showed excellent stability in aqueous and biological media, unlike organic photosensitizers that tend to degrade over time. The in vitro PDT against human melanoma cell line (A2058) and Staphylococcus aureus shows about 30% inhibition rate upon 20min light irradiation. Cell staining images clearly demonstrated that co-treatment with ZAIS QDs and light irradiation effectively killed A2058 cells, demonstrating the potential of ZAIS QDs as novel and versatile photosensitizers for PDT in cancer and bacterial treatment, and provides useful information for future designing of QD-based photosensitizers.

Anti-apoptotic, anti-inflammatory, and anti-melanogenic effects of the ethanol extract of Picrasma quassioides (D. Don) Benn

Ethnopharmacological relevancePicrasma quassioides (D. Don) Benn is a vascular plant belonging to the genus Picrasma of Simaroubaceae family and grows in Korea, China, India, Taiwan, and Japan. Picrasma quassioides extract has been reported to have anti-inflammatory, anti-bacterial, and anti-cancer properties. Moreover, this plant has been also traditionally used to alleviate symptoms of eczema, atopic dermatitis, psoriasis, scabies, and boils in skin. Aim of the studyThe Pq-EE has been reported in Chinese pharmacopoeia for its pharmacological effects on skin. However, the detailed mechanism on alleviating skin conditions is not understood. Hence, we investigated the skin improvement potential of Pq-EE against skin damage. Materials and methodsWe used the human keratinocyte cell line (HaCaT) and mouse melanoma cell line (B16F10) to study the effects of Pq-EE on the epidermis. Additionally, in vitro antioxidant assays were performed using a solution that included either metal ions or free radicals. ResultsIn colorimetric antioxidant assays, Pq-EE inhibited free radicals in a dose-dependent manner. The Pq-EE did not affect cell viability and promoted cell survival under UVB exposure conditions in the MTT assay. The Pq-EE downregulated the mRNA levels of apoptotic factors. Moreover, MMP1 and inflammatory cytokine iNOS mRNA levels decreased with Pq-EE treatment. With regard to protein levels, caspases and cleaved caspases were more powerfully inhibited by Pq-EE than UVB-irritated conditions. p53 and Bax also decreased with Pq-EE treatment. The melanin contents and secretion were decreased at nontoxic concentrations of Pq-EE. The pigmentation pathway genes also were inhibited by treatment with Pq-EE. ConclusionsIn summary, we suggest the cell protective potential of Pq-EE against UVB and ROS, indicating its use in UV-protective cosmeceutical materials.

Extracellular vesicles promote migration despite BRAF inhibitor treatment in malignant melanoma cells.

Extracellular vesicles (EVs) constitute a vital component of intercellular communication, exerting significant influence on metastasis formation and drug resistance mechanisms. Malignant melanoma (MM) is one of the deadliest forms of skin cancers, because of its high metastatic potential and often acquired resistance to oncotherapies. The prevalence of BRAF mutations in MM underscores the importance of BRAF-targeted therapies, such as vemurafenib and dabrafenib, alone or in combination with the MEK inhibitor, trametinib. This study aimed to elucidate the involvement of EVs in MM progression and ascertain whether EV-mediated metastasis promotion persists during single agent BRAF (vemurafenib, dabrafenib), or MEK (trametinib) and combined BRAF/MEK (dabrafenib/trametinib) inhibition.Using five pairs of syngeneic melanoma cell lines, we assessed the impact of EVs - isolated from their respective supernatants - on melanoma cell proliferation and migration. Cell viability and spheroid growth assays were employed to evaluate proliferation, while migration was analyzed through mean squared displacement (MSD) and total traveled distance (TTD) measurements derived from video microscopy and single-cell tracking.Our results indicate that while EV treatments had remarkable promoting effect on cell migration, they exerted only a modest effect on cell proliferation and spheroid growth. Notably, EVs demonstrated the ability to mitigate the inhibitory effects of BRAF inhibitors, albeit they were ineffective against a MEK inhibitor and the combination of BRAF/MEK inhibitors. In summary, our findings contribute to the understanding of the intricate role played by EVs in tumor progression, metastasis, and drug resistance in MM.

DEHP (di(2-ethylhexyl)phthalate) stimulates skin pigmentation by perturbing cytoskeletal homeostasis.

Phthalates are extensively employed plasticizers crucial for conferring flexibility and plasticity to polyvinyl chloride. Phthalates, including DEHP (di(2-ethylhexyl)phthalate), present in diverse products, have been identified in fine dust and are capable of infiltrating the body, potentially posing health hazards. Importantly, melanocytes, existing at the basal layer of the epidermis, are susceptible to toxic substances. In our study, we employed the 3D human pigmented epidermis model, MelanoDerm™, along with the B16F10 murine melanoma cell line, to examine the influence of DEHP exposure on melanocytes. The exposure to low concentrations of DEHP (~ 5μM), resulted in the extension of melanocyte dendrites, indicating the stimulation of melanocytes. Analysis of gene expression and protein profiles unveiled the up-regulation of MITF, Arpc2, and TRP1 genes subsequent to DEHP exposure, indicating alterations in cytoskeletal and melanosome-related genetic and protein components in melanocytes. Notably, increased pigmentation was observed in MelanoDerm™ following DEHP exposure. DEHP-stimulated reactive oxygen species generation appeared to be involved in these events since the antioxidant, ascorbic acid attenuated ROS generation and MITF upregulation. Collectively, our study demonstrated that DEHP exposure can induce cytoskeletal disturbance and skin pigmentation through oxidative stress.

The effect of gD-derived peptides on T cell immune response mediated by BTLA-HVEM protein complex in melanoma patients.

The effector function of T cells is regulated via immune checkpoints, activating or inhibiting the immune response. The BTLA-HVEM complex, the inhibitory immune checkpoint, may act as one of the tumor immune escape mechanisms. Therefore, interfering with the binding of these proteins can prove beneficial in cancer treatment. Our study focused on peptides interacting with HVEM at the same place as BTLA, thus disrupting the BTLA-HVEM interaction. These peptides' structure and amino acid sequences are based on the gD protein, the ligand of HVEM. Here, we investigated their immunomodulatory potential in melanoma patients. Flow cytometry analyses of activation, proliferation, and apoptosis of T cells from patients were performed. Additionally, we evaluated changes within the T cell memory compartment. The most promising compound - Pep(2), increased the percentages of activated T cells and promoted their proliferation. Additionally, this peptide affected the proliferation rate and apoptosis of melanoma cell line in co-culture with T cells. We conclude that the examined peptide may act as a booster for the immune system. Moreover, the adjuvant and activating properties of the gD-derived peptide could be used in a combinatory therapy with currently used ICI-based treatment. Our studies also demonstrate that even slight differences in the amino acid sequence of peptides and any changes in the position of the disulfide bond can strongly affect the immunomodulatory properties of compounds.

Investigation of Anticancer Properties of 2-benzylidene-1-indanone and Its Derivatives by DFT and Molecular Docking

In this study, 2-benzylidene-1-indanone and its derivatives, which is a chalcone compound and contains indanone in its structure, were examined. Quantum chemical parameters for these compounds were calculated with the B3LYP method and the 6-31G(d) basis set and evaluated for their biological activity. The effect of different functional groups (F, Cl, Br, CF3, CH3 and OCH3) attached to the 2-benzylidene-1-indanone compound on biological activity was investigated. Some quantum chemical parameters such as highest energy filled molecule orbital energy (EHOMO), lowest non-bonding empty molecule orbital energy (ELUMO), energy gap (ΔE), hardness (η), softness (σ), global molecular electrophilicity (ω) index, global molecular nucleophilicity (ɛ) index, electron-accepting (ω+) and electron-donating (ω-) electrophilicity index were calculated for the biological activities of the compounds. Frontier molecular orbitals and molecular electrostatic potential (MEP) maps were interpreted. The biological activities of 2-benzylidine-1-indanone and some of its derivatives bearing the 1-indanone skeleton were evaluated by performing molecular docking studies with the target protein PDB ID = 1HJD corresponding to the melanoma cell line. The activity ranking obtained with quantum chemical parameters was found to be compatible with the binding energies obtained from docking results.

Neratinib, a pan ERBB/HER inhibitor, restores sensitivity of PTEN-null, BRAFV600E melanoma to BRAF/MEK inhibition

IntroductionApproximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy.MethodsTo compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition.ResultsRNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells.ConclusionsThe findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.

Bcl-2 dependent modulation of Hippo pathway in cancer cells

IntroductionBcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2.MethodsWe performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts.ResultsWe demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation.ConclusionsWe discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

F127/chlorin e6-nanomicelles to enhance Ce6 solubility and PDT-efficacy mitigating lung metastasis in melanoma.

Photodynamic Therapy (PDT) is a promising paradigm for treating cancer, especially superficial cancers, including skin and oral cancers. However, the effectiveness of PDT is hindered by the hydrophobicity of photosensitizers. Here, chlorin e6 (Ce6), a hydrophobic photosensitizer, was loaded into pluronic F127 micelles to enhance solubility and improve tumor-specific targeting efficiency. The resulting Ce6@F127 Ms demonstrated a significant increase in solubility and singlet oxygen generation (SOG) efficiency in aqueous media compared to free Ce6. The confocal imaging and fluorescence-activated cell sorting (FACS) analysis confirmed the enhanced internalization rate of Ce6@F127 Ms in murine melanoma cell lines (B16F10) and human oral carcinoma cell lines (FaDu). Upon laser irradiation (666nm), the cellular phototoxicity of Ce6@F127 Ms against B16F10 and FaDu was approximately three times higher than the free Ce6 treatment. The in vivo therapeutic investigations conducted on a murine model of skin cancer demonstrated the ability of Ce6@F127 Ms, when combined with laser treatment, to penetrate solid tumors effectively, which resulted in a significant reduction in tumor volume compared to free Ce6. Further, the Ce6@F127 Ms demonstrated upregulation of TUNEL-positive cells, downregulation of proliferation markers in tumor tissues, and prevention of lung metastasis with insignificant levels of proliferating cells and collagenase, as validated through immunohistochemistry. Subsequent analysis of serum and blood components affirmed the safety and efficacy of Ce6@F127 Ms in mice. Consequently, the developed Ce6@F127 Ms exhibits significant potential for concurrently treating solid tumors and preventing metastasis. The photodynamic formulation holds great clinical translation potential for treating superficial tumors.

Designing JAK2 Inhibitors Beyond Myeloproliferative Neoplasms ‐ Theoretical and Experimental Analysis for Solid Cancers

AbstractSomatic mutations in JAK2 have been reported to cause myeloproliferative neoplasms, autoimmune disorders, rheumatoid arthritis, and inflammation. JAK2 protein with PDB id 3FUP was selected for docking analysis via cross‐docking. Molecular docking of JAK2 with ATP and crystal ligand revealed their binding affinities and interactions. 3D QSAR models with R2=0.997 and Q2=0.984 having high stability and predictivity were generated. The biological activity of the hit compound obtained from virtual screening, LAS 26879206, was predicted to be 4.21 using the 3D QSAR model. Modification of LAS 26879206 utilizing 3D QSAR contours for modulating its JAK2 inhibitory potential yielded two novel inhibitors with a 2‐aminopyrimidine core – ACP1 and ACPM1. Molecular docking, molecular dynamics simulations, and ADMET analysis indicated improvements in the efficiency of these compounds compared to the hit compound. Synthesis of these compounds was carried out using a two‐step reaction‐ first Buchwald – Hartwig coupling reaction to yield an intermediate ACB1 which was then coupled with appropriate boronic acids via Suzuki reaction to get ACP1 and ACPM1. The effects of these designed compounds as anticancer agents were analyzed in hCT‐116 (human colorectal carcinoma), HeLa (cervical carcinoma), A549 (human lung adenocarcinoma), and A375 (human melanoma) cell lines. ACPM1 was found to be active in all the cell lines with a pIC50 of 5 which was in concordance with the in silico studies. Western blot analysis validated the JAK2 inhibitory property of ACPM1 which can be further ensured by in vivo studies.

Hexagon Gold Nanoring-Mediated Combined Phototherapy for the Treatment of B16 Melanoma Cell Line

Hexagon Gold Nanoring-Mediated Combined Phototherapy for the Treatment of B16 Melanoma Cell Line