Mouse Melanoma Cell Line Research Articles

Abstract 1256: Transglutaminase-2 is elevated in the invasive cell state and modulates the tumor immune microenvironment in cutaneous melanoma

Abstract Cutaneous melanoma is the most fatal skin cancer; resistance to targeted therapies contributes to poor prognosis. In some cases, resistance arises from pre-existing tumor heterogeneity, which allows subpopulations of drug-tolerant cells with distinct transcriptional states to survive in the presence of drug. The loss of SOX10, a lineage-specific transcription factor, is known to drive melanoma phenotype switching from a proliferative to an invasive drug-tolerant state. Here, we found that knocking out SOX10 in human and mouse melanoma cell lines leads to the upregulation of transglutaminase-2 (TGM2), a calcium-dependent cross-linking enzyme. Analysis of publicly available bulk and single-cell RNA-sequencing data showed that TGM2 is highly expressed in the invasive cell state in melanoma cell lines and patient-derived melanoma cultures, respectively. Furthermore, knockdown of SOX10 in patient-derived melanoma cultures increases TGM2 mRNA expression. 3D spheroid assays and extracellular matrix production/remodeling assays showed that knockdown of TGM2 has no effect on the invasiveness of SOX10-deficient cells. However, we found that TGM2 is secreted by SOX10-deficient cells and hypothesized that it could modulate the tumor immune microenvironment (TIME) given that TGM2 has been associated with increased immune infiltration in melanoma patient samples. To investigate the effect of TGM2 on the TIME, we stably overexpressed TGM2/Tgm2 in syngeneic mouse melanoma cell lines and allowed tumors to grow in immune-competent C57BL/6 mice. As compared to empty vector, TGM2/Tgm2-overexpression led to an increase in the percentage and number of CD4+ T cells and B cells, and a decrease in the number of myeloid cells, in the tumor immune compartment by flow cytometry analysis. Future studies will assess which subsets of CD4+ T cells and B cells infiltrate TGM2/Tgm2-overexpressing tumors, how these changes to the TIME affect melanoma invasiveness, and if TGM2 expression affects response to targeted therapy in vivo. Overall, our data suggest that TGM2 is highly expressed in the invasive melanoma cell state, is regulated by the SOX10 transcription factor, and can modulate adaptive immune cells in the TIME. Citation Format: Signe Caksa, Nicole A. Wilski, Erica L. Kitterman, McKenna Q. Glasheen, Jacob S. Heilizer, Timothy J. Purwin, Claudia Capparelli, Andrew E. Aplin. Transglutaminase-2 is elevated in the invasive cell state and modulates the tumor immune microenvironment in cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1256.

Abstract 2536: Intracellular Ctla4 overexpression promotes melanoma progression and metastasis

Abstract Cytotoxic T Lymphocyte Antigen 4 (CTLA4) expressed on the surface of T cells is an important immunotherapeutic target in many cancers, including melanoma. However, we previously found highly upregulated expression of CTLA4 in BRAF/NRAS-mutant human melanoma cell lines and tissues. Although CTLA4 is almost entirely expressed intracellularly in melanoma cells, its intracellular functions have remained elusive. Ectopic expression of Ctla4 (Ctla4-ee) in mouse melanoma cell lines significantly promoted lung colonization in syngeneic mice as well as in immunocompromised mice, suggesting that Ctla4 had pro-tumorigenic effects independent of its extracellular immune checkpoint function. Proteomics analysis identified apoptosis to be a major affected pathway and we found that Ctla4 ectopic-expressing mouse melanoma cells (B2905-Ctla4-ee) were significantly resistant to doxorubicin-induced apoptosis as compared to empty vector (EV) controls. In a reciprocal experiment, knocking out CTLA4 in the Hs936T human melanoma cells, that exhibit high endogenous CTLA4 expression, showed significantly increased apoptosis than the parental cells. Moreover, significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) were observed in CTLA4-ee cells as compared to EV controls. CTLA4-ee cells also showed significantly higher invasion in Matrigel-coated Transwell assay. Interestingly, we also found that CTLA4 translocated to mitochondria, where it downregulated oxidative phosphorylation and increased glycolysis. These findings lead us to hypothesize that CTLA4 plays a significant role in regulating apoptosis and mitochondrial metabolic functions, leading to the promotion of melanoma progression and metastasis. Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sarah Preston Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, John Elrod, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. Intracellular Ctla4 overexpression promotes melanoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2536.

Abstract 5188: Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models

Abstract Brain metastases (BrM) remain an intractable, deadly complication for advanced melanoma patients and efficient therapeutic strategies are desperately needed. The tumor microenvironment (TME) plays an important role in response to therapy. However, studies addressing the contribution of the TME to therapy efficacy for BrM are lacking, mostly due to limited access to human samples and scarcity of appropriate preclinical models. Here, we describe two novel isogenic immunocompetent BrM models generated by intracardiac injection of UV-induced mouse melanoma cell lines, representative of mutant-RAS human melanoma subtypes. We used these models to test immune checkpoint blockade (ICB) therapy and to interrogate the role of the TME in therapeutic efficacy. To evaluate response, we developed and applied a new machine-learning method to quantify metastatic burden. We investigated the TME by high-parametric flow cytometry and single-cell RNA sequencing (scRNA-seq). We showed that the models have distinct metastatic behaviors, with BR1 being mostly brain tropic and BR3 displaying widespread metastases. Notably, BR1 BrM were sensitive to ICB with a better response to anti-PD-L1/anti-CTLA-4 combination therapy as compared to monotherapies. In contrast, BR3 BrM were resistant to both mono- and combination therapies. Interestingly, we found that ICB efficacy on extracranial BR3 metastases is organ-dependent. Characterization of the BrM immune microenvironment before and after treatment revealed dramatic differences between the models. Untreated BR1 BrM showed significant recruitment of T cells, dendritic cells, and natural killer cells, while neutrophils were enriched in untreated ICB-resistant BR3 BrM. Moreover, we uncovered phenotypically distinct microglia populations exclusively present in ICB-sensitive BR1 BrM that positively correlated with T cell infiltration. Consistent with this finding, scRNA-seq showed upregulation of genes encoding for T cell-attracting chemokines and antigen presentation uniquely in the BR1-associated microglia. Post-treatment analysis of the brain TME highlighted beneficial changes induced by ICB in the responsive BR1 model, including increased recruitment of CD8 T cells with an activated phenotype, while a mild recruitment of exhausted T cells was observed in the resistant BR3 model. Altogether, our data emphasize the importance of interrogating the BrM TME to understand therapeutic response. Our unique BrM models, mirroring the diversity of ICB response observed in patients, provide a robust platform for the much-needed mechanistic studies to optimize BrM therapy. Deciphering the contribution of the newly identified BR1 BrM-associated microglia to ICB efficacy will be crucial to the identification of novel therapeutic targets. Citation Format: Amélie Lopès, Jessica Rappaport, Eva Pérez Guijarro, Quanyi Chen, Emily Wu, Isabella Church, April Huang, Jessica Bridge, Sung Chin, Cari Smith, Charli Gruen, Khiem C. Lam, Romina E. Araya, Antonella Sassano, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid. Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5188.

Derivation and Use of Cell Lines from Mouse Models of Melanoma

The establishment of consistent genetically modified mouse melanoma models and cell lines is of paramount importance for prevention and treatment. In this study, we review the different mouse melanoma cell lines that have been established. After careful molecular characterization of the established mouse melanoma cell lines, modification of the genome, microenvironment, or even the environment using appropriate in cellulo and invivo assays may reveal novel genetic and nongenetic changes. These murine melanoma cell lines with defined genetic mutations allow the testing of innovative therapies based on chemistry, physics, and biology using alternative methods. In addition to the fundamental aspects, these results are important for humans because of the relevance of these murine melanoma cell lines to human disease.

흰색 무궁화꽃과 분홍색 무궁화꽃 추출물의 항산화, 멜라닌 합성 저해 및 항균활성 비교

This study investigates the bioactivities of Hibiscus syriacus (H. syriacus) L. flower extracts on dermal cells and bacteria. The white and pink H. syriacus L. flower extracts showed no or minimal cytotoxicity on HaCaT (human keratinocyte) and B16F10 (mouse melanoma) cell lines. H. syriacus L. flower extract inhibited oxidative stress, and its effect was superior to the effect of glutathione. The antioxidant efficacy of pink H. syriacus L. flower extract was higher than the white flower extract. The chloroform layer of the white H. syriacus L. flower extract (40 μg/mL) inhibited melanin synthesis and showed remarkable antibacterial effects against Staphylococcus aureus. The extracted layer also exhibited antibacterial effects on Staphylococcus epidermidis and Pseudomonas aeruginosa. Based on the bioactivity experiment, the white H. syriacus L. flower extract was more effective than the pink flower extract in inhibiting melanin synthesis and antibacterial efficacy. The results of this study indicate potential applications of H. syriacus L. flowers in cosmetics.

Anti-Melanogenesis Effects of a Cyclic Peptide Derived from Flaxseed via Inhibition of CREB Pathway.

Linosorbs (Los) are cyclic peptides from flaxseed oil composed of the LO mixture (LOMIX). The activity of LO has been reported as being anti-cancer and anti-inflammatory. However, the study of skin protection has still not proceeded. In particular, there are poorly understood mechanisms of melanogenesis to LO. Therefore, we investigated the anti-melanogenesis effects of LOMIX and LO, and its activity was examined in mouse melanoma cell lines. The treatment of LOMIX (50 and 100 μg/mL) and LO (6.25-50 μM) suppressed melanin secretion and synthesis, which were 3-fold increased, in a dose-dependent manner, up to 95%. In particular, [1-9-NαC]-linusorb B3 (LO1) and [1-9-NαC]-linusorb B2 (LO2) treatment (12.5 and 25 μM) highly suppressed the synthesis of melanin in B16F10 cell lines up to 90%, without toxicity. LOMIX and LOs decreased the 2- or 3-fold increased mRNA levels, including the microphthalmia-associated transcription factor (MITF), Tyrosinase, tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2) at the highest concentration (25 μM). Moreover, the treatment of 25 μM LO1 and LO2 inhibited the expression of MITF and phosphorylation of upper regulatory proteins such as CREB and PKA. Taken together, these results suggested that LOMIX and its individual LO could inhibit melanin synthesis via downregulating the CREB-dependent signaling pathways, and it could be used for novel therapeutic materials in hyperpigmentation.

Biophysical informatics reveals distinctive phenotypic signatures and functional diversity of single-cell lineages.

In this work, we present an analytical method for quantifying both single-cell morphologies and cell network topologies of tumor cell populations and use it to predict 3D cell behavior. We utilized a supervised deep learning approach to perform instance segmentation on label-free live cell images across a wide range of cell densities. We measured cell shape properties and characterized network topologies for 136 single-cell clones derived from the YUMM1.7 and YUMMER1.7 mouse melanoma cell lines. Using an unsupervised clustering algorithm, we identified six distinct morphological subclasses. We further observed differences in tumor growth and invasion dynamics across subclasses in an in vitro 3D spheroid model. Compared to existing methods for quantifying 2D or 3D phenotype, our analytical method requires less time, needs no specialized equipment and is capable of much higher throughput, making it ideal for applications such as high-throughput drug screening and clinical diagnosis. https://github.com/trevor-chan/Melanoma_NetworkMorphology. Supplementary data are available at Bioinformatics online.

MeVa2.1.dOVA and MeVa2.2.dOVA: two novel BRAFV600E-driven mouse melanoma cell lines to study tumor immune resistance.

While immunotherapy has become standard-of-care for cutaneous melanoma patients, primary and acquired resistance prevent long-term benefits for about half of the late-stage patients. Pre-clinical models are essential to increase our understanding of the resistance mechanisms of melanomas, aiming to improve the efficacy of immunotherapy. Here, we present two novel syngeneic transplantable murine melanoma cell lines derived from the same primary tumor induced on BrafV600E Pten-/- mice: MeVa2.1 and MeVa2.2. Derivatives of these cell lines expressing the foreign antigen ovalbumin (dOVA) showed contrasting immune-mediated tumor control. MeVa2.2.dOVA melanomas were initially controlled in immune-competent hosts until variants grew out that had lost their antigens. By contrast, MeVa2.1.dOVA tumors were not controlled despite presenting the strong OVA antigen, as well as infiltration of tumor-reactive CD8+ T cells. MeVa2.1.dOVA displayed reduced sensitivity to T cell-mediated killing and growth inhibition in vitro by both IFN-γ and TNF-α. MeVa2.1.dOVA tumors were transiently controlled in vivo by either targeted therapy, adoptive T cell transfer, regulatory T cell depletion, or immune checkpoint blockade. MeVa2.1.dOVA could thus become a valuable melanoma model to evaluate novel immunotherapy combinations aiming to overcome immune resistance mechanisms.

Rolling Circle Amplification-Based DNA Nano-Assembly for Targeted Drug Delivery and Gene Therapy.

Combining the killing ability of chemotherapy drugs on tumor cells with the inhibiting ability of genetic drugs on tumor cell growth, a dual drug delivery system loaded with therapy drugs and siRNA has gradually received more and more research and extensive attention. In this paper, we designed a DNA nano-assembly based on rolling circle amplification that can co-deliver doxorubicin (Dox) and siRNA simultaneously. In order to fully exploit the potential of the dual loading system in cancer treatment, we selected STAT3 gene as a target and used siRNA to target STAT3 of mRNA and reduce the STAT3 expression in mouse melanoma cell line (B16); meanwhile, Dox as a chemotherapy drug was combined with multivalent aptamers specifically targeting B16 to achieve efficient delivery of siRNA and Dox. The results showed that the synergistic delivery system could achieve high efficiency in targeting and inhibiting proliferation in mouse melanoma cells. In addition, the synergistic effect of the dual delivery system on apoptosis of cancer cells was significantly better than that of single drug delivery systems.

Interferon-gamma signaling promotes melanoma progression and metastasis.

Interferon-gamma (IFNG) has long been regarded as the flag-bearer for the anti-cancer immunosurveillance mechanisms. However, relatively recent studies have suggested a dual role of IFNG, albeit there is no direct experimental evidence for its potential pro-tumor functions. Here we provide in vivo evidence that treatment of mouse melanoma cell lines with Ifng enhances their tumorigenicity and metastasis in lung colonization allograft assays performed in immunocompetent syngeneic host mice, but not in immunocompromised host mice. We also show that this enhancement is dependent on downstream signaling via Stat1 but not Stat3, suggesting an oncogenic function of Stat1 in melanoma. The experimental results suggest that melanoma cell-specific Ifng signaling modulates the tumor microenvironment and its pro-tumorigenic effects are partially dependent on the γδ T cells, as Ifng-enhanced tumorigenesis was inhibited in the TCR-δ knockout mice. Overall, these results show that Ifng signaling may have tumor-promoting effects in melanoma by modulating the immune cell composition of the tumor microenvironment.

Phenolic from apple blossom "Hongro" inhibits the expression of proteins related to melanogenesis in B16F10 melanoma cells.

This study aimed to assess apple blossom extracts as potential natural whitening agents due to their ability to inhibit melanogenesis. Ethanol extracts of apple blossom (ABE) were assessed for biological activity in the B16F10 mouse melanoma cell line. ABE toxicity was assessed by thiazolyl blue tetrazolium bromide (MTT) assay. Levels of melanogenic enzyme expression in response to ABE supplementation were assessed by western blotting. Also assessed purified kaempferol, one of the phenolic compounds extracted from apple blossom, was evaluated using western blot analysis. The expression levels of cellular tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase-related protein (TRP)-1, and TRP-2 proteins related to melanogenesis decreased in a dose-dependent manner with ABE treatment of cells. Using nuclear magnetic resonance, we identified kaempferol in the ABE. Treatment of cells with purified kaempferol decreased the expression levels of tyrosinase and the MITF protein to a similar degree as that observed with ABE treatment. This suggests that the efficacy of melanogenesis-related inhibition demonstrated by ABE was due to kaempferol. ABE has an inhibitive effect on melanogenic enzymes and potentially can be applied to functional foods and cosmetics having a whitening effect as a natural material.

Raptinal Induces Gasdermin E-Dependent Pyroptosis in Naïve and Therapy-Resistant Melanoma.

Raptinal can rapidly induce pyroptosis in naïve and BRAFi plus MEKi-resistant melanoma, which may be beneficial for patients who have developed acquired resistance to targeted therapies.

Imiquimod-induced ROS production causes lysosomal membrane permeabilization and activates caspase-8-mediated apoptosis in skin cancer cells

BackgroundLysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteolytic enzymes, including cathepsins (CTSs), which results in mitochondrial dysfunction and apoptosis. Imiquimod (IMQ), a synthetic TLR7 ligand, has both antiviral and antitumor activity against various skin malignancies in clinical treatment. Previously, we demonstrated IMQ not only caused lysosomal dysfunction but also triggered lysosome biogenesis to achieve lysosomal adaptation in cancer cells. ObjectiveTo determine whether lysosomes are involved in IMQ-induced apoptosis. MethodsThe human skin cancer cell lines BCC, A375 and mouse melanoma cell line B16F10 were used in all experiments. Cell death was determined by the Cell Counting Kit-8 (CCK-8) assay and DNA content assay. Protein expression was determined by immunoblotting. Caspase-8 activity was assessed using a fluorescence caspase-8 kit and determined by flow cytometry and confocal microscopy. ResultsIMQ not only induced lysosome damage but also abrogated lysosome function in skin cancer cells. IMQ-induced caspase-8 activation contributed to the processes of lysosomal cell death. Moreover, the use of ROS scavengers significantly abolished caspase-8 activation and inhibited IMQ-induced LMP. Additionally, pharmacological inhibition of CTSD not only abrogated caspase-8 activation but also rescued IMQ-induced cell death. Finally, lysosome-alkalizing agents enhanced the cytotoxicity of IMQ in vitro and in vivo. ConclusionsIMQ-induced ROS accumulation promotes LMP, releases CTSs into the cytosol, stimulates caspase-8 activation and finally causes lysosomal cell death. Lysosomal cell death and the CTSD/caspase-8 axis may play a crucial role in IMQ-induced cell death.

Unique enantiopure camphor-based neutral arene–ruthenium(II) complexes: DNA/BSA binding, kinetic and cytotoxic studies

Two neutral enantiopure arene-ruthenium(II) complexes, [(arene)RuII(κ2(1S,3R)-Ar)Cl2] (1, 2), arene = p-cymene, Ar = o-tolyl or mesityl, derived from the reaction of arene-ruthenium(II) dimers and 1,3-diamino camphor based derivatives, were synthesized. The N1-mesityl-2,2,3-trimethylcyclopentane-1,3-diamine ligand and complex 2 are characterized via X-ray crystallography. Single crystal X-ray structure analysis revealed that 2 crystallizes in monoclinic space group P21 with Z = 4, a = 7.7724(14), b = 14.262(3), c = 14.433(3) Å. Reactivity measurements using UV-Vis of the RuII-arene complexes with guanosine-5′-monophosphate (5′-GMP), 9-methylguanine (9MeG), and L-methionine (L-Met) showed a high affinity towards these nucleophiles. Also, DFT calculations with 2 showed that the reaction with a guanine derivative is thermodynamically favored. Furthermore, the RuII-arene complexes were found to interact with CT-DNA and HT-DNA, and protein BSA. The cytotoxicities of 1 and 2 have been evaluated against human breast cancer cell line (MDA-MB), human cancer colon cell line (HCT-116), human lung carcinoma epithelial cell line (A549), mouse melanoma cell line (B16F10), and mesenchymal stem cells (MSCs) and compared to cisplatin as standard analog. The complexes display relevant activities against all cancer cell lines.

Peripheral benzodiazepine receptor TSPO needs to be reconsidered before using as a drug target for a pigmentary disorder.

The peripheral benzodiazepine receptor (TSPO/PBR) is highly conserved among different species but with perplexing biochemical functions. Multiple ligands of TSPO show commendable regulatory activities in lots of biological functions, such as neuro-protection, cholesterol transport, and so on. These researches support that TSPO may be a potential target for disease treatment and drug development. Previous studies have shown that its ligands benzodiazepines show a satisfactory effect on melanogenic promotion. However, the potential application of TSPO in drug development for pigmentary disorder needs further investigation. In this study, we confirmed the melanogenesis induction of TSPO ligand, Ro5-4864 in mouse melanoma cell lines, human skin tissue, and zebrafish embryos by inducing melanin synthesis and melanosome transport. Molecular genetics and pharmacological studies showed that TSPO deficiency did not affect melanin production in B16F10 cells and zebrafish embryos, nor did it affect the melanin promotion effect of Ro5-4864. Whether or not TSPO exists, the expression of lots of melanogenesis-related proteins, such as TYR, TRP-1, DCT, Mlph, and Rab27 was upregulated with the Ro5-4864 administration. These results indicated that Ro5-4864 induces melanogenesis in a TSPO-independent manner, which is inconsistent with previous research. This research is a reminder that we need to be very careful during target validation in drug development.

7-Hydroxy-2-octenoic acid-ethyl ester mixture as an UV protectant secondary metabolite of an endolichenic fungus isolated from Menegazzia terebrata.

Enodolichenic fungi (ELF) are considered a promising bio-resource since they produce a variety of novel secondary metabolites with bioactivities. Ultraviolet (UV) radiation in sunlight containing UVA and UVB can cause acute and chronic skin diseases, and the demand for UV protectants in sunscreens has been increasing. Such situations evoke the strong interest of researchers in seeking effective UV protectants from natural products. In this study, we obtained partially purified 7-hydroxy-2-octenoic acid-ethyl ester (7E) from the secondary metabolites of ELF000548, which has UVA absorption activity. The antioxidant properties were performed by in vitro tests. The superoxide anion scavenging activity and inhibition of linoleic acid peroxidation of the 7E mixture were higher than ascorbic acid (ASA) and butyl hydroxyl anisole (BHA). Furthermore, the compound recovered the damage caused by UVB irradiation and inhibited melanin synthesis. Additionally, the 7E mixture exhibited no cytotoxicity toward the mouse melanoma cell lines, B16F1 and B16F10, except for the normal cell line, HaCaT. In general, these results are the first report about bioactivities of 7E, and those demonstrated that this compound might be a UV protectant to go further study.

The Growth Inhibitory Effect on B16F10 Melanoma Cells by 4-BPCA, an Amide Derivative of Caffeic Acid

Caffeic acid (CA) is a phenolic compound found naturally in plants and foods. CA and its natural derivatives are reported to have anti-cancer effects on many cancers, including melanoma. (E)-N-(4-Butylphenyl)-3-(3,4-dihydroxyphenyl)acrylamide (4-BPCA) is an amide derivative of CA. Thus far, the anti-cancer effect and mechanism of 4-BPCA in melanoma cells remain unknown. Here we investigated whether 4-BPCA inhibits the growth of B16F10 cells, a mouse melanoma cell line. Of note, treatment of 4-BPCA at 5 µM for 24 or 48 h significantly reduced the growth (survival) of B16F10 cells. On mechanistic levels, treatment with 4-BPCA for 24 h led to the activation of caspase-9/3, but not caspase-8, in B16F10 cells. 4-BPCA treatment for 2 or 4 h also decreased the expression levels of myeloid B-cell lymphoma 1 (Mcl-1) in B16F10 cells. However, 4-BPCA treatment for the times tested did not influence the expression levels of X-linked inhibitor of apoptosis protein (XIAP) in B16F10 cells. Of interest, treatment of 4-BPCA for 2 or 4 h greatly reduced the phosphorylation levels of JAK-2 and STAT-5 without altering their total protein expression levels. 4-BPCA also had abilities to increase the expression and phosphorylation levels of glucose-regulated protein-78 (GRP-78) and eukaryotic translation initiation factor-2α (eIF-2α) in B16F10 cells. In summary, these results demonstrate firstly that 4-BPCA has a strong growth-inhibitory effect on B16F10 melanoma cells, mediated via activation of the intrinsic caspase pathway, inhibition of JAK-2 and STAT-5, and triggering endoplasmic reticulum (ER) stress. Keywords: 4-BPCA, B16F10, Caspase-9, Endoplasmic reticulum stress, JAK-2, STAT-5

Abstract 5371: Repurposing erbb2 (HER2/neu) as a neoantigen target for neu-negative tumors using a novel erbb2-encoding lentivirus

Abstract INTRODUCTION: HER2/neu is a potent biomarker for which anti-HER2/neu antibodies offer significant responses. However, many cancers, such as melanoma, have less than 5% expression of HER2/neu. The purpose of this study was to determine whether induced expression of erbb2 (mouse HER2/neu analogue) could function as a neoantigen target for anti-erbb2 antibody. METHODS: RT-PCR was used to construct a rat erbb2-encoding vector using the live, non-replicating lentivirus pLenti6.3. erbb2-encoding pLenti6.3 was transfected in vitro into the B16 mouse melanoma cell line, which has less than 5% endogenous expression of erbb2. The generated erbb2+ B16 cell line was designated B16/neu. Anti-erbb2 antibody (7.16.4) was used to treat C57Bl/6 male and female mice bearing B16/neu (200 ug intraperitoneally three times per week for 3 weeks). To confirm activity of 7.16.4, this antibody (100 ug per T75 flask) was co-cultured with B16/neu in vitro, and cell growth/confluence was measured. RESULTS: The novel B16/neu cell line expressed approximately 50% erbb2 in vitro on flow cytometry analysis, compared to 2% errb2 expression in naïve B16. Erbb2/neu expression was confirmed on fluorescent Western blot. Anti-erbb2 antibody (7.16.4) slowed the growth of B16/neu in vitro, confirming the suppressive effects of 7.16.4. In B16/neu-bearing mice, 7.16.4 resulted in effective control of tumor growth and a complete tumor response rate of 35.7%, (5/14 mice) compared to placebo controls (p < 0.01). Interestingly, B16/neu tumors demonstrated slower growth kinetics compared to naïve B16 tumors (p = 0.013), suggesting immune recognition of erbb2 in naïve C57Bl/6 mice as a neoantigen target. No adverse reactions were observed with 7.16.4. In vivo inoculation of erbb2-encoding pLenti6.3 in naïve B16 tumors and treatment of in vivo transfected B16/neu tumors with 7.16.4 comprise ongoing experiments. CONCLUSIONS: erbb2 expression on B16 melanoma was successfully achieved using a novel erbb2-encoding lentivirus. Induced erbb2 served as a neoantigen target for anti-erbb2 antibody. This preclinical study provides translational evidence that induced HER2/neu expression may be utilized to repurpose anti-HER2 antibodies for human malignancies that do not normally express high levels of HER2/neu. Citation Format: Emmanuel M. Gabriel, Brian Necela, Deborah Bahr, Jamie Kaplan, Kristopher Attwood, Keith Knutson. Repurposing erbb2 (HER2/neu) as a neoantigen target for neu-negative tumors using a novel erbb2-encoding lentivirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5371.

Abstract 5652: A novel anti apoptotic function of CTLA4 in melanoma

Abstract Cytotoxic T Lymphocyte Antigen 4 (CTLA4) is an important immune checkpoint protein and has been utilized as an immunotherapeutic target against melanoma. Our previous studies have shown that CTLA4 is expressed at low levels in primary human melanocytes but is upregulated in mutant-BRAF/NRAS melanoma cells and tissues. Despite predominantly intracellular localization of CTLA4, its intracellular function remains highly contradictory and unsubstantiated. We found that ectopic expression of Ctla4 in mouse melanoma cell lines substantially promoted lung colonization in allograft model systems in syngeneic mice. Moreover, similar results were observed in immunocompromised recipient mice, suggesting an intracellular cell-autonomous tumorigenic role of Ctla4 in melanoma. These finding led us to investigated intracellular functions of CTLA4 and its potential roles in melanomagenesis. Through the Ingenuity Pathway Analysis of our proteomics data, we found apoptosis as a major affected pathway in Ctla4-expressing mouse melanoma cells. We treated the cells (B2905-Ctla4-ee and EV control) with doxorubicin to induce apoptosis, followed by assessment by Annexin V assay. We found that Ctla4 expressing mouse melanoma cells were significantly resistant to doxorubicin-induced apoptosis. In reciprocal experiment, we generated (by CRISPR-Cas9) CTLA4-knockout A2058 human melanoma cells that exhibit high endogenous CTLA4 expression and we found that knockout cells showed significantly increased apoptosis than the parental cells. Moreover, we also observed significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) in CTLA4-expressing human melanoma cell lines. CTLA4-expressing cells also showed significantly higher invasion in Matrigel-coated transwell assay. These findings lead us to conclude that CTLA4 plays a significant role in regulating apoptosis and promoting melanoma progression in BRAF/NRAS mutant cells. Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sara Preston-Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. A novel anti apoptotic function of CTLA4 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5652.

Serotonin (5-HT) 2A Receptor Involvement in Melanin Synthesis and Transfer via Activating the PKA/CREB Signaling Pathway

The 5-HT2A serotonin receptor (HTR2A) has been reported to be involved in the serotonin- or serotonin receptor 2A agonist-induced melanogenesis in human melanoma cells. However, the molecular mechanisms underlying HTR2A in regulating melanogenesis remain poorly understood. In this research, cultured mouse melanoma cell line B16F10, human skin, and zebrafish embryos were used to elucidate the downstream signaling of HTR2A in regulating melanogenesis and to verify the potential application of HTR2A in the treatment of pigment-associated cutaneous diseases. We demonstrated that HTR2A antagonists (AT1015 and ketanserin) attenuated the melanogenesis induction of serotonin in both mouse melanoma cells and zebrafish embryos. The agonists of HTR2A (DOI and TCB-2) increased melanin synthesis and transfer in B16F10 cells, human skin tissue, and zebrafish embryos. Furthermore, the HTR2A agonists increased the expression of proteins related to melanosome organization and melanocyte dendrites to facilitate the melanocyte migration and melanosome transport. HTR2A antagonists and genetic knockout of zebrafish htr2aa (the homologue of mammalian HTR2A gene) were also used to clarify that HTR2A mediates serotonin and DOI in regulating melanogenesis. Finally, through small scale screening of the candidate downstream pathway, we demonstrated that HTR2A mediates the melanogenesis induction of its ligands by activating the PKA/CREB signaling pathway. In this research, we further confirmed that HTR2A is a crucial protein to mediate melanocyte function. Meanwhile, this research supports that HTR2A could be designed as a drug target for the development of chemicals to treat cutaneous diseases with melanocytes or melanogenesis abnormality.