Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level. Here we show that melanoma cells exhibit an oxidative stress phenotype compared with normal melanocytes, as evidenced by increased total cellular ROS, KEAP1/NRF2 pathway activity, protein damage, and elevated oxidized glutathione. Our overall objective was to test whether augmenting this high oxidative stress level in melanoma cells would inhibit their dependence on oncogenic PI3K/AKT/mTOR-mediated survival. We report that NexrutineR augmented the constitutively elevated oxidative stress markers in melanoma cells, which was abrogated by N-acetyl cysteine (NAC) pre-treatment. NexrutineR disrupted growth homeostasis by inhibiting proliferation, survival, and colony formation in melanoma cells without affecting melanocyte cell viability. Increased oxidative stress in melanoma cells inhibited PI3K/AKT/mTOR pathway through disruption of mTORC1 formation and phosphorylation of downstream targets p70S6K, 4EBP1 and rpS6. NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism. Overall, our results illustrate the importance of disruption of the intrinsically high oxidative stress in melanoma cells to selectively inhibit their survival mediated by PI3K/AKT/mTOR.

Novel agents are desperately needed for improving the quality of life and 5-year survival to more than 30% for metastatic castrate-resistant prostate cancer. Previously we showed that Nexrutine, Phellodendron amurense bark extract, inhibits prostate tumor growth in vitro and in vivo. Subsequently using biochemical fractionation we identified butanol fraction contributes to the observed biological activities. We report here that palmatine, which is present in the butanol fraction, selectively inhibits growth of prostate cancer cells without significant effect on non-tumorigenic prostate epithelial cells. By screening receptor tyrosine kinases in a protein kinase array, we identified ribosomal protein S6, a downstream target of p70S6K and the Akt/mTOR signaling cascade as a potential target. We further show that palmatine treatment is associated with decreased activation of NFκB and its downstream target gene FLIP. These events led to inhibition of invasion. Similar results were obtained using parent extract Nexrutine (Nx) suggesting that palmatine either in the purified form or as one of the components in Nx is a potent cytotoxic agent with tumor invasion inhibitory properties. Synergistic inhibition of rpS6/NFκB/FLIP axis with palmatine may have therapeutic potential for the treatment of prostate cancer and possibly other malignancies with their constitutive activation. These data support a biological link between rpS6/NFκB/FLIP in mediating palmatine-induced inhibitory effects and warrants additional preclinical studies to test its therapeutic efficacy.

Two prominent features of tumors that contribute to oncogenic survival signaling are redox disruption, or oxidative stress phenotype, and high autophagy signaling, making both phenomena ideal therapeutic targets. However, the relationship between redox disruption and autophagy signaling is not well characterized and the clinical impact of reactive oxygen species (ROS)-generating chemotherapeutics on autophagy merits immediate attention as autophagy largely contributes to chemotherapeutic resistance. In this commentary we focus on melanoma, using it as an example to provide clarity to current literature regarding the roles of autophagy and redox signaling which can be applicable to initiation and maintenance of most tumor types. Further, we address the crosstalk between ROS and autophagy signaling during pharmacological intervention and cell fate decisions. We attempt to elucidate the role of autophagy in regulating cell fate following treatment with ROS-generating agents in preclinical and clinical settings and discuss the emerging role of autophagy in cell fate decisions and as a cell death mechanism. We also address technical aspects of redox and autophagy evaluation in experimental design and data interpretation. Lastly, we present a provocative view of the clinical relevance, emerging challenges in dual targeting of redox and autophagy pathways for therapy, and the future directions to be addressed in order to advance both basic and translational aspects of this field.

We sought to determine the subcellular distribution of 30+ immune-related markers including PD-L1 in stage III melanoma tumor tissues (nuclear, cytoplasmic, surface) using novel imaging mass cytometry technology that enables simultaneous detection of over 30 immune and tumor markers. Metal-tagged antibodies were used to stain stage III melanoma tissues similarly to an IHC-based protocol, followed by laser ablation and mass cytometry using the Helios® and Hyperion® technology (Fluidigm). Markers for immune subsets included CD45, CD3, CD4, CD8, PD-1, Granzyme B, FoxP3, CD11b, CD11c, CD107, CD20, Vista, and CD68. Markers for tumor cell phenotyping included PD-L1 (intracellular and extracellular domain specific), E-cadherin, B-catenin, PD-L2, and keratin 8/18. Structural, inflammatory, survival, and signaling markers included Ki67, p53, p-tyrosine, Bcl-2, Cl-caspase 3, histone H3, collagen-1, actin, and arginase-1. We further validated subcellular distribution of PD-L1 in the same patient tumor cores using fluorochrome-labeled antibodies and 100X confocal microscopy to show difference in surface-only, cytoplasmic-only, and nuclear PD-L1. We then assessed the association between subcellular PD-L1 and immune populations in the tumor microenvironment, along with overall and recurrence-free survival. We wrote custom algorithms for data processing to identify novel immune subsets, ran SPADE and viSNE analyses for cross-validation, and employed Imaris Bitplane software to validate our own algorithms for immune population identification. Overall, we found that intracellular-only PD-L1 in tumor tissues has the highest correlation (Pearson r) to CD11b, FoxP3, PD-1, arginase, Ki67, PD-L2 and nuclear stain iridium, while extracellular (surface) PD-L1 in tumors has the highest correlation (Pearson r) to FoxP3, PD-1, arginase, Ki67, PD-L2 and Vista. We identified two novel immunophenotypes (histone-3+/p-tyrosinehi /Vistalo / PD-L1-ICDlo / PD-L1-ECDlo and CD11bhi / PD-L1-ICDhi / PD-L1-ECDhi) that are positively associated with overall patient survival. Of note, only phospho-Tyrhi was significantly associated with RFS. This work highlights the use of cutting-edge technology that can employ the highest multiplexing available for assessing novel interactions of 30+ tumor tissue markers for identification of novel immunophenotypes and biomarker in stage III melanoma patients. Our novel study showcases a novel methodologic approach to high-throughput analysis of retrospective profiling of the melanoma tumor immune microenvironment on a single IHC slide, followed by appropriate and rigorous cross-validation using Fluidigm’s novel CyTOF imaging technology. Our study provides a groundwork for this powerful, novel technology in translational and clinical research for biomarker identification and identification of novel combinatorial markers that may predict overall and recurrence-free survival in stage III melanoma. Citation Format: Heather G. Hambright, Anand V.R. Kornepati, Dai Ogata, Roland R.L. Bassett, Suhendan Ekmekcioglu, Elizabeth A. Grimm, Tyler J. Curiel. High-dimensional (30-plex) imaging mass cytometry on tissue microarray identifies novel PD-L1-inclusive immunophenotypes associated with overall survival in stage III melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B10.

The primary objectives of this study were i) systematic evaluation of the differences in basal autophagic activity between melanocytes and malignant melanoma cells to determine whether these differences may be selectively targeted using the ROS inducer, Nexrutine (NX); ii) determination of NX selectivity in inhibiting melanoma cell survival and iii) specific involvement of autophagy signaling protein, SQSTM1/p62 in NX-mediated autophagic cell survival. NX is obtained from the bark of the cork tree, Phellodendron amurense. A panel of melanocyte and melanoma cells was evaluated for basal autophagy level and effect of NX treatment, including protein levels and turnover of p62 and LC3B (+/- CQ), and number of autophagic puncta per cell. Trypan blue, MTT, and Annexin V-APC were used to evaluate the effect of NX on cell death, proliferation and apoptosis. Genetic approach using siRNA for p62 and overexpression using HA-p62 followed by survival, proliferation, and autophagy measurements were used to further evaluate the possibility of p62 as a molecular target for inhibition of autophagy seen using NX. We found that i) melanoma cells displayed high autophagic flux compared with melanocytes, ii) NX treatment a) inhibited autophagy in melanoma cells, b) inhibited melanoma cell survival, and c) induced apoptosis in melanoma cells, iii) knockdown of p62 resulted in a lesser inhibition of cell viability and autophagy after NX treatment in high p62-expressing melanoma cells, and iv) overexpression of p62 sensitized low p62-expressing normal melanocytes and melanoma cells to NX-mediated cell death and autophagy inhibition, suggesting that p62 is a bonafide molecular target of NX. Overall, we show that Nexrutine is able to inhibit the inherent high level of pro-survival autophagy in melanoma cells, leading to loss of cell viability and induction of apoptosis, while melanocytes remain unaffected by NX at the same doses. Further, SQSTM1/p62 was found to be a critical mediator of the NX-mediated inhibition of autophagy and cell viability. This study demonstrates that NX maybe a promising novel, non-toxic agent for melanoma. Supported by R21 CA125719 & ACRCF (RG); NIDCR T32 DE14318/COSTAR (HGH) Citation Format: Heather G. Hambright, Addanki P. Kumar, Rita Ghosh. Inhibition of melanoma cell survival through p62/LC3B autophagic signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3534.

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Metastatic melanoma has a dismal survival rate for patients, despite recent advances in targeted and immune-therapies. Recent studies show that elevated autophagy and redox imbalance in melanoma cells contributes to chemotherapeutic resistance, making these biological processes ideal therapeutic targets. However, gaps still exist regarding the mechanism by which pro-oxidant therapeutics affect autophagic signaling and the ultimate impact on melanoma cell fate. We previously demonstrated that pro-oxidant compound Nexrutine (NX) selectively inhibits melanoma cell survival and autophagy through autophagy scaffolding protein SQSTM1/p62. However, the mechanism by which NX-induced oxidative stress inhibits autophagy through p62 remained unknown. Thus, the primary objective of this study was to determine how p62 regulates cell fate during oxidative damage. We used a panel of primary melanocyte and melanoma cells to evaluate i) proteasomal degradation of p62 during oxidative stress-induced autophagy blockade, ii) the requirement for p62 during NX-induced apoptosis, iii) effect of NX-induced superoxide on autophagy and cell survival, iv) association of p62 with caspase-8 during oxidative damage, and v) subcellular localization of NX. Proteasomal degradation was evaluated using MG132 and western blotting for p62 following NX. We used genetic approaches (RNAi and overexpression using HA-p62) to determine the role of p62 in cell fate as determined by survival, apoptosis, and autophagy measurements. Fluorescence microscopy was used to determine the subcellular localization of NX. Generation of superoxide was determined using mitoSOX and mitoTEMPO pre-treatment was used to determine specificity. We found that i) NX-induced ROS inhibits p62 protein by stimulating proteasomal degradation, ii) p62 mediates apoptosis in response to high levels of oxidative damage through caspase-8 iii) NX generates superoxide which inhibits autophagy and melanoma cell survival, and iv) NX accumulates selectively in mitochondria. Overall, we show that oxidative stress induced by NX occurs upstream of its inhibitory effect on p62, autophagy and cell survival. Further, we demonstrate that NX induces superoxide, which leads to autophagy inhibition through increased p62 degradation, that ultimately inhibits melanoma cell survival, while not affecting primary melanocytes. We also show that p62 mediates apoptosis through increased association with caspase-8 in melanoma cells during superoxide generated by NX. We also demonstrate for the first time that NX localizes to mitochondria of melanoma cells and results in membrane potential loss due to its generation of high amounts of superoxide. This study demonstrates that pro-oxidants like NX can successfully target two pro-survival melanoma features simultaneously and may be useful melanoma therapeutic agents. Supported by R21 CA125719 & ACRCF (RG); NIDCR T32 DE14318/COSTAR (HGH) Citation Format: Heather G. Hambright, Addanki P. Kumar, Rita Ghosh. Mitochondrial superoxide inhibits autophagy and induces apoptosis through SQSTM1-mediated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5466. doi:10.1158/1538-7445.AM2017-5466

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