Strikingly High Activity of 15-Lipoxygenase Towards Di-Polyunsaturated Arachidonoyl/Adrenoyl-Phosphatidylethanolamines Generates Peroxidation Signals of Ferroptotic Cell Death.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.

Redox phospholipidomics discovers pro-ferroptotic death signals in A375 melanoma cells in vitro and in vivo.

Growing cancer cells effectively evade most programs of regulated cell death, particularly apoptosis. This necessitates a search for alternative therapeutic modalities to cause cancer cell's demise, among them - ferroptosis. One of the obstacles to using pro-ferroptotic agents to treat cancer is the lack of adequate biomarkers of ferroptosis. Ferroptosis is accompanied by peroxidation of polyunsaturated species of phosphatidylethanolamine (PE) to hydroperoxy- (-OOH) derivatives, which act as death signals. We demonstrate that RSL3-induced death of A375 melanoma cells in vitro was fully preventable by ferrostatin-1, suggesting their high susceptibility to ferroptosis. Treatment of A375 cells with RSL3 caused a significant accumulation of PE-(18:0/20:4-OOH) and PE-(18:0/22:4-OOH), the biomarkers of ferroptosis, as well as oxidatively truncated products - PE-(18:0/hydroxy-8-oxo-oct-6-enoic acid (HOOA) and PC-(18:0/HOOA). A significant suppressive effect of RSL3 on melanoma growth was observed in vivo (utilizing a xenograft model of inoculation of GFP-labeled A375 cells into immune-deficient athymic nude mice). Redox phospholipidomics revealed elevated levels of 18:0/20:4-OOH in RSL3-treated group vs controls. In addition, PE-(18:0/20:4-OOH) species were identified as major contributors to the separation of control and RSL3-treated groups, with the highest variable importance in projection predictive score. Pearson correlation analysis revealed an association between tumor weight and contents of PE-(18:0/20:4-OOH) (r = -0.505), PE-18:0/HOOA (r = -0.547) and PE 16:0-HOOA (r = -0.503). Thus, LC-MS/MS based redox lipidomics is a sensitive and precise approach for the detection and characterization of phospholipid biomarkers of ferroptosis induced in cancer cells by radio- and chemotherapy.

Melanoma-associated repair-like Schwann cells suppress anti-tumor T-cells via 12/15-LOX/COX2-associated eicosanoid production.

Peripheral glia, specifically the Schwann cells (SCs), have been implicated in the formation of the tumor microenvironment (TME) and in cancer progression. However, in vivo and ex vivo analyses of how cancers reprogram SC functions in different organs of tumor-bearing mice are lacking. We generated Plp1-CreERT/tdTomato mice which harbor fluorescently labeled myelinated and non-myelin forming SCs. We show that this model enables the isolation of the SCs with high purity from the skin and multiple other organs. We used this model to study phenotypic and functional reprogramming of the SCs in the skin adjacent to melanoma tumors. Transcriptomic analyses of the peritumoral skin SCs versus skin SCs from tumor-free mice revealed that the former existed in a repair-like state typically activated during nerve and tissue injury. Peritumoral skin SCs also downregulated pro-inflammatory genes and pathways related to protective anti-tumor responses. In vivo and ex vivo functional assays confirmed immunosuppressive activities of the peritumoral skin SCs. Specifically, melanoma-reprogrammed SCs upregulated 12/15-lipoxygenase (12/15-LOX) and cyclooxygenase (COX)-2, and increased production of anti-inflammatory polyunsaturated fatty acid (PUFA) metabolites prostaglandin E2 (PGE2) and lipoxins A4/B4. Inhibition of 12/15-LOX or COX2 in SCs, or EP4 receptor on lymphocytes reversed SC-dependent suppression of anti-tumor T-cell activation. Therefore, SCs within the skin adjacent to melanoma tumors demonstrate functional switching to repair-like immunosuppressive cells with dysregulated lipid oxidation. Our study suggests the involvement of the melanoma-associated repair-like peritumoral SCs in the modulation of locoregional and systemic anti-tumor immune responses.

Tumor-Induced T Cell Polarization by Schwann Cells.

Nerve-cancer crosstalk resulting in either tumor neurogenesis or intratumoral neurodegeneration is critically controlled by Schwann cells, the principal glial cells of the peripheral nervous system. Though the direct stimulating effect of Schwann cells on malignant cell proliferation, motility, epithelial-mesenchymal transition, and the formation of metastases have been intensively investigated, the ability of Schwann cells to affect the effector and regulatory immune cells in the tumor environment is significantly less studied. Here, we demonstrated that tumor cells could stimulate Schwann cells to produce high levels of prostaglandin E, which could be blocked by COX-2 inhibitors. This effect was mediated by tumor-derived TGF-ß as neutralization of this cytokine in the tumor-conditioned medium completely blocked the inducible prostaglandin E production by Schwann cells. Similar protective effects were also induced by the Schwann cell pretreatment with TGF-ßR1/ALK4/5/7 and MAPK/ERK kinase inhibitors of the canonical and non-canonical TGF-ß signaling pathways, respectively. Furthermore, prostaglandin E derived from tumor-activated Schwann cells blocked the proliferation of CD3/CD28-activated T cells and upregulated the expression of CD73 and PD-1 on both CD4+ and CD8+ T cells, suggesting T cell polarization to the exhausted phenotype. This new pathway of tumor-induced T cell inhibition via the activation of neuroglial cells represents new evidence of the importance of nerve-cancer crosstalk in controlling tumor development and progression. A better understanding of the tumor-neuro-immune axis supports the development of efficient targets for harnessing this axis and improving the efficacy of cancer therapy.

Sensory Nerves Impede the Formation of Tertiary Lymphoid Structures and Development of Protective Antimelanoma Immune Responses.

Peripheral neurons comprise a critical component of the tumor microenvironment (TME). The role of the autonomic innervation in cancer has been firmly established. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Specifically, sensory ablation led to improved leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Cutaneous sensory nerves hindered the maturation of intratumoral high endothelial venules and limited the formation of mature tertiary lymphoid-like structures containing organized clusters of CD4+ T cells and B cells. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Importantly, CD8a depletion prevented denervation-dependent antitumor effects. Finally, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby defining a novel target for therapeutic intervention in the melanoma setting.

Keratinocyte death by ferroptosis initiates skin inflammation after UVB exposure.

The ultraviolet B radiation (UVB) causes skin inflammation, which contributes to the causality and the exacerbation of a number of cutaneous diseases. However, the mechanism of UVB-driven inflammation in the skin remains poorly understood. We show that ferroptosis, a non-apoptotic programmed cell death pathway that is promoted by an excessive phospholipid peroxidation, is activated in the epidermal keratinocytes after their exposure to UVB. The susceptibility of the keratinocytes to UVB-induced ferroptosis depends on the extent of pro-ferroptosis death signal generation and the dysregulation of the glutathione system. Inhibition of ferroptosis prevents the release of HMGB1 from the human epidermal keratinocytes, and blocks necroinflammation in the UVB-irradiated mouse skin. We show that while apoptosis and pyroptosis are also detectable in the keratinocytes after UVB exposure, ferroptosis plays a significant role in initiating UVB-induced inflammation in the skin. Our results have important implications for the prevention and the treatment of a broad range of skin diseases which are fostered by UVB-induced inflammation.

Combined carvedilol and gabapentin treatment induces a rapid response in red scrotum syndrome.

Red scrotum syndrome (RSS) is a rare dermatologic condition characterized by persistent erythema and analgesia of the male genitalia that cannot be attributed to contact or atopic dermatitis or acute or chronic infections. Treatment of RSS is challenging since it often fails to respond to corticosteroids, antifungals, antivirals, and antibiotics. Several reports described RSS patients who responded to gabapentin, pregabalin, and ?-adrenergic receptor blockers, suggesting a neuropathic etiology. Here we present a refractory RSS case with rapid clinical improvement on a combined carvedilol plus gabapentin therapy. We suggest that RSS manifestations are driven by neurogenic inflammation and that the efficacy of gabapentin/carvedilol relates to the suppression of the neuro-immuno-epidermal axis.

Melanoma Ex Blue Nevus With GNA11 Mutation and BAP1 Loss: Case Report and Review of the Literature.

Cutaneous melanomas may demonstrate a variety of histopathological features and genetic abnormalities. Melanomas that arise in the setting of blue nevi, also known as "malignant blue nevus" or melanoma ex blue nevus (MBN), share a similar histopathological and mutational profile with uveal melanoma. Most uveal melanomas show characteristic GNA11 or GNAQ mutations; additional BAP1 mutation or loss is associated with the highest risk of metastasis and worst prognosis. However, the significance of BAP1 loss in melanomas ex blue nevus remains unclear. We present a case of MBN arising from the scalp of a 21-year-old woman. The diagnosis was established on histopathological findings demonstrating a markedly atypical melanocytic proliferation with increased mitotic activity, necrosis, and a focus of angiolymphatic invasion. Immunohistochemical analysis demonstrated the absence of BAP1 nuclear expression within tumor cells. Next generation sequencing detected GNA11 Q209L mutation and BAP1 loss (chromosome 3p region loss), supporting the diagnosis. We reviewed another 21 MBN cases with reported BAP1 status from the literature. MBN with BAP1 loss presented at a younger average age (41 vs. 61 years), demonstrated larger average lesion thickness (9.0 vs. 7.3 mm), and had a higher rate of metastasis (50% vs. 33%) compared with BAP1-retained MBN. BAP1 expression studies may assist in the diagnosis and management of MBN, but further research is needed.

The Neuroimmune Axis in the Tumor Microenvironment.

Cancer is a complex ecosystem and should be considered in the context of its cellular and molecular microenvironment, which includes the nerves. Peripheral nerves can modulate phenotype and behavior of the malignant cells and thus affect tumor growth and metastasis. Only recently has the role of neuroimmune cross-talk surfaced as a key contributor to cancer progression. However, little is known about the immunomodulatory role of the neuroglial cells in cancer progression and metastasis and the response to therapy. Schwann cells, the principal glial cells of the peripheral nervous system, are now considered to be important players in the tumor microenvironment. They can directly accelerate malignant cell migration and the formation of metastases. Better understanding of the neuroimmune circuits in the tumor milieu will be instrumental in the development of novel therapeutic approaches for the malignancies known to be associated with inflammation and dysregulated immune responses.

Immunomodulation by Schwann cells in disease.

Schwann cells are the principal glial cells of the peripheral nervous system which maintain neuronal homeostasis. Schwann cells support peripheral nerve functions and play a critical role in many pathological processes including injury-induced nerve repair, neurodegenerative diseases, infections, neuropathic pain and cancer. Schwann cells are implicated in a wide range of diseases due, in part, to their ability to interact and modulate immune cells. We discuss the accumulating examples of how Schwann cell regulation of the immune system initiates and facilitates the progression of various diseases. Furthermore, we highlight how Schwann cells may orchestrate an immunosuppressive tumor microenvironment by polarizing and modulating the activity of the dendritic cells.

Melanoma-Induced Reprogramming of Schwann Cell Signaling Aids Tumor Growth.

The tumor microenvironment has been compared with a nonhealing wound involving a complex interaction between multiple cell types. Schwann cells, the key regulators of peripheral nerve repair, have recently been shown to directly affect nonneural wound healing. Their role in cancer progression, however, has been largely limited to neuropathic pain and perineural invasion. In this study, we showed that melanoma activated otherwise dormant functions of Schwann cells aimed at nerve regeneration and wound healing. Such reprogramming of Schwann cells into repair-like cells occurred during the destruction and displacement of neurons as the tumor expanded and via direct signaling from melanoma cells to Schwann cells, resulting in activation of the nerve injury response. Melanoma-activated Schwann cells significantly altered the microenvironment through their modulation of the immune system and the extracellular matrix in a way that promoted melanoma growth in vitro and in vivo. Local inhibition of Schwann cell activity following cutaneous sensory nerve transection in melanoma orthotopic models significantly decreased the rate of tumor growth. Tumor-associated Schwann cells, therefore, can have a significant protumorigenic effect and may present a novel target for cancer therapy. SIGNIFICANCE: These findings reveal a role of the nerve injury response, particularly through functions of activated Schwann cells, in promoting melanoma growth.

Schwann cells shape the neuro-immune environs and control cancer progression.

At present, significant experimental and clinical data confirm the active involvement of the peripheral nervous system (PNS) in different phases of cancer development and progression. Most of the research effort focuses on the impact of distinct neuronal types, e.g., adrenergic, cholinergic, dopaminergic, etc. in carcinogenesis, generally ignoring neuroglia. The very fact that these cells far outnumber the other cellular types may also play an important role worthy of study in this context. The most prevalent neuroglia within the PNS consists of Schwann cells (SCs). These cells play a substantial role in maintaining homeostasis within the nervous system. They possess distinct immunomodulatory, inflammatory and regenerative capacities-also, one should consider their broad distribution throughout the body; this makes them a perfect target for malignant cells during the initial stages of cancer development and the very formation of the tumor microenvironment itself. We show that SCs in the tumor milieu attract different subsets of immune regulators and augment their ability to suppress effector T cells. SCs may also up-regulate invasiveness of tumor cells and support metastatic disease. We outline the interactive potential of SCs juxtaposed with cancerous cells, referring to data from various external sources alongside data of our own.

The nervous system: a new target in the fight against cancer.

During cancer progression, tumor cells interact with the neighboring environment, including neuronal tissue. The important influence of the nervous system on growth and metastasis of cancer is now widely accepted. As such, using medications that traditionally target the nervous system may be an avenue toward treating cancer. The focus of this review is to detail how several classes of medications, traditionally used to treat nervous system disorders, impact cancer. Specifically, we review the preclinical and clinical evidence that support the use of anti-ß-adrenergic, anticholinergic, antipsychotic, and antidepressant medications to treat some cancers. In addition, we discuss the use of ablative modalities, such as physical and chemical denervation, to treat cancer or protect against cancer development. Using the medications that target the nervous system to treat cancer is a promising addition to an existing therapy or an alternative treatment strategy. Furthermore, rapidly expanding basic science research in this area will likely yield novel cancer therapies that work by targeting the nervous system.

Schwann Cells Augment Cell Spreading and Metastasis of Lung Cancer.

Although lungs are densely innervated by the peripheral nervous system (PNS), the role of the PNS in the progression of lung cancer is unknown. In this study, we report that mouse adult Schwann cells (SC), the principal glial cells of the PNS, can regulate the motility of lung cancer cells in vitro and the formation of metastases in vivo SCs promoted epithelial-to-mesenchymal transition (EMT) and the motility of two lung cancer cell lines by increasing expression of Snail and Twist in tumor cells; blocking of Snail and Twist expression abolished SC-induced motility of tumor cells. SC-derived CXCL5 was responsible for EMT in lung cancer cells, as the inhibition of CXCL5 or its receptor CXCR2 reduced SC-induced expression of Snail and Twist and reduced motility in tumor cells. CXCL5/CXCR2 binding activated the PI3K/AKT/GSK-3ß/Snail-Twist signaling pathway in lung cancer cells, and the PI3K inhibitor blocked CXCL5-dependent phosphorylation of AKT and GSK-3ß, reduced expression of Snail/Twist, and limited tumor cell invasiveness. SC conditioning of tumor cells prior to their injection into mice significantly increased the formation of metastases in the regional lymph nodes. In summary, SCs can regulate the CXCL5/CXCR2/PI3K/AKT/GSK-3ß/Snail-Twist pathway to promote EMT, invasiveness, and metastatic potential of lung cancer cells. Our results reveal a new role of the PNS in the functional organization of the tumor microenvironment and tumor progression.Significance: This study increases our understanding of how nerves and, in particular, specific glial cells, Schwann cells, in the peripheral nervous system, may help promote tumor growth and metastasis. Cancer Res; 78(20); 5927-39. ©2018 AACR.

Schwann cells: a new player in the tumor microenvironment.

Cancerous cells must cooperate with the surrounding stroma and non-malignant cells within the microenvironment to support the growth and invasion of the tumor. The nervous system is a component of every organ system of the body, and therefore, is invariably at the front line of the tumor invasion. Due to the complexity of the nervous system physiology, this review separately discusses the contributions of the central and peripheral nervous systems to the tumorigenesis and tumor progression. We further focus the discussion on the evidence that Schwann cells aid in tumor growth and invasion. Schwann cells, a largely unexplored element of the tumor microenvironment, may participate in the creation of tumor-favorable conditions through both bi-directional interaction with cancer cells and the facilitation of the immune-suppressive microenvironment through the mechanism of neural repair and immunomodulation.

Deoxycytidine kinase augments ATM-Mediated DNA repair and contributes to radiation resistance.

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [(18)F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.

Quantitative real-time measurements of DNA hybridization with alkylated nonoxidized silicon nanowires in electrolyte solution.

The quantitative, real-time detection of single-stranded oligonucleotides with silicon nanowires (SiNWs) in physiologically relevant electrolyte solution is demonstrated. Debye screening of the hybridization event is circumvented by utilizing electrostatically adsorbed primary DNA on an amine-terminated NW surface. Two surface functionalization chemistries are compared: an amine-terminated siloxane monolayer on the native SiO2 surface of the SiNW, and an amine-terminated alkyl monolayer grown directly on a hydrogen-terminated SiNW surface. The SiNWs without the native oxide exhibit improved solution-gated field-effect transistor characteristics and a significantly enhanced sensitivity to single-stranded DNA detection, with an accompanying 2 orders of magnitude improvement in the dynamic range of sensing. A model for the detection of analyte by SiNW sensors is developed and utilized to extract DNA-binding kinetic parameters. Those values are directly compared with values obtained by the standard method of surface plasmon resonance (SPR) and demonstrated to be similar. The nanowires, however, are characterized by higher detection sensitivity. The implication is that SiNWs can be utilized to quantitate the solution-phase concentration of biomolecules at low concentrations. This work also demonstrates the importance of surface chemistry for optimizing biomolecular sensing with silicon nanowires.

Electrochemical fabrication of conducting polymer nanowires in an integrated microfluidic system.

In this paper, we introduce a new approach for the in situ electrochemical fabrication of an individually addressable array of conducting polymer nanowires (CPNWs) positioned within an integrated microfluidic device and also demonstrate that such an integrated device can be used as a chemical sensor immediately after its construction.