As a protective mechanism, the cornea is sensitive to noxious stimuli. Here, we show that in mice, a high proportion of corneal TRPM8+ cold-sensing fibers express the heat-sensitive TRPV1 channel. Despite its insensitivity to cold, TRPV1 enhances membrane potential changes and electrical firing of TRPM8+ neurons in response to cold stimulation. This elevated neuronal excitability leads to augmented ocular cold nociception in mice. In a model of dry eye disease, the expression of TRPV1 in TRPM8+ cold-sensing fibers is increased, and results in severe cold allodynia. Overexpression of TRPV1 in TRPM8+ sensory neurons leads to cold allodynia in both corneal and non-corneal tissues without affecting their thermal sensitivity. TRPV1-dependent neuronal sensitization facilitates the release of the neuropeptide substance P from TRPM8+ cold-sensing neurons to signal nociception in response to cold. Our study identifies a mechanism underlying corneal cold nociception and suggests a potential target for the treatment of ocular pain.

Itch and pain are refractory symptoms of many ocular conditions. Ocular itch is generated mainly in the conjunctiva and is absent from the cornea. In contrast, most ocular pain arises from the cornea. However, the underlying mechanisms remain unknown. Using genetic axonal tracing approaches, we discover distinct sensory innervation patterns between the conjunctiva and cornea. Further genetic and functional analyses in rodent models show that a subset of conjunctival-selective sensory fibers marked by MrgprA3 expression, rather than corneal sensory fibers, mediates ocular itch. Importantly, the actions of both histamine and nonhistamine pruritogens converge onto this unique subset of conjunctiva sensory fibers and enable them to play a key role in mediating itch associated with allergic conjunctivitis. This is distinct from skin itch, in which discrete populations of sensory neurons cooperate to carry itch. Finally, we provide proof of concept that selective silencing of conjunctiva itch-sensing fibers by pruritogen-mediated entry of sodium channel blocker QX-314 is a feasible therapeutic strategy to treat ocular itch in mice. Itch-sensing fibers also innervate the human conjunctiva and allow pharmacological silencing using QX-314. Our results cast new light on the neural mechanisms of ocular itch and open a new avenue for developing therapeutic strategies.

Antigen-specific immune diseases such as rheumatoid arthritis are often accompanied by pain and hyperalgesia. Our previous studies have demonstrated that Fc-gamma-receptor type I (FcγRI) is expressed in a subpopulation of rat dorsal root ganglion (DRG) neurons and can be directly activated by IgG immune complex (IgG-IC). In this study we investigated whether neuronal FcγRI contributes to antigen-specific pain in the naïve and rheumatoid arthritis model rats. In vitro calcium imaging and whole-cell patch clamp recordings in dissociated DRG neurons revealed that only the small-, but not medium- or large-sized DRG neurons responded to IgG-IC. Accordingly, in vivo electrophysiological recordings showed that intradermal injection of IgG-IC into the peripheral receptive field could sensitize only the C- (but not A-) type sensory neurons and evoke action potential discharges. Pain-related behavioral tests showed that intradermal injection of IgG-IC dose-dependently produced mechanical and thermal hyperalgesia in the hindpaw of rats. These behavioral effects could be alleviated by localized administration of non-specific IgG or an FcγRI antibody, but not by mast cell stabilizer or histamine antagonist. In a rat model of antigen-induced arthritis (AIA) produced by methylated bovine serum albumin, FcγRI were found upregulated exclusively in the small-sized DRG neurons. In vitro calcium imaging revealed that significantly more small-sized DRG neurons responded to IgG-IC in the AIA rats, although there was no significant difference between the AIA and control rats in the magnitude of calcium changes in the DRG neurons. Moreover, in vivo electrophysiological recordings showed that C-nociceptive neurons in the AIA rats exhibited a greater incidence of action potential discharges and stronger responses to mechanical stimuli after IgG-IC was injected to the receptive fields. These results suggest that FcγRI expressed in the peripheral nociceptors might be directly activated by IgG-IC and contribute to antigen-specific pain in pathological conditions.

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Abbreviations: AR, androgen receptor; ARE, androgen-responsive element; DUB, deubiquitinating enzyme; H3K9, histone H3 on lysine 9; H3K36, histone H3 on lysine 36; HA-ub, HA-tagged ubiquitin; IB, immunoblot; IHC, immunohistochemistry; IP, immunoprecipitation; KDM4A, lysine-specific demethylase 4A; PC, prostate cancer; PTEN, phosphatase and tensin homolog; qPCR, quantitative PCR. AbstractThe histone demethylase lysine-specific demethylase 4A (KDM4A) is reported to be overexpressed and plays a vital in multiple cancers through controlling gene expression by epigenetic regulation of H3K9 or H3K36 methylation marks. However, the biological role and mechanism of KDM4A in prostate cancer (PC) remain unclear.Herein, we reported KDM4A expression was upregulation in phosphatase and tensin homolog knockout mouse prostate tissue. Depletion of KDM4A in PC cells inhibited their proliferation and survival in vivo and vitro. Further studies reveal that USP1 is a deubiquitinase that regulates KDM4A K48-linked deubiquitin and stability.Interestingly, we found c-Myc was a key downstream effector of the USP1-KDM4A/ androgen receptor axis in driving PC cell proliferation. Notably, upregulation of KDM4A expression with high USP1 expression was observed in most prostate tumors and inhibition of USP1 promotes PC cells response to therapeutic agent enzalutamide. Our studies propose USP1 could be an anticancer therapeutic target in PC.

Fibroblast growth factor‐2 (FGF‐2) is one of the most important angiogenic factors to promote tumor growth, progression and metastasis. Neutralizing antibodies against FGF‐2 may suppress the growth of tumor cells by blocking the FGF‐2 signaling pathway. In this study, a disulfide‐stabilized diabody (ds‐Diabody) that specifically targets FGF‐2 was designed. Compared to its parent antibody, the introduction of disulphide bonds in the diabody could significantly increase the stability of ds‐Diabody and maintain its antigen binding activity. The ds‐Diabody against FGF‐2 could effectively inhibit the tube formation and migration of vascular endothelial cells and block the proliferation and invasion of human breast cancer cells. In the mouse model of breast cancer xenograft tumors, the ds‐Diabody against FGF‐2 could significantly inhibit the growth of tumor cells. Moreover, the densities of microvessels stained with CD31 and lymphatic vessels stained with LYVE1 in tumors showed a significant decrease following treatment with the ds‐Diabody against FGF‐2. Our data indicated that the ds‐Diabody against FGF‐2 could inhibit tumor angiogenesis, lymphangiogenesis and tumor growth.

The histone demethylase KDM4B functions as a key co‐activator for the androgen receptor (AR) and plays a vital in multiple cancers through controlling gene expression by epigenetic regulation of H3K9 methylation marks. Constitutively active androgen receptor confers anti‐androgen resistance in advanced prostate cancer. However, the role of KDM4B in resistance to next‐generation anti‐androgens and the mechanisms of KDM4B regulation are poorly defined. Here we found that KDM4B is overexpressed in enzalutamide‐resistant prostate cancer cells. Overexpression of KDM4B promoted recruitment of AR to the c‐Myc (MYC) gene enhancer and induced H3K9 demethylation, increasing AR‐dependent transcription of c‐Myc mRNA, which regulates the sensitivity to next‐generation AR‐targeted therapy. Inhibition of KDM4B significantly inhibited prostate tumor cell growth in xenografts, and improved enzalutamide treatments through suppression of c‐Myc. Clinically, KDM4B expression was found upregulated and to correlate with prostate cancer progression and poor prognosis. Our results revealed a novel mechanism of anti‐androgen resistance via histone demethylase alteration which could be targeted through inhibition of KDM4B to reduce AR‐dependent c‐Myc expression and overcome resistance to AR‐targeted therapies. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Yes‐associated protein (YAP) is a component of the canonical Hippo signaling pathway that is known to play essential roles in modulating organ size, development, and tumorigenesis. Activation or upregulation of YAP1, which contributes to cancer cell survival and chemoresistance, has been verified in different types of human cancers. However, the molecular mechanism of YAP1 upregulation in cancer is still unclear. Here we report that the E3 ubiquitin ligase STUB1 ubiquitinates and destabilizes YAP1, thereby inhibiting cancer cell survival. Low levels of STUB1 expression were correlated with increased protein levels of YAP1 in human gastric cancer cell lines and patient samples. Moreover, we revealed that STUB1 ubiquitinates YAP1 at the K280 site by K48‐linked polyubiquitination, which in turn increases YAP1 turnover and promotes cellular chemosensitivity. Overall, our study establishes YAP1 ubiquitination and degradation mediated by the E3 ligase STUB1 as an important regulatory mechanism in gastric cancer, and provides a rationale for potential therapeutic interventions.