SUMMARY High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit diminished estrogen receptor β (ERβ) expression. We report that a key function of ERβ and its specific ligand 5α-androstane-3β,17β-diol (3β-adiol) is to maintain an epithelial phenotype and repress mesenchymal characteristics in prostate carcinoma. Stimuli (TGF-β and hypoxia) that induce an epithelial-mesenchymal transition (EMT) diminish ERβ expression, and loss of ERβ is sufficient to promote an EMT. The mechanism involves ERβ-mediated destabilization of HIF-1α and transcriptional repression of VEGF-A. The VEGF-A receptor neuropilin-1 drives the EMT by promoting Snail1 nuclear localization. Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF-1α and VEGF expression, and Snail1 nuclear localization compared to low Gleason grade cancers.

Voltage-gated sodium channels are crucial determinants of neuronal excitability and signaling. Trafficking of the voltage-gated sodium channel NaV1.7 is dysregulated in neuropathic pain. We identify a trafficking program for NaV1.7 driven by hierarchical interactions with posttranslationally modified versions of the binding partner collapsin response mediator protein 2 (CRMP2). The binding described between CRMP2 and NaV1.7 was enhanced by conjugation of CRMP2 with small ubiquitin-like modifier (SUMO) and further controlled by the phosphorylation status of CRMP2. We determined that CRMP2 SUMOylation is enhanced by prior phosphorylation by cyclin-dependent kinase 5 and antagonized by Fyn phosphorylation. As a consequence of CRMP2 loss of SUMOylation and binding to NaV1.7, the channel displays decreased membrane localization and current density, and reduces neuronal excitability. Preventing CRMP2 SUMOylation with a SUMO-impaired CRMP2-K374A mutant triggered NaV1.7 internalization in a clathrindependent manner involving the E3 ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) and endocytosis adaptor proteins Numb and epidermal growth factor receptor pathway substrate 15. Collectively, our work shows that diverse modifications of CRMP2 cross-talk to control NaV1.7 activity and illustrate a general principle for regulation of NaV1.7.NaV1.7 sodium channel | trafficking | CRMP2 | SUMOylation | phosphorylation

Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2–CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca2+, but not Na+, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3—the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.

Chronic pain affects the life of millions of people. Current treatments have deleterious side effects. We have advanced a strategy for targeting protein interactions which regulate the N-type voltage-gated calcium (CaV2.2) channel as an alternative to direct channel block. Peptides uncoupling CaV2.2 interactions with the axonal collapsin response mediator protein 2 (CRMP2) were antinociceptive without effects on memory, depression, and reward/addiction. A search for small molecules that could recapitulate uncoupling of the CaV2.2–CRMP2 interaction identified (S)-lacosamide [(S)-LCM], the inactive enantiomer of the Food and Drug Administration–approved antiepileptic drug (R)-lacosamide [(R)-LCM, Vimpat]. We show that (S)-LCM, but not (R)-LCM, inhibits CRMP2 phosphorylation by cyclin dependent kinase 5, a step necessary for driving CaV2.2 activity, in sensory neurons. (S)-lacosamide inhibited depolarization-induced Ca2+ influx with a low micromolar IC50. Voltage-clamp electrophysiology experiments demonstrated a commensurate reduction in Ca2+ currents in sensory neurons after an acute application of (S)-LCM. Using constellation pharmacology, a recently described high content phenotypic screening platform for functional fingerprinting of neurons that uses subtype-selective pharmacological agents to elucidate cell-specific combinations (constellations) of key signaling proteins that define specific cell types, we investigated if (S)-LCM preferentially acts on certain types of neurons. (S)-lacosamide decreased the dorsal root ganglion neurons responding to mustard oil, and increased the number of cells responding to menthol. Finally, (S)-LCM reversed thermal hypersensitivity and mechanical allodynia in a model of postoperative pain, and 2 models of neuropathic pain. Thus, using (S)-LCM to inhibit CRMP2 phosphorylation is a novel and efficient strategy to treat pain, which works by targeting specific sensory neuron populations.

Treatments for chronic pain are inadequate, and new options are needed. Nonpharmaceutical approaches are especially attractive with many potential advantages including safety. Light therapy has been suggested to be beneficial in certain medical conditions such as depression, but this approach remains to be explored for modulation of pain. We investigated the effects of light-emitting diodes (LEDs), in the visible spectrum, on acute sensory thresholds in naive rats as well as in experimental neuropathic pain. Rats receiving green LED light (wavelength 525 nm, 8 h/d) showed significantly increased paw withdrawal latency to a noxious thermal stimulus; this antinociceptive effect persisted for 4 days after termination of last exposure without development of tolerance. No apparent side effects were noted and motor performance was not impaired. Despite LED exposure, opaque contact lenses prevented antinociception. Rats fitted with green contact lenses exposed to room light exhibited antinociception arguing for a role of the visual system. Antinociception was not due to stress/anxiety but likely due to increased enkephalins expression in the spinal cord. Naloxone reversed the antinociception, suggesting involvement of central opioid circuits. Rostral ventromedial medulla inactivation prevented expression of light-induced antinociception suggesting engagement of descending inhibition. Green LED exposure also reversed thermal and mechanical hyperalgesia in rats with spinal nerve ligation. Pharmacological and proteomic profiling of dorsal root ganglion neurons from green LED-exposed rats identified changes in calcium channel activity, including a decrease in the N-type (CaV2.2) channel, a primary analgesic target. Thus, green LED therapy may represent a novel, nonpharmacological approach for managing pain.