Up-Regulation of Activating Transcription Factor 3 in Human Fibroblasts Inhibits Melanoma Cell Growth and Migration Through a Paracrine Pathway

Front Oncol

2020 Apr 21

Zu T, Wen J, Xu L, Li H, Mi J, Li H, Brakebusch C, Fisher DE, Wu X
PMID: 32373541 | DOI: 10.3389/fonc.2020.00624

The treatment of melanoma has remained a difficult challenge. Targeting the tumor stroma has recently attracted attention for developing novel strategies for melanoma therapy. Activating transcription factor 3 (ATF3) plays a crucial role in regulating tumorigenesis and development, but whether the expression of ATF3 in human dermal fibroblasts (HDFs) can affect melanoma development hasn't been studied. Our results show that ATF3 expression is downregulated in stromal cells of human melanoma. HDFs expressing high levels of ATF3 suppressed the growth and migration of melanoma cells in association with downregulation of different cytokines including IL-6 in vitro. In vivo, HDFs with high ATF3 expression reduced tumor formation. Adding recombinant IL-6 to melanoma cells reversed those in vitro and in vivo effects, suggesting that ATF3 expression by HDFs regulates melanoma progression through the IL-6/STAT3 pathway. More importantly, HDFs pretreated with cyclosporine A or phenformin to induce ATF3 expression inhibited melanoma cell growth in vitro and in vivo. In summary, our study reveals that ATF3 suppresses human melanoma growth and that inducing the expression of ATF3 in HDFs can inhibit melanoma growth, a new potential melanoma therapeutic approach

Mechanisms and treatments of neuropathic itch in a mouse model of lymphoma

The Journal of clinical investigation

2022 Dec 15

Chen, O;He, Q;Han, Q;Furutani, K;Gu, Y;Olexa, M;Ji, RR;
PMID: 36520531 | DOI: 10.1172/JCI160807

Our understanding of neuropathic itch is limited, due to the lack of relevant animal models. Patients with cutaneous T-cell lymphoma (CTCL) suffer from severe itching. Here we characterize a mouse model of chronic itch with remarkable lymphoma growth, immune cell accumulation, and persistent pruritus. Intradermal CTCL inoculation produces time-dependent changes in nerve innervations in lymphoma-bearing skin. In the early-phase (20 days), CTCL causes hyper-innervations in the epidermis. However, chronic itch is associated with loss of epidermal nerve fibers in the late-phases (40 and 60 days). CTCL is also characterized by marked nerve innervations in mouse lymphoma. Blockade of C-fibers reduced pruritus at early- and late-phases, whereas blockade of A-fibers only suppressed late-phase itch. Intrathecal gabapentin injection reduced late-phase but not early-phase pruritus. IL-31 is upregulated in mouse lymphoma, while its receptor Il31ra was persistently upregulated in Trpv1-expressing sensory neurons in CTCL mice. Intratumoral anti-IL-31 treatment effectively suppressed CTCL-induced scratching and alloknesis (mechanical itch). Finally, intrathecal administration of TLR4 antagonist attenuated pruritus in early and late phases and in both sexes. Collectively, we have established a mouse model of neuropathic and cancer itch with relevance to human disease. Our findings also suggest distinct mechanisms underlying acute, chronic, and neuropathic itch.

Longitudinal single-cell RNA-seq analysis reveals stress-promoted chemoresistance in metastatic ovarian cancer

Science advances

2022 Feb 25

Zhang, K;Erkan, EP;Jamalzadeh, S;Dai, J;Andersson, N;Kaipio, K;Lamminen, T;Mansuri, N;Huhtinen, K;Carpén, O;Hietanen, S;Oikkonen, J;Hynninen, J;Virtanen, A;Häkkinen, A;Hautaniemi, S;Vähärautio, A;
PMID: 35196078 | DOI: 10.1126/sciadv.abm1831

Chemotherapy resistance is a critical contributor to cancer mortality and thus an urgent unmet challenge in oncology. To characterize chemotherapy resistance processes in high-grade serous ovarian cancer, we prospectively collected tissue samples before and after chemotherapy and analyzed their transcriptomic profiles at a single-cell resolution. After removing patient-specific signals by a novel analysis approach, PRIMUS, we found a consistent increase in stress-associated cell state during chemotherapy, which was validated by RNA in situ hybridization and bulk RNA sequencing. The stress-associated state exists before chemotherapy, is subclonally enriched during the treatment, and associates with poor progression-free survival. Co-occurrence with an inflammatory cancer-associated fibroblast subtype in tumors implies that chemotherapy is associated with stress response in both cancer cells and stroma, driving a paracrine feed-forward loop. In summary, we have found a resistant state that integrates stromal signaling and subclonal evolution and offers targets to overcome chemotherapy resistance.

Promoting regeneration while blocking cell death preserves motor neuron function in a model of ALS

Brain : a journal of neurology

2022 Nov 07

Wlaschin, JJ;Donahue, C;Gluski, J;Osborne, JF;Ramos, LM;Silberberg, H;Le Pichon, CE;
PMID: 36342754 | DOI: 10.1093/brain/awac415

Amyotrophic lateral sclerosis or ALS is a devastating and fatal neurodegenerative disease of motor neurons with very few treatment options. We had previously found that motor neuron degeneration in a mouse model of ALS can be delayed by deleting the axon damage sensor MAP3K12 or Dual Leucine Zipper Kinase (DLK)1. However, DLK is also involved in axon regeneration2-5, prompting us to ask whether combining DLK deletion with a way to promote axon regeneration would result in greater motor neuron protection. To achieve this, we used a mouse line that constitutively expresses ATF3, a master regulator of regeneration in neurons6,7. Although there is precedence for each individual strategy in the SOD1G93A mouse model of ALS1,8, these have not previously been combined. By several lines of evidence including motor neuron electrophysiology, histology and behavior, we observed a powerful synergy when combining DLK deletion with ATF3 expression. The combinatorial strategy resulted in significant protection of motor neurons with fewer undergoing cell death, reduced axon degeneration, and preservation of motor function and connectivity to muscle. This study provides a demonstration of the power of combinatorial therapy to treat neurodegenerative disease.

Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain

Nat Commun

2020 Jan 14

Yu X, Liu H, Hamel KA, Morvan MG, Yu S, Leff J, Guan Z, Braz JM, Basbaum AI
PMID: 31937758 | DOI: 10.1038/s41467-019-13839-2

Paralleling the activation of dorsal horn microglia after peripheral nerve injury is a significant expansion and proliferation of macrophages around injured sensory neurons in dorsal root ganglia (DRG). Here we demonstrate a critical contribution of DRG macrophages, but not those at the nerve injury site, to both the initiation and maintenance of the mechanical hypersensitivity that characterizes the neuropathic pain phenotype. In contrast to the reported sexual dimorphism in the microglial contribution to neuropathic pain, depletion of DRG macrophages reduces nerve injury-induced mechanical hypersensitivity and expansion of DRG macrophages in both male and female mice. However, fewer macrophages are induced in the female mice and deletion of colony-stimulating factor 1 from sensory neurons, which prevents nerve injury-induced microglial activation and proliferation, only reduces macrophage expansion in male mice. Finally, we demonstrate molecular cross-talk between axotomized sensory neurons and macrophages, revealing potential peripheral DRG targets for neuropathic pain management

Single cell atlas of spinal cord injury in mice reveals a pro-regenerative signature in spinocerebellar neurons

Nature communications

2022 Sep 26

Matson, KJE;Russ, DE;Kathe, C;Hua, I;Maric, D;Ding, Y;Krynitsky, J;Pursley, R;Sathyamurthy, A;Squair, JW;Levi, BP;Courtine, G;Levine, AJ;
PMID: 36163250 | DOI: 10.1038/s41467-022-33184-1

After spinal cord injury, tissue distal to the lesion contains undamaged cells that could support or augment recovery. Targeting these cells requires a clearer understanding of their injury responses and capacity for repair. Here, we use single nucleus RNA sequencing to profile how each cell type in the lumbar spinal cord changes after a thoracic injury in mice. We present an atlas of these dynamic responses across dozens of cell types in the acute, subacute, and chronically injured spinal cord. Using this resource, we find rare spinal neurons that express a signature of regeneration in response to injury, including a major population that represent spinocerebellar projection neurons. We characterize these cells anatomically and observed axonal sparing, outgrowth, and remodeling in the spinal cord and cerebellum. Together, this work provides a key resource for studying cellular responses to injury and uncovers the spontaneous plasticity of spinocerebellar neurons, uncovering a potential candidate for targeted therapy.

Engram-specific transcriptome profiling of contextual memory consolidation.

Nat Commun

2019 May 20

Rao-Ruiz P, Couey JJ, Marcelo IM, Bouwkamp CG, Slump DE, Matos MR, van der Loo RJ, Martins GJ, van den Hout M, van IJcken WF, Costa RM, van den Oever MC, Kushner SA.
PMID: 31110186 | DOI: 10.1038/s41467-019-09960-x

Sparse populations of neurons in the dentate gyrus (DG) of the hippocampus are causally implicated in the encoding of contextual fear memories. However, engram-specific molecular mechanisms underlying memory consolidation remain largely unknown. Here we perform unbiased RNA sequencing of DG engram neurons 24 h after contextual fear conditioning to identify transcriptome changes specific to memory consolidation. DG engram neurons exhibit a highly distinct pattern of gene expression, in which CREB-dependent transcription features prominently (P = 6.2 × 10-13), including Atf3 (P = 2.4 × 10-41), Penk (P = 1.3 × 10-15), and Kcnq3 (P = 3.1 × 10-12). Moreover, we validate the functional relevance of the RNAseq findings by establishing the causal requirement of intact CREB function specifically within the DG engram during memory consolidation, and identify a novel group of CREB target genes involved in the encoding of long-term memory

A cell-type-specific atlas of the inner ear transcriptional response to acoustic trauma

Cell reports

2021 Sep 28

Milon, B;Shulman, ED;So, KS;Cederroth, CR;Lipford, EL;Sperber, M;Sellon, JB;Sarlus, H;Pregernig, G;Shuster, B;Song, Y;Mitra, S;Orvis, J;Margulies, Z;Ogawa, Y;Shults, C;Depireux, DA;Palermo, AT;Canlon, B;Burns, J;Elkon, R;Hertzano, R;
PMID: 34592158 | DOI: 10.1016/j.celrep.2021.109758

Noise-induced hearing loss (NIHL) results from a complex interplay of damage to the sensory cells of the inner ear, dysfunction of its lateral wall, axonal retraction of type 1C spiral ganglion neurons, and activation of the immune response. We use RiboTag and single-cell RNA sequencing to survey the cell-type-specific molecular landscape of the mouse inner ear before and after noise trauma. We identify induction of the transcription factors STAT3 and IRF7 and immune-related genes across all cell-types. Yet, cell-type-specific transcriptomic changes dominate the response. The ATF3/ATF4 stress-response pathway is robustly induced in the type 1A noise-resilient neurons, potassium transport genes are downregulated in the lateral wall, mRNA metabolism genes are downregulated in outer hair cells, and deafness-associated genes are downregulated in most cell types. This transcriptomic resource is available via the Gene Expression Analysis Resource (gEAR; https://umgear.org/NIHL) and provides a blueprint for the rational development of drugs to prevent and treat NIHL.

Dual leucine zipper kinase is required for mechanical allodynia and microgliosis after nerve injury.

Elife.

2018 Jul 03

Wlaschin JJ, Gluski JM, Nguyen E, Silberberg H, Thompson JH, Chesler AT, Le Pichon CE.
PMID: 29968565 | DOI: 10.7554/eLife.33910

Neuropathic pain resulting from nerve injury can become persistent and difficult to treat but the molecular signaling responsible for its development remains poorly described. Here, we identify the neuronal stress sensor dual leucine zipper kinase (DLK; Map3k12) as a key molecule controlling the maladaptive pathways that lead to pain following injury. Genetic or pharmacological inhibition of DLK reduces mechanical hypersensitivity in a mouse model of neuropathic pain. Furthermore, DLK inhibition also prevents the spinal cord microgliosis that results from nerve injury and arises distant from the injury site. These striking phenotypes result from the control by DLK of a transcriptional program in somatosensory neurons regulating the expression of numerous genes implicated in pain pathogenesis, including the immune gene Csf1. Thus, activation of DLK is an early event, or even the master regulator, controlling a wide variety of pathways downstream of nerve injury that ultimately lead to chronic pain.

Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration

PLoS biology

2021 Nov 01

Udagawa, T;Atkinson, PJ;Milon, B;Abitbol, JM;Song, Y;Sperber, M;Huarcaya Najarro, E;Scheibinger, M;Elkon, R;Hertzano, R;Cheng, AG;
PMID: 34758021 | DOI: 10.1371/journal.pbio.3001445

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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