Dedifferentiation Of Cells Research Articles

Innate immune receptor signaling induces transient melanoma dedifferentiation while preserving immunogenicity

BackgroundImmune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor...

Phase I/II trial of RVU120 (SEL120), a CDK8/CDK19 inhibitor in patients with relapsed/refractory metastatic or advanced solid tumors.

e15091 Background: CDK 8 /CDK19 are kinases involved in transcriptional control of embryonic development and cellular homeostasis via the mediator complex. They are also described to maintain tumor dedifferentiation and stem cell properties. A variety of cancer cells hijack the mediator complex and targeting cancer-specific gene transcription via CDK8/19 inhibition has potential for treatment of solid tumors. RVU120 (SEL120) is a selective CDK8/19 inhibitor with preclinical efficacy in hematologic malignancies and a variety of solid tumor types (Rzymski et al. 2017). RVU120 showed strong anticancer activity in preclinical triple-negative breast cancer (TNBC) models, especially in the mesenchymal stem-like (MSL) molecular subtype (Rzymski et al SABCS 2021). Methods: A Phase I/II clinical trial of RVU120 in solid tumors is currently ongoing (NCT05052255). Patients with metastatic or advanced solid tumors without available treatments receive increasing doses of RVU120 as a single oral dose every other day (QOD) for a total of 7 doses on Days 1, 3, 5, 7, 9, 11 and 13, in a 3-week treatment cycle. Primary objectives of the study are safety / tolerability of RVU120 as single agent and determination of the recommended Phase II dose (RP2D) via a 3+3 dose escalation design. Secondary objectives include objective response rate (ORR) (RECIST criteria v1.1) and PK of RVU120. Results: As of 11FEB 2022, 5 pts have been enrolled into the trial: 3 into cohort 1, 75 mg dose, 2 into cohort 2, 100 mg. None of the pts experienced a DLT, SAEs or > Grade 3 AE. Treatment-related AEs were all G1 except for G2 diarrhea and weakness. Cohort 1 patients had at least 1 post-baseline tumor assessment. A 62 YO patient, stage IV esophago-gastric junction carcinoma, metastases to the liver, retroperitoneum, adrenal gland and portocaval lymph nodes, progressing after 4 lines of previous therapy, achieved a SD with a +4% change of target lesion size and progressed in cycle 6. A 65 YO patient, stage IV adenoid cystic carcinoma of the trachea, metastases to the lung and refractory to antiandrogens and radiotherapy, achieved a SD with -4% change of target lesion size after 3 cycles and is currently ongoing in cycle 5. A 43 YO pt stage IV thymic carcinoma, metastases to the lung, mediastinum, pleura and lymph nodes, progressing under cisplatin/etoposide, stopped study drug at the end of cycle 3 for PD in non-target lesions, target lesions increased by 11%. Cohort 2 patients, a 65 YO pt with pancreatic cancer stage IV, metastases to liver and lung, and a 55 YO pt with TNBC stage IV, metastases to the lung, will be evaluated end of cycle 3. Conclusions: In the first dose escalation level with single agent RVU120, no DLTs or ≥G3 AEs were observed in patients with previously progressive solid tumors. Initial assessments in 3 pts demonstrated stable disease in 2 patients. Collection of further data at higher doses is ongoing with 2 patients enrolled at 100 mg. Clinical trial information: NCT05052255.

In preprints: the fast-paced field of single-cell lineage tracing

In preprints: the fast-paced field of single-cell lineage tracing

The RNA signature of immune-related genes and distinct spatial distribution of immune cells are closely associated with the treatment outcomes in the malignant melanoma patients receiving immune checkpoint inhibitors.

e21504 Background: The immune checkpoint inhibitors (ICIs), have improved survival with durable response for advanced malignant melanomas, but, only a minority of patients has achieved. To stay sway failure of triage patients, considerable side effects and costs of ICB therapies, the development of robust signatures predictive of response to ICB therapies is warranted. Methods: Clinical data were collected at Seoul St. Mary’s Hospitals. Among them, we examined RNA expression profile by QuantSeq 3’ mRNA-Sequencing and the composition, and spatial distance of immune cells by multiplex immunofluorescent staining. Results: The gene sets with relatively higher RNA expression in the good response group, were as follows; hematopoietic cell dedifferentiation gene, T-cell related adaptive immunity gene, IFN-related cytokine gene, and macrophage (TAM), NK cell, innate immunity gene set. Meanwhile, in the poor response group, inflammatory response, fibrosis response genes was increased.The cell densities of T cells, cytotoxic T cells, Treg cells, CD8+Treg cells, and exhausted T cells in the good response group tended to be higher, which was clearer in the peritumoral (PT) area than the intratumoral (IT) area. For the good response group, M1/M2 ratio in the IT area was significantly higher compared to that of poor response group (p = 0.045). According to the nearest neighbor analysis for % of melanoma cells within 15um distance present a close distance from M1, M2, and NK cells tended to be higher in the IT area than PT area. For the cases with higher spatial distribution from M1 in the IT area, PFS and OS was significantly extended (PFS, p = 0.0021; OS, p = 0.036). The gene sets with relatively higher mRNA expression in high neighborhood macrophage group, were cell death, apoptosis process gene set. In low neighboring M1 macrophage group, relatively higher mRNA expressions of extracellular component inflammation and fibrosis gene, cell migration, and cell de-differentiation gene set were observed. Conclusions: Taken together, our results suggest 4 key points. First, good and poor responder groups for ICB show marked differences from mRNA profiling to protein expression according to central dogma. Second, immune cells can be distributed sporadically in many places, but the actionable ground of the adaptive immune cells of the T cell family is the PT, and the playground of innate immune cells such as TAM and NK cells is the IT. In particular, the anti-tumor response of M1 macrophage and M2 macrophage was oppositely in IT. Third, high TAMs in IT are interpreted to have a tumoricidal effect because they show a stationery state while coding the killing effect gene set. In addition, topologically, spatial analysis as well as bulk genomics are important for predicting tumor response for ICIs.

Redox Homeostasis in Thyroid Cancer: Implications in Na+/I- Symporter (NIS) Regulation.

Radioiodine therapy (RAI) is a standard and effective therapeutic approach for differentiated thyroid cancers (DTCs) based on the unique capacity for iodide uptake and accumulation of the thyroid gland through the Na+/I− symporter (NIS). However, around 5–15% of DTC patients may become refractory to radioiodine, which is associated with a worse prognosis. The loss of RAI avidity due to thyroid cancers is attributed to cell dedifferentiation, resulting in NIS repression by transcriptional and post-transcriptional mechanisms. Targeting the signaling pathways potentially involved in this process to induce de novo iodide uptake in refractory tumors is the rationale of “redifferentiation strategies”. Oxidative stress (OS) results from the imbalance between ROS production and depuration that favors a pro-oxidative environment, resulting from increased ROS production, decreased antioxidant defenses, or both. NIS expression and function are regulated by the cellular redox state in cancer and non-cancer contexts. In addition, OS has been implicated in thyroid tumorigenesis and thyroid cancer cell dedifferentiation. Here, we review the main aspects of redox homeostasis in thyrocytes and discuss potential ROS-dependent mechanisms involved in NIS repression in thyroid cancer.

Spatial transcriptomics reveals metabolic changes underly age-dependent declines in digit regeneration

De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema. Using this technique, we generated a gene signature with high specificity for the blastema in both our spatial data, as well as other previously published single-cell RNA-sequencing transcriptomic studies. To elucidate potential mechanisms distinguishing regenerative from non-regenerative healing, we applied spatial transcriptomics to an aging model. Consistent with other forms of repair, our digit amputation mouse model showed a significant impairment in regeneration in aged mice. Contrasting young and aged mice, spatial analysis revealed a metabolic shift in aged blastema associated with an increased bioenergetic requirement. This enhanced metabolic turnover was associated with increased hypoxia and angiogenic signaling, leading to excessive vascularization and altered regenerated bone architecture in aged mice. Administration of the metabolite oxaloacetate decreased the oxygen consumption rate of the aged blastema and increased WNT signaling, leading to enhanced in vivo bone regeneration. Thus, targeting cell metabolism may be a promising strategy to mitigate aging-induced declines in tissue regeneration.

Notch3 expression in capillary pericytes predicts worse graft outcome in human renal grafts with antibody-mediated rejection.

Microvasculature consisting of endothelial cells and pericytes is the main site of injury during antibody‐mediated rejection (ABMR) of renal grafts. Little is known about the mechanisms of activation of pericytes in this pathology. We have found recently that activation of Notch3, a mediator of vascular smooth muscle cell proliferation and dedifferentiation, promotes renal inflammation and fibrosis and aggravates progression of renal disease. Therefore, we studied the pericyte expression of Notch3 in 49 non‐selected renal graft biopsies (32 for clinical cause, 17 for graft surveillance). We analysed its relationship with patients’ clinical and morphological data, and compared with the expression of partial endothelial mesenchymal transition (pEndMT) markers, known to reflect endothelial activation during ABMR. Notch3 was de novo expressed in pericytes of grafts with ABMR, and was significantly correlated with the microcirculation inflammation scores of peritubular capillaritis and glomerulitis and with the expression of pEndMT markers. Notch3 expression was also associated with graft dysfunction and proteinuria at the time of biopsy and in the long term. Multivariate analysis confirmed pericyte expression of Notch3 as an independent risk factor predicting graft loss. These data suggest that Notch3 is activated in the pericytes of renal grafts with ABMR and is associated with poor graft outcome.

Perioperative Suppression of Schwann Cell Dedifferentiation Reduces the Risk of Adenomyosis Resulting from Endometrial-Myometrial Interface Disruption in Mice.

We have recently demonstrated that endometrial–myometrial interface (EMI) disruption (EMID) can cause adenomyosis in mice, providing experimental evidence for the well-documented epidemiological finding that iatrogenic uterine procedures increase the risk of adenomyosis. To further elucidate its underlying mechanisms, we designed this study to test the hypothesis that Schwann cells (SCs) dedifferentiating after EMID facilitate the genesis of adenomyosis, but the suppression of SC dedifferentiation perioperatively reduces the risk. We treated mice perioperatively with either mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated protein kinase (ERK) or c-Jun N-terminal kinase (JNK) inhibitors or a vehicle 4 h before and 24 h, 48 h and 72 h after the EMID procedure. We found that EMID resulted in progressive SCs dedifferentiation, concomitant with an increased abundance of epithelial cells in the myometrium and a subsequent epithelial–mesenchymal transition (EMT). This EMID-induced change was abrogated significantly with perioperative administration of JNK or MEK/ERK inhibitors. Consistently, perioperative administration of a JNK or a MEK/ERK inhibitor reduced the incidence by nearly 33.5% and 14.3%, respectively, in conjunction with reduced myometrial infiltration of adenomyosis and alleviation of adenomyosis-associated hyperalgesia. Both treatments significantly decelerated the establishment of adenomyosis and progression of EMT, fibroblast-to-myofibroblast trans-differentiation and fibrogenesis in adenomyotic lesions. Thus, we provide the first piece of evidence strongly implicating the involvement of SCs in the pathogenesis of adenomyosis induced by EMID.

Sbk2, a Newly Discovered Atrium-Enriched Regulator of Sarcomere Integrity.

Heart development relies on tight spatiotemporal control of cardiac gene expression. Genes involved in this intricate process have been identified using animals and pluripotent stem cell-based models of cardio(myo)genesis. Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal line of conditionally immortalized neonatal rat atrial myocytes (NRAMs), which allows toggling between proliferative and differentiated (ie, excitable and contractile) phenotypes in a synchronized and homogenous manner. In this study, the unique properties of conditionally immortalized NRAMs (iAMs) were exploited to identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation. Transcriptome analysis of iAM-1 cells at different stages during one cycle of differentiation and subsequent dedifferentiation identified ≈13 000 transcripts, of which the dynamic changes in expression upon cardiomyogenic differentiation mostly opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many with known (lineage-specific) functions in cardiac muscle formation. Filtering for cardiac-enriched low-abundance transcripts, identified multiple genes with an uncharacterized role during cardio(myo)genesis including Sbk2 (SH3 domain binding kinase family member 2). Sbk2 encodes an evolutionarily conserved putative serine/threonine protein kinase, whose expression is strongly up- and downregulated during iAM-1 cell differentiation and dedifferentiation, respectively. In neonatal and adult rats, the protein is muscle-specific, highly atrium-enriched, and localized around the A-band of cardiac sarcomeres. Knockdown of Sbk2 expression caused loss of sarcomeric organization in NRAMs, iAMs and their human counterparts, consistent with a decrease in sarcomeric gene expression as evinced by transcriptome and proteome analyses. Interestingly, co-immunoprecipitation using Sbk2 as bait identified possible interaction partners with diverse cellular functions (translation, intracellular trafficking, cytoskeletal organization, chromatin modification, sarcomere formation). iAM-1 cells are a relevant and suitable model to identify (lowly expressed) genes with a hitherto unidentified role in cardiomyocyte differentiation as exemplified by Sbk2: a regulator of atrial sarcomerogenesis.

RNA splicing is a key mediator of tumour cell plasticity and a therapeutic vulnerability in colorectal cancer

Tumour cell plasticity is a major barrier to the efficacy of targeted cancer therapies but the mechanisms that mediate it are poorly understood. Here, we identify dysregulated RNA splicing as a key driver of tumour cell dedifferentiation in colorectal cancer (CRC). We find that Apc-deficient CRC cells have dysregulated RNA splicing machinery and exhibit global rewiring of RNA splicing. We show that the splicing factor SRSF1 controls the plasticity of tumour cells by controlling Kras splicing and is required for CRC invasion in a mouse model of carcinogenesis. SRSF1 expression maintains stemness in human CRC organoids and correlates with cancer stem cell marker expression in human tumours. Crucially, partial genetic downregulation of Srsf1 does not detrimentally affect normal tissue homeostasis, demonstrating that tumour cell plasticity can be differentially targeted. Thus, our findings link dysregulation of the RNA splicing machinery and control of tumour cell plasticity.

Prohaptoglobin inhibits the transforming growth factor-β-induced epithelial-to-mesenchymal transition in vitro by increasing Smad1/5 activation and suppressing the Smad2/3 signaling pathway in SK-Hep1 liver cancer cells.

Transforming growth factor-β (TGF-β) is an important inducer of the epithelial-to-mesenchymal transition (EMT) in various cancers. Our previous study demonstrated that prohaptoglobin (proHp) stimulates Smad1/5 activation via ALK1, a TGF-β type I receptor, in endothelial cells, suggesting that proHp plays a role in TGF-β signaling. However, the function of proHp in cellular events downstream of Smads remains unclear. The current study investigated the effects of proHp on TGF-β-mediated Smad-dependent EMT induction and cell invasion in vitro using proHp-overexpressing SK-Hep1 liver cancer cells. The results of Western blotting, quantitative real-time RT-PCR, and immunocytochemistry indicated that proHp downregulated expression of mesenchymal marker and EMT regulator such as N-cadherin, vimentin, and twist, and upregulated expression of the epithelial marker E-cadherin. Compared with control cells, proHp-overexpressing cells exhibited high levels of ALK1/2/3 receptors and markedly increased Smad1/5 phosphorylation. Interestingly, proHp attenuated TGF-β-induced expression of mesenchymal markers and Smad2/3 phosphorylation. It also significantly suppressed cell invasion and migration. Knockdown of Smad1/5 abolished the inhibitory effects of proHp on TGF-β-stimulated Smad2/3 phosphorylation and mesenchymal marker expression. These findings indicate that proHp suppresses the TGF-β-induced EMT and cell invasion in vitro by enhancing Smad1/5 activation via ALK1/2/3 receptors and thus suppressing the Smad2/3 signaling pathway in SK-Hep1 cells. This study suggests that proHp may prevent a de-differentiation of hepatic cells and induce a cell differentiation by regulating the Smad signaling pathway.

Remodeling Osteoarthritic Articular Cartilage under Hypoxic Conditions.

Osteoarthritis (OA) is one of the leading joint diseases induced by abnormalities or inflammation in the synovial membrane and articular cartilage, causing severe pain and disability. Along with the cartilage malfunction, imbalanced oxygen uptake occurs, changing chondrocytes into type I collagen- and type X collagen-producing dedifferentiated cells, contributing to OA progression. However, mounting evidence suggests treating OA by inducing a hypoxic environment in the articular cartilage, targeting the inhibition of several OA-related pathways to bring chondrocytes into a normal state. This review discusses the implications of OA-diseased articular cartilage on chondrocyte phenotypes and turnover and debates the hypoxic mechanism of action. Furthermore, this review highlights the new understanding of OA, provided by tissue engineering and a regenerative medicine experimental design, modeling the disease into diverse 2D and 3D structures and investigating hypoxia and hypoxia-inducing biomolecules and potential cell therapies. This review also reports the mechanism of hypoxic regulation and highlights the importance of activating and stabilizing the hypoxia-inducible factor and related molecules to protect chondrocytes from mitochondrial dysfunction and apoptosis occurring under the influence of OA.

Molecular Determinants of in vitro Plant Regeneration: Prospects for Enhanced Manipulation of Lettuce (Lactuca sativa L.).

In vitro plant regeneration involves dedifferentiation and molecular reprogramming of cells in order to regenerate whole organs. Plant regeneration can occur via two pathways, de novo organogenesis and somatic embryogenesis. Both pathways involve intricate molecular mechanisms and crosstalk between auxin and cytokinin signaling. Molecular determinants of both pathways have been studied in detail in model species, but little is known about the molecular mechanisms controlling de novo shoot organogenesis in lettuce. This review provides a synopsis of our current knowledge on molecular determinants of de novo organogenesis and somatic embryogenesis with an emphasis on the former as well as provides insights into applying this information for enhanced in vitro regeneration in non-model species such as lettuce (Lactuca sativa L.).

A Novel Estrogen Receptor β Agonist Diminishes Ovarian Cancer Stem Cells via Suppressing the Epithelial-to-Mesenchymal Transition.

Simple SummaryEstrogen receptors α (ERα) and β (ERβ) show distinct contributions to tumor initiation and progression. Given that ERβ mainly functions as a “tumor suppressor”, activation of ERβ using its specific agonist should be able to inhibit tumor progression. In this study, we show that a newly developed ERβ agonist, OSU-ERb-12, can not only impede ovarian cancer cell expansion and tumor growth but also reduce the cancer stem cell (CSC) population. OSU-ERb-12 could inhibit epithelial-to-mesenchymal transition (EMT) in ovarian cancer cells by increasing Snail expression, thereby, blocking EMT-mediated cancer cell dedifferentiation. We also found that OSU-ERb-12 inhibits ovarian cancer expansion in an ERα-independent manner while limiting the CSC population in an ERα-dependent manner. Taken together, our data suggest that the newly developed ERβ agonist OSU-ERb-12 can be used not only to hinder tumor growth but also has the potential to prevent tumor relapse and metastasis by depleting CSCs.Epithelial ovarian cancer is the most lethal malignancy of the female reproductive tract. A healthy ovary expresses both Estrogen Receptor α (ERα) and β (ERβ). Given that ERα is generally considered to promote cell survival and proliferation, thereby, enhancing tumor growth, while ERβ shows a protective effect against the development and progression of tumors, the activation of ERβ by its agonists could be therapeutically beneficial for ovarian cancer. Here, we demonstrate that the activation of ERβ using a newly developed ERβ agonist, OSU-ERb-12, can impede ovarian cancer cell expansion and tumor growth in an ERα-independent manner. More interestingly, we found that OSU-ERb-12 also reduces the cancer stem cell (CSC) population in ovarian cancer by compromising non-CSC-to-CSC conversion. Mechanistically, we revealed that OSU-ERb-12 decreased the expression of Snail, a master regulator of the epithelial-to-mesenchymal transition (EMT), which is associated with de novo CSC generation. Given that ERα can mediate EMT and facilitate maintenance of the CSC subpopulation and that OSU-ERb-12 can block the transactivity of ERα, we conclude that OSU-ERb-12 reduces the CSC subpopulation by inhibiting EMT in an ERα-dependent manner. Taken together, our data indicate that the ERβ agonist OSU-ERb-12 could be used to hinder tumor progression and limit the CSC subpopulation with the potential to prevent tumor relapse and metastasis in patients with ovarian cancer.

Experimental Aristolochic Acid Nephropathy: A Relevant Model to Study AKI-to-CKD Transition.

Aristolochic acid nephropathy (AAN) is a progressive tubulointerstitial nephritis caused by the intake of aristolochic acids (AA) contained in Chinese herbal remedies or contaminated food. AAN is characterized by tubular atrophy and interstitial fibrosis, characterizing advanced kidney disease. It is established that sustained or recurrent acute kidney injury (AKI) episodes contribute to the progression of CKD. Therefore, the study of underlying mechanisms of AA-induced nephrotoxicity could be useful in understanding the complex AKI-to-CKD transition. We developed a translational approach of AKI-to-CKD transition by reproducing human AAN in rodent models. Indeed, in such models, an early phase of acute tubular necrosis was rapidly followed by a massive interstitial recruitment of activated monocytes/macrophages followed by cytotoxic T lymphocytes, resulting in a transient AKI episode. A later chronic phase was then observed with progressive tubular atrophy related to dedifferentiation and necrosis of tubular epithelial cells. The accumulation of vimentin and αSMA-positive cells expressing TGFβ in interstitial areas suggested an increase in resident fibroblasts and their activation into myofibroblasts resulting in collagen deposition and CKD. In addition, we identified 4 major actors in the AKI-to-CKD transition: (1) the tubular epithelial cells, (2) the endothelial cells of the interstitial capillary network, (3) the inflammatory infiltrate, and (4) the myofibroblasts. This review provides the most comprehensive and informative data we were able to collect and examines the pending questions.

Limbal Niche Cells and Three-Dimensional Matrigel-Induced Dedifferentiation of Mature Corneal Epithelial Cells.

PurposeTo investigate the phenotypic changes of mature corneal epithelial cells (MCECs) that cocultured with limbal niche cells (LNCs) in three-dimensional Matrigel (3D Matrigel) in vitro.MethodsMCECs were isolated from central corneas, and limbal epithelial progenitor cells (LEPCs) were isolated from limbal segments with Dispase II. LNCs were isolated and cultured from limbal niche using the collagenase A digestion method and identified with PCK/VIM/CD90/CD105/SCF/PDGFRβ. MCECs were cultured on 3D Matrigel (50%, v/v) with or without LNCs for 10 days. Expression of CK12 and p63α and clone formation test were used to compare the progenitor phenotypic changes for MCECs before and after induction using LEPCs as control.ResultsHomogeneous LNCs were isolated and identified as spindle shape and adherent to a plastic surface coated with 5% Matrigel. Double immunostaining of the fourth-passage LNCs was uniformly PCK−/VIM+/CD90+/CD105+/SCF+/PDGFRβ+. Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) revealed the decrease of PCK expression from the second passage and elevation of Vim, CD90, CD105, SCF, and PDGFRβ transcripts from the third passage, and the transcription level of Vim, CD90, CD105, SCF, and PDGFRβ was elevated statistically in the fourth passage compared to the first passage (P < 0.01). Both immunofluorescence (IF) staining for cross section and cytospin cells demonstrated that MCECs expressed higher CK12 while lower p63α than LEPCs (P < 0.01). Sphere growth formation was noticed as early as 24 hours in the MCEC + LNC group, 48 hours in the LEPC group, and 72 hours in the MCEC group. The diameters of the spheres were the biggest in the MCEC + LNC group (182.24 ± 57.91 µm), smaller in the LEPC group (125.71 ± 41.20 µm), and smallest in the MCEC group (109.39 ± 34.85 µm) by the end of the 10-day culture (P < 0.01). Double immunostaining with CK12/p63α showed that cells in the sphere formed from MCECs expressed CK12 but not p63α; in contrast, some cells in the MCEC + LNC group expressed CK12, but most of them expressed p63α. RT-qPCR revealed a significant reduction of CK12 transcript but elevation of p63α, Oct4, Nanog, Sox2, and SSEA4 (P < 0.05). Holoclone composed of cubic epithelial cells could be generated in the MCEC + LNC group but not in the other two groups.ConclusionsThe data shows that human MCEC cell phenotype could be induced to the dedifferentiation stage when cocultured with LNCs in 3D Matrigel that simulated the microenvironment of limbal stem cells in vitro.

The sialyltransferase ST6Gal1 is upregulated in a cytokine‐mediated manner during pancreatitis

Pancreatitis is a life‐threatening medical disorder that occurs when abnormally activated pancreatic enzymes damage surrounding pancreatic acinar cells, leading to the release of several pro‐inflammatory cytokines. Subsequently, inflammation of the pancreas promotes the dedifferentiation of acinar cells towards a ductal‐like, progenitor phenotype, to heal from pancreatic injury, in a process known as acinar to ductal metaplasia (ADM). Recent studies by our group suggest that the sialyltransferase, ST6Gal1 promotes ADM. ST6Gal1 adds sialic acid to N‐glycosylated surface receptors and thereby regulates cell signaling and phenotype. In the current study, we report the novel finding that ST6Gal1 is upregulated in the pancreatic tissues of patients with chronic pancreatitis and mice with experimental pancreatitis, whereas ST6Gal1 expression is undetectable in normal acinar cells. These results are the first to show stark modulation of ST6Gal1 within pancreatic inflammation. While the upregulation of ST6Gal1 has been noted across multiple cancers, the mechanisms by which ST6Gal1 expression is modulated within an inflammatory milieu are not well understood. To evaluate inflammation‐induced ST6Gal1 upregulation, we injected mice with cerulein to evoke pancreatitis. We first examined levels of pro‐inflammatory cytokines through immunohistochemistry in cerulein‐treated mice and observed striking increases in the release of both IL‐6 and IL‐1β. Accordingly, we proceeded to treat multiple pancreatic acinar cell line models with IL‐6 and IL‐1β to assess their respective capability to upregulate ST6Gal1. These two cytokines stimulated increases in ST6Gal1 mRNA and protein in all cell models. It's known that there are three major isoforms of ST6Gal1, each driven by a different promoter; thus, we investigated which of the three isoforms was increased following cytokine treatment. We found that the isoform driven by the P3 promoter, a ubiquitously expressed isoform known as “YZ”, was the only isoform seen to be upregulated following cytokine treatment. To further understand the mechanisms by which IL‐6 and IL‐1β mediate their upregulation of ST6Gal1, we used an online transcription factor binding predictor to assess which transcription factors bind to the P3 promoter of the YZ isoform of ST6Gal1. On this P3 promoter, we found binding sites for STAT3, which is activated by IL‐6, and NF‐κB, which is activated by IL‐1β. Furthermore, we observed STAT3 and NF‐κB pathway activation following cytokine treatment. Importantly, STAT3 and NF‐κB inhibitors blocked cytokine‐induced ST6Gal1 upregulation in multiple cell models, thus confirming that IL‐6 and IL‐1β mediated their upregulation of the YZ isoform through the STAT3 and NF‐κB pathways, respectively. Taken together, these results not only show ST6Gal1 modulation within pancreatitis but detail the mechanisms by which this occurs, thus providing significant insight into an area of research that has not been well characterized.

Diminished noncanonical NF-κB signaling induces colitis-associated colorectal cancer susceptibility upon de-differentiation of epithelial cells

Abstract Patients with a medical history of Inflammatory Bowel Disease are predisposed to developing colitis-associated colorectal cancer (CAC), with greater than 20% developing CAC within 30 years of disease onset. CAC is a form of inflammation-induced tumorigenesis, where unregulated inflammation causes cells in the colon to accumulate mutations poising them for neoplasia. We demonstrate that NF-κB inducing kinase (NIK) is a critical regulator of intestinal epithelial cell (IEC) regeneration. We hypothesize that IECs exposed to chronic inflammatory conditions mitigated by dysregulated noncanonical NF-κB signaling are most prone to mutagenesis and cancer-initiating events termed Top-down tumorigenesis. In CAC, NIK depletion results in the accumulation of mature IECs that are increasingly susceptible to mutation and neoplastic transformation. This suggests that terminally differentiated IECs dedifferentiate into a stem-like phenotype. Our work has clinical relevancy, where human CAC biopsy samples have diminished noncanonical NF-κB signaling and NIK (MAP3K14) gene expression. Murine models with NIK deletion in IECs have increased susceptibility to inflammation-induced tumorigenesis following the chemical induction of AOM/DSS to induce colitis. Our mechanistic studies evaluating crypts demonstrate accumulation of mature, non-dividing IECs, which are more susceptible to mutations and malignant transformation. Here, we present a novel role for noncanonical NF-κB signaling in regulating intestinal epithelial cell turnover in protecting against CAC development. Focusing on the key niche signaling interplay following noncanonical NF-κB dysregulation gives greater insight into the CAC tumor microenvironment. Supported by grants from the iTHRIVE Pilot Translational and Clinical Studies Program, VCOM Center for One Health Research, Virginia Tech Carilion Research Acceleration Program, and the Virginia-Maryland College of Veterinary Medicine.

4‐Aminopyridine promotes cutaneous reinnervation and accelerates wound healing

Skin wound healing is a tightly orchestrated process between epithelial, dermal, neuronal and vascular cells, all working in concert to restore tissue integrity and skin barrier function. Here, we report the therapeutic regenerative effects of FDA‐approved 4‐aminopyridine (4‐AP) to promote skin wound healing by accelerating re‐epithelialization, wound induced hair neogenesis (WIHN), dermal collagen deposition, angiogenesis, and reinnervation. We demonstrate that 4‐AP enhanced wound closure by promoting keratinocyte proliferation and migration as well as collagen deposition through myofibroblast differentiation. 4‐AP treatment increased both epidermal thickness, as well as the number of hair follicles and blood vessels in healed wounds, suggesting better restoration of skin function and architecture compared to simple wound scar formation. Moreover, 4‐AP enhanced reinnervation of healed wounds by promoting Schwann cell (SC) de‐differentiation and secretion of neuromediators (NGF, SP) associated with regeneration. In‐vitro studies using human cells demonstrated that 4‐AP enhanced proliferation and migration of keratinocytes, and SCs and that 4‐AP enhances cellular interactions between neuronal and non‐neuronal cells to further accelerate wound healing. 4‐AP induced secretion of NGF from both SCs and keratinocytes. 4‐AP enhances many of the key attributes of successful wound healing and is a promising therapeutic adjuvant for skin wound healing and tissue regeneration.

Castration mediated schwann cell dedifferentiation leads to slower nerve conduction, decreased neuritogenesis, and nitrergic neuron loss

ABSTRACT Introduction/Objectives Low levels of androgens due to androgen deprivation therapy for prostate cancer treatment or due to aging lead to erectile dysfunction (ED). Castration, a preclinical model of low androgens, induces ED due to damage to pelvic nerves and a loss of erectogenic nitrergic nerves. Schwann cells (SC) release neurotrophic factors to promote pelvic nerve growth and survival. It is unknown how testosterone deficiency impacts SC function. This study determines how testosterone deficiency influences SC phenotype and trophic support in neuronal regeneration. We hypothesize testosterone deficiency will decrease myelinated pelvic SC populations, delay penile nerve conduction and reduce pelvic nitrergic neurons. Methods Male Sprague Dawley rats (12 wks) were divided into control (CON) and surgical castration (CAST) groups. All experiments were performed six weeks following castration. Dorsal penile nerves (DPN; 15-16 mm) were excised, placed in a sylgard dish containing oxygenated buffer and conduction velocity of myelinated fast fibers was assessed (n=8 per group). The major pelvic ganglia (MPG) were collected and dissociated into SC or neurons. Dissociated neurons were grown on laminin coated coverslips. To determine whether SCs could provide trophic support, we co-cultured neurons on top of confluent SCs. We assessed the following co-culture groups: CON SC + CON MPG, CON SC + CAST MPG, CAST SC + CON MPG (n=4 per group, cultured in triplicate). After 72 hours, all cultures were fixed and stained with beta tubulin class III, neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH) to assess neurite length and branching, nitrergic and sympathetic neuron populations, respectively. Dissociated SC were characterized via qPCR for markers of all SC (Sox10), myelinated SC (Krox20), and dedifferentiated SC (glial fibrillary acidic protein; GFAP). Results CAST slows DPN myelinated fast fiber conduction velocity. In dissociated cultured MPG neurons, castration reduces both neurite length and number of branches. Interestingly, CAST MPG neurons co-cultured with CON SC restore neurite length and branching to same level as CON MPG neurons co-cultured with CON SC; however, CAST SC do not impact CON MPG neurite length. CAST MPG neurons co-cultured with CON SC and CON MPG neurons co-cultured with CAST SC both have less nitrergic neurons than CON SC + CON MPG neurons. CAST does not impact the number of TH neurons in single or co-cultured conditions. CAST reduces gene expression of Sox10, myelinated SC, and dedifferentiated SC. Conclusions CAST markedly impairs penile nerve conduction, reduces neurite outgrowth and the proportion of pro-erectile nitrergic neurons. Healthy SC are able to rescue neuritogenesis but not the survival of nitrergic neurons. Additionally, healthy MPG neurons plated on CAST SC have a reduced number of nitrergic neurons. We believe CAST SC are undergoing a phenotypic switch from a myelinated neurotrophic form into dedifferentiated SC promoting neuritogenesis and loss of nitrergic neurons. Preserving the myelinated SC phenotype may prevent impaired nerve damage and subsequent ED in patients with testosterone deficiency. Disclosure Work supported by industry: no.