Reciprocal Crosstalk Research Articles

Resveratrol induces apoptosis by modulating the reciprocal crosstalk between p53 and Sirt-1 in the CRC tumor microenvironment.

P53 represents a key player in apoptosis-induction in cancers including colorectal cancer (CRC) that ranks third worldwide in cancer prevalence as well as mortality statistics. Although a pro-apoptotic effect of resveratrol has been repeatedly proven in CRC cells, its pathway mechanisms are not completely understood, as there are controversial statements in the literature regarding its activation or inhibition of the counteracting proteins Sirt-1 and p53. CRC cells as wild-type (HCT-116 WT) or p53-deficient (HCT-116 p53-/-) were cultured using multicellular tumor microenvironment (TME) cultures containing T-lymphocytes and fibroblasts to elucidate the role of p53/Sirt-1 modulation in resveratrol's concentration-dependent, pro-apoptotic, and thus anti-cancer effects. Resveratrol dose-dependently inhibited viability, proliferation, plasticity as well as migration, and induced apoptosis in HCT-116 WT more effectively than in HCT-116 p53-/- cells. Moreover, resveratrol stimulated Sirt-1 expression when administered at low concentrations (<5µM) but suppressed it when added at high concentrations (>10µM) to CRC-TME. In parallel, similar to the knockdown of Sirt-1 at the mRNA level, treatment with high-concentration resveratrol boosted the acetylation of p53, the expression of p21, Bax, cytochrome C, caspase-3, and ultimately induced apoptosis in CRC WT but not in CRC p53-/- cells. Notably, increasing concentrations of resveratrol were found to promote hyperacetylation of p53 and FOXO3a as post-translational substrates of Sirt-1, indicating a negative regulatory loop between Sirt-1 and p53. These results demonstrate for the first time, a negative reciprocal crosstalk between the regulatory circuits of p53 and Sirt-1, consequently, apoptosis induction by higher resveratrol concentrations in CRC-TME.

Growth factors and mechano-regulated reciprocal crosstalk with extracellular matrix tune the keratocyte–fibroblast/myofibroblast transition

Improper healing of the cornea after injury, infections or surgery can lead to corneal scar formation, which is associated with the transition of resident corneal keratocytes into activated fibroblasts and myofibroblasts (K–F/M). Myofibroblasts can create an extracellular matrix (ECM) niche in which fibrosis is promoted and perpetuated, resulting in progressive tissue opacification and vision loss. As a reversion back to quiescent keratocytes is essential to restore corneal transparency after injury, we characterized how growth factors with demonstrated profibrotic effects (PDGF, FGF, FBS, TGFβ1) induce the K–F/M transition, and whether their withdrawal can revert it. Indeed, the upregulated expression of αSMA and the associated changes in cytoskeletal architecture correlated with increases in cell contractility, fibronectin (Fn) and collagen matrix density and Fn fiber strain, as revealed by 2D cell culture, nanopillar cellular force mapping and a FRET-labeled Fn tension probe. Substrate mechanosensing drove a more complete K–F/M transition reversal following growth factor withdrawal on nanopillar arrays than on planar glass substrates. Using decellularized ECM scaffolds, we demonstrated that the K–F/M transition was inhibited in keratocytes reseeded onto myofibroblast-assembled, and/or collagen-1-rich ECM. This supports the presence of a myofibroblast-derived ECM niche that contains cues favoring tissue homeostasis rather than fibrosis.

Abstract A002: A reciprocal signaling crosstalk between the AR and BCl2-AKT pathways induces castration resistance in castration sensitive prostate cancer

Abstract Introduction: A leading contributor to the significant mortality burden of prostate cancer (PC) is the resistance to androgen deprivation therapy (ADT). Although most PCs are initially sensitive to ADT, the duration of response is variable, and relapse invariably occurs in the transition to metastatic castration-resistant prostate cancer (mCRPC), the lethal form of the disease. Therefore, it is essential to discover and validate early resistance mechanisms that initially protect castration sensitive prostate cancer (CSPC) cells from ADT and to develop novel combination therapies that can block or delay the ADT-induced shift from CSPC to mCRPC. Overexpression of the hallmark antiapoptotic gene B-cell lymphoma 2 (BCL2) is a potential mechanism of resistance to ADT. Methods: To discover the molecular drivers of castration resistance, we analyzed the tumor transcriptomic profile of a cohort of localized PC patients (N=58) treated with neoadjuvant-intensive ADT (NCT00924469; Taplin et al, 2014, Sowalsky et al, 2018). Experimentally, we treated CSPC cells and genetically engineered mice (GEM) prostate tumors with ADT and analyzed the signaling pathways, which were enriched post-ADT treatment compared untreated samples. Results: We discovered ~10-fold increased mRNA expression of BCL2 (p&amp;lt;0.001) in ADT-treated localized PC patients compared to untreated (standard of care surgery) patients in NCT00924469 samples. Our experimental study showed that treatment with AR inhibitors strongly augments BCL2 expression in human CSPC cell lines and GEM PC model, indicating possible direct negative regulation of BCL2 by the AR-signaling pathway. Mechanistically, our preliminary data reveal a striking induction of cell metabolism pathways (fatty acid and xenobiotic metabolism), including activation of PI3K/AKT signaling in BCL2-overexpressing CSPC cells. We used siRNA-mediated BCL2 knockdown in LNCaP cells and showed that loss of BCL2 strongly suppressed ADT-induced AKT- phosphorylation. We also showed increased Bcl2 mRNA expression in PTEN-knockout mice-derived prostate organoids compared to the wild-type control. Furthermore, we observed that AKT kinase -inhibitor MK2206 strongly inhibits enzalutamide-induced BCL2 expression in LNCaP cells. Conclusion: Together our data indicate a non-canonical role of BCL2 in activating PI3K/AKT signaling in prostate cancer. For the first time, we demonstrate the crucial importance of AR-BCL2-AKT signaling pathway crosstalk in CSPC biology and reveal this complex signaling crosstalk may act as a key driver of CSPC transformation to lethal CRPC. Citation Format: Goutam Chakraborty, Rahim Hirani, Subhiksha Nandakumar, Nabila Zaman, Teja Muralidhar Kalidindi, Sai Harisha Rajanala, Gwo-Shu M. Lee, Konrad H. Stopsack, Naga Vara Kishore Pillarsetty, Lorelei A. Mucci, Daniel C. Danila, Philip W. Kantoff. A reciprocal signaling crosstalk between the AR and BCl2-AKT pathways induces castration resistance in castration sensitive prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A002.

Abstract B065: Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer

Abstract The phosphoinositide 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is dysregulated in the majority of advanced prostate cancer patients, often linked to phosphate and tensin homolog (PTEN) inactivation. An increasing number of studies demonstrates the reciprocal crosstalk between the PI3K/AKT/mTOR and the androgen receptor (AR) pathways with the inhibition of one of them leading to the compensatory activation of the other one. We compared the effects of different PI3K/AKT/mTOR pathway inhibitors as single agents or in combination with the AR inhibitor darolutamide.In vitro impact of the compounds was evaluated in prostate cancer cell lines by proliferation and apoptosis assays, and by RNA-seq analysis. In vivo effects were determined in a patient-derived (PDX) prostate cancer model in an efficacy study followed by immunohistochemistry analysis. Analysis of VCaP cells treated with the androgen R1881 showed that beside androgen response, the PI3K/AKT/mTOR pathway was an essential hallmark characteristic of androgen action. Additional darolutamide treatment opposed androgen effects on both pathways. We therefore evaluated the impact of selective inhibitors of PI3K or AKT and saw strong in vitro antiproliferative effects in the prostate cancer cell lines tested. We then focused on the pan-PI3K inhibitor copanlisib and observed pronounced synergistic effects with darolutamide in the VCaP model. This was accompanied by induction of PARP cleavage and activation of caspase 3/7. A detailed transcriptomic analysis revealed that the combination of both inhibitors resulted in more pronounced transcript changes, compared to individual treatments. Principal component analysis furthermore showed the combination to be closer to the DMSO control along the axis that represents androgen regulation. We observed a pronounced downregulation of cell proliferation and cell division genes following combination treatment compared to individual treatments, a prominent example being the proliferation marker KI67. In vivo studies performed with the PDX LuCaP 35 model revealed a superior inhibitory effect of the combination treatment with darolutamide and copanlisib when compared to single agents. Immunohistochemistry analysis revealed a significant upregulation of a pro-apoptotic pathway in tumors treated with darolutamide and copanlisib which was not observed in single treatment arms, when compared to vehicle control. In summary, we found that different PI3K and AKT inhibitors impaired the in vitro proliferation of prostate cancer cell lines. Combining the AR inhibitor darolutamide with the pan-PI3K inhibitor copanlisib led to increased apoptosis and down-regulation of cell division and proliferation genes. Importantly, the improved tumor inhibition by the combination was also observed in vivo. These results warrant further analysis of the impact of combined AR and PI3K pathway inhibition in prostate cancer, especially when aberrations in the PI3K/AKT/mTOR pathway or PTEN loss are observed. Citation Format: Simon Heller, Tatsuo Sugawara, Ekaterina Nevedomskaya, Simon J. Baumgart, Holly Nguyen, Eva Corey, Annika Böhme, Oliver von Ahsen, Oliver Politz, Bernard Haendler. Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B065.

Integration analysis of single-cell multi-omics in the prostate cancer ecosystem.

e17046 Background: Prostate cancer (PCa) is characterized as a extensive heterogeneous disease with the complex cellular ecosystem in the tumor microenvironment (TME). However, the content of how heterogeneity is shaped by tumors and stromal cells or vice versa remains poorly understood. Methods: Single-cell RNA sequencing, spatial transcriptomics and bulk ATAC-sequence from a series of human PCa and normal control were integrated. Results: We identified a stemness subset of club cells marked with SOX9highARlow expression, which enriched significantly after neoadjuvant androgen-deprivation therapy (ADT). Furthermore, a subset of CD8+CXCR6+ T cell, functioned as effector T cell, was observed to be markedly reduced in the malignant PCa patients. Notably, immunosuppressive microenvironment in advanced PCa was associated with infiltration of regulatory T cells (Tregs), which is potentially induced by FAP+ fibroblasts (cancer-associated fibroblasts, CAFs subset). Moreover, for spatial transcriptome analysis, we comprehensively utilized machine learning and computational intelligence to identify cellular diversity of prostate and cell-cell communication with each other in situ.Importantly, we also depicted macrophage and neutrophil state transition under different biological conditions in vivo. Conclusions: We delineate the cellular heterogeneity in the stage-specific PCa microenvironment at single-cell resolution, uncover their reciprocal crosstalk with disease progression and these results will be helpful in exploring potential target for PCa therapy.

Osteocytes: New Kids on the Block for Cancer in Bone Therapy.

The tumor microenvironment plays a central role in the onset and progression of cancer in the bone. Cancer cells, either from tumors originating in the bone or from metastatic cancer cells from other body systems, are located in specialized niches where they interact with different cells of the bone marrow. These interactions transform the bone into an ideal niche for cancer cell migration, proliferation, and survival and cause an imbalance in bone homeostasis that severely affects the integrity of the skeleton. During the last decade, preclinical studies have identified new cellular mechanisms responsible for the dependency between cancer cells and bone cells. In this review, we focus on osteocytes, long-lived cells residing in the mineral matrix that have recently been identified as key players in the spread of cancer in bone. We highlight the most recent discoveries on how osteocytes support tumor growth and promote bone disease. Additionally, we discuss how the reciprocal crosstalk between osteocytes and cancer cells provides the opportunity to develop new therapeutic strategies to treat cancer in the bone.

The Impact of microRNAs on Mitochondrial Function and Immunity: Relevance to Parkinson's Disease.

Parkinson's Disease (PD), the second most common neurodegenerative disorder, is characterised by the severe loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc) and by the presence of Lewy bodies. PD is diagnosed upon the onset of motor symptoms, such as bradykinesia, resting tremor, rigidity, and postural instability. It is currently accepted that motor symptoms are preceded by non-motor features, such as gastrointestinal dysfunction. In fact, it has been proposed that PD might start in the gut and spread to the central nervous system. Growing evidence reports that the gut microbiota, which has been found to be altered in PD patients, influences the function of the central and enteric nervous systems. Altered expression of microRNAs (miRNAs) in PD patients has also been reported, many of which regulate key pathological mechanisms involved in PD pathogenesis, such as mitochondrial dysfunction and immunity. It remains unknown how gut microbiota regulates brain function; however, miRNAs have been highlighted as important players. Remarkably, numerous studies have depicted the ability of miRNAs to modulate and be regulated by the host's gut microbiota. In this review, we summarize the experimental and clinical studies implicating mitochondrial dysfunction and immunity in PD. Moreover, we gather recent data on miRNA involvement in these two processes. Ultimately, we discuss the reciprocal crosstalk between gut microbiota and miRNAs. Studying the bidirectional interaction of gut microbiome-miRNA might elucidate the aetiology and pathogenesis of gut-first PD, which could lead to the application of miRNAs as potential biomarkers or therapeutical targets for PD.

Epigenetic control is involved in molecular dialogue in plant-microbe symbiosis.

This article is a Commentary on Vigneaud et al. (2023), 238: 2561–2577.

In silico analysis of the possible crosstalk between O-linked β-GlcNAcylation and phosphorylation sites of Disabled 1 adaptor protein in vertebrates.

Disabled 1 (Dab1) is an adaptor protein with essential functions regulated by reelin signaling and affects many biological processes in the nervous system, including cell motility, adhesion, cortical development, maturation, and synaptic plasticity. Posttranslational modifications directly guide the fates of cytoplasmic proteins to complete their functions correctly. Reciprocal crosstalk between O-GlcNAcylation and phosphorylation is a dynamic modification in cytoplasmic proteins. It modulates the functions of the proteins by regulating their interactions with other molecules in response to the continuously changeable cell microenvironment. Although Dab1 contains conserved recognition sites for phosphorylation in their N-terminal protein interaction domain, the O-β-GlcNAcylation and phosphorylation sites of human Dab1 sequence, their reciprocal crosstalk, and potential kinases catalyzing the phosphorylation remain unknown. In this study, we determined potential thirty-seven O-β-GlcNAcylation and sixty-seven phosphorylation sites. Conserved twenty-one residues of these glycosylated sites were also phosphorylated with various kinases, including ATM, CKI, DNAPK, GSK3, PKC, PKG, RSK, cdc2, cdk5, and p38MAPK. In addition, we analyzed these conserved sites at our constructed two- and three-dimensional structures of human Dab1 protein. Dab1 protein models were frequently composed of coil structures as well as α-helix and β-strands. Many of these conserved crosstalk sites between O-β-GlcNAcylation and phosphorylation were localized at the coil region of the protein model. These findings may guide biochemical, genetic, and glyco-biology based on further experiments about the Dab1 signaling process. Understanding these modifications might change the point of view of the Dab1 signaling process and treatment for pathological conditions in neurodegenerative diseases such as Alzheimer's disease.

How the mechanobiology orchestrates the iterative and reciprocal ECM-cell cross-talk that drives microtissue growth.

Controlled tissue growth is essential for multicellular life and requires tight spatiotemporal control over cell proliferation and differentiation until reaching homeostasis. As cells synthesize and remodel extracellular matrix, tissue growth processes can only be understood if the reciprocal feedback between cells and their environment is revealed. Using de novo-grown microtissues, we identified crucial actors of the mechanoregulated events, which iteratively orchestrate a sharp transition from tissue growth to maturation, requiring a myofibroblast-to-fibroblast transition. Cellular decision-making occurs when fibronectin fiber tension switches from highly stretched to relaxed, and it requires the transiently up-regulated appearance of tenascin-C and tissue transglutaminase, matrix metalloprotease activity, as well as a switch from α5β1 to α2β1 integrin engagement and epidermal growth factor receptor signaling. As myofibroblasts are associated with wound healing and inflammatory or fibrotic diseases, crucial knowledge needed to advance regenerative strategies or to counter fibrosis and cancer progression has been gained.

Mechanisms of Organ-Specific Metastasis of Breast Cancer.

Cancer metastasis, or the development of secondary tumors in distant tissues, accounts for the vast majority of fatalities in patients with breast cancer. Breast cancer cells show a striking proclivity to metastasize to distinct organs, specifically the lung, liver, bone, and brain, where they face unique environmental pressures and a wide variety of tissue-resident cells that together create a strong barrier for tumor survival and growth. As a consequence, successful metastatic colonization is critically dependent on reciprocal cross talk between cancer cells and host cells within the target organ, a relationship that shapes the formation of a tumor-supportive microenvironment. Here, we discuss the mechanisms governing organ-specific metastasis in breast cancer, focusing on the intricate interactions between metastatic cells and specific niche cells within a secondary organ, and the remarkable adaptations of both compartments that cooperatively support cancer growth. More broadly, we aim to provide a framework for the microenvironmental prerequisites within each distinct metastatic site for successful breast cancer metastatic seeding and outgrowth.

Intestinal barrier dysfunction as a key driver of severe COVID-19.

The intestinal lumen harbors a diverse consortium of microorganisms that participate in reciprocal crosstalk with intestinal immune cells and with epithelial and endothelial cells, forming a multi-layered barrier that enables the efficient absorption of nutrients without an excessive influx of pathogens. Despite being a lung-centered disease, severe coronavirus disease 2019 (COVID-19) affects multiple systems, including the gastrointestinal tract and the pertinent gut barrier function. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can inflict either direct cytopathic injury to intestinal epithelial and endothelial cells or indirect immune-mediated damage. Alternatively, SARS-CoV-2 undermines the structural integrity of the barrier by modifying the expression of tight junction proteins. In addition, SARS-CoV-2 induces profound alterations to the intestinal microflora at phylogenetic and metabolomic levels (dysbiosis) that are accompanied by disruption of local immune responses. The ensuing dysregulation of the gut-lung axis impairs the ability of the respiratory immune system to elicit robust and timely responses to restrict viral infection. The intestinal vasculature is vulnerable to SARS-CoV-2-induced endothelial injury, which simultaneously triggers the activation of the innate immune and coagulation systems, a condition referred to as "immunothrombosis" that drives severe thrombotic complications. Finally, increased intestinal permeability allows an aberrant dissemination of bacteria, fungi, and endotoxin into the systemic circulation and contributes, to a certain degree, to the over-exuberant immune responses and hyper-inflammation that dictate the severe form of COVID-19. In this review, we aim to elucidate SARS-CoV-2-mediated effects on gut barrier homeostasis and their implications on the progression of the disease.

Immune determinants of the pre-metastatic niche.

Primary tumors actively and specifically prime pre-metastatic niches (PMNs), the future sites of organotropic metastasis, preparing these distant microenvironments for disseminated tumor cell arrival. While initial studies of the PMN focused on extracellular matrix alterations and stromal reprogramming, it is increasingly clear that the far-reaching effects of tumors are in great part achieved through systemic and local PMN immunosuppression. Here, we discuss recent advances in our understanding of the tumor immune microenvironment and provide a comprehensive overview of the immune determinants of the PMN's spatiotemporal evolution. Moreover, we depict the PMN immune landscape, based on functional pre-clinical studies as well as mounting clinical evidence, and the dynamic, reciprocal crosstalk with systemic changes imposed by cancer progression. Finally, we outline emerging therapeutic approaches that alter the dynamics of the interactions driving PMN formation and reverse immunosuppression programs in the PMN ensuring early anti-tumor immune responses.

A Channel Imbalance Calibration Scheme with Distributed Targets for Circular Quad-Polarization SAR with Reciprocal Crosstalk

As polarimetric antennas can be isolated through excellent electronic frameworks in circular quad-polarization (CQP) synthetic aperture radar (SAR) systems, cross-polarization (x-pol) and co-polarization (co-pol) channel imbalances are more challenging and essential to calibrate than crosstalk in polarimetric calibration (PolCAL). In uncalibrated CQP SAR images without corner reflectors (CRs), the reciprocity and reflection symmetry assumptions of the distributed targets are commonly used to estimate the x-pol and co-pol channel imbalances, respectively. To suppress the influence of additive noise on determining channel imbalances through distributed targets, high signal-to-noise ratio (SNR) distributed targets should be obtained from the x-pol and co-pol channels of the CQP SAR images: namely, surface-dominated and volume-dominated targets. However, some reflection symmetry assumptions used in the existing calibration literature have poor applicability with volume-dominated targets, resulting in unsatisfactory estimation results for the co-pol channel imbalance phase. In this paper, we assess the priority of the reflection symmetry properties of volume-dominated targets used to calibrate the co-pol channel imbalance phase in CQP SAR data synthesized from linear quad-polarization data of ALOS, GF-3, and RADARSAT-2. In the theoretical part, high-priority reflection symmetry (termed semireflection symmetry) assumptions are confirmed as the most suitable for estimating the co-pol channel imbalance phase, and were selected to develop an algorithm for estimating the co-pol channel imbalance phase. Furthermore, based on the novel method for estimating the co-pol channel imbalance phase, a channel imbalance calibration scheme is proposed for CQP SAR systems with reciprocal crosstalk, including extracting surface-dominated and volume-dominated targets, and estimating and filtering channel imbalances. We demonstrate the effectiveness of our proposed scheme with CRs in simulated CQP SAR images. The experimental results show that the calibration scheme is an effective workflow for estimating channel imbalances in CQP SAR systems with reciprocal crosstalk.

The Role of Tumor Epithelial-Mesenchymal Transition and Macrophage Crosstalk in Cancer Progression.

The purpose of this review is to summarize the recently published findings regarding the role of epithelial to mesenchymal transition (EMT) in tumor progression, macrophages in the tumor microenvironment, and crosstalk that exists between tumor cells and macrophages. EMT is a crucial process in tumor progression. In association with EMT changes, macrophage infiltration of tumors occurs frequently. A large body of evidence demonstrates that various mechanisms of crosstalk exist between macrophages and tumor cells that have undergone EMT resulting in a vicious cycle that promotes tumor invasion and metastasis. Tumor-associated macrophages and tumor cells undergoing EMT provide reciprocal crosstalk which leads to tumor progression. These interactions provide potential targets to exploit for therapy.

Neutrophils and their friends.

Neutrophils and their friends.

Addressing the Reciprocal Crosstalk between the AR and the PI3K/AKT/mTOR Signaling Pathways for Prostate Cancer Treatment.

The reduction in androgen synthesis and the blockade of the androgen receptor (AR) function by chemical castration and AR signaling inhibitors represent the main treatment lines for the initial stages of prostate cancer. Unfortunately, resistance mechanisms ultimately develop due to alterations in the AR pathway, such as gene amplification or mutations, and also the emergence of alternative pathways that render the tumor less or, more rarely, completely independent of androgen activation. An essential oncogenic axis activated in prostate cancer is the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, as evidenced by the frequent alterations of the negative regulator phosphatase and tensin homolog (PTEN) and by the activating mutations in PI3K subunits. Additionally, crosstalk and reciprocal feedback loops between androgen signaling and the PI3K/AKT/mTOR signaling cascade that activate pro-survival signals and play an essential role in disease recurrence and progression have been evidenced. Inhibitors addressing different players of the PI3K/AKT/mTOR pathway have been evaluated in the clinic. Only a limited benefit has been reported in prostate cancer up to now due to the associated side effects, so novel combination approaches and biomarkers predictive of patient response are urgently needed. Here, we reviewed recent data on the crosstalk between AR signaling and the PI3K/AKT/mTOR pathway, the selective inhibitors identified, and the most advanced clinical studies, with a focus on combination treatments. A deeper understanding of the complex molecular mechanisms involved in disease progression and treatment resistance is essential to further guide therapeutic approaches with improved outcomes.

Androgens and NGF Mediate the Neurite-Outgrowth through Inactivation of RhoA.

Steroid hormones and growth factors control neuritogenesis through their cognate receptors under physiological and pathological conditions. We have already shown that nerve growth factor and androgens induce neurite outgrowth of PC12 cells through a reciprocal crosstalk between the NGF receptor, TrkA and the androgen receptor. Here, we report that androgens or NGF induce neuritogenesis in PC12 cells through inactivation of RhoA. Ectopic expression of the dominant negative RhoA N19 promotes, indeed, the neurite-elongation of unchallenged and androgen- or NGF-challenged PC12 cells and the increase in the expression levels of βIII tubulin, a specific neuronal marker. Pharmacological inhibition of the Ser/Thr kinase ROCK, an RhoA effector, induces neuritogenesis in unchallenged PC12 cells, and potentiates the effect of androgens and NGF, confirming the role of RhoA/ROCK axis in the neuritogenesis induced by androgen and NGF, through the phosphorylation of Akt. These findings suggest that therapies based on new selective androgen receptor modulators and/or RhoA/ROCK inhibitors might exert beneficial effects in the treatment of neuro-disorders, neurological diseases and ageing-related processes.

The Polo-Like Kinase 1–Mammalian Target of Rapamycin Axis Regulates Autophagy to Prevent Intestinal Barrier Dysfunction During Sepsis

The intestines play a crucial role in the development of sepsis. The balance between autophagy and apoptosis in intestinal epithelial cells is dynamic and determines intestinal permeability. The present study focused on the potential role of autophagy in sepsis-induced intestinal barrier dysfunction and explored the mechanisms invivo and invitro. Excessive apoptosis in intestinal epithelia and a disrupted intestinal barrier were observed in septic mice. Promoting autophagy with rapamycin reduced intestinal epithelial apoptosis and restored intestinal barrier function, presenting as decreased serum diamine oxidase (DAO) and fluorescein isothiocyanate-dextran 40 (FD40) levels and increased expression of zonula occludens-1 (ZO-1) and Occludin. Polo-like kinase 1 (PLK1) knockdown in mice ameliorated intestinal epithelial apoptosis and the intestinal barrier during sepsis, whereas these effects were reduced with chloroquine and enhanced with rapamycin. PLK1 also promoted cell autophagy and improved lipopolysaccharide-induced apoptosis and high permeability invitro. Moreover, PLK1 physically interacted with mammalian target of rapamycin (mTOR) and participated in reciprocal regulatory crosstalk in intestinal epithelial cells during sepsis. This study provides novel insight into the role of autophagy in sepsis-induced intestinal barrier dysfunction and indicates that the PLK1-mTOR axis may be a promising therapeutic target for sepsis.

Tumor-derived exosomes elicit cancer-associated fibroblasts shaping inflammatory tumor microenvironment in head and neck squamous cell carcinoma

ObjectivesExosome-mediated reciprocal crosstalk between tumor and stromal cells plays a crucial role in tumor development and progression. This study investigated whether exosomes released from head and neck squamous cell carcinoma (HNSCC) tumor cells can convert normal fibroblasts into cancer-associated fibroblasts (CAF)-like cells and further analyzed the functional characterization of fibroblasts educated by tumor-derived exosomes. Materials and methodsExosomes secreted from HNSCC cell lines were isolated and normal fibroblasts were established from normal oropharyngeal mucosa. The effects of the exosomes on fibroblasts were examined by proliferation and migration assays, and exosome-educated fibroblasts were analyzed for the expression of eight genes (IL1B, IL6, CXCL8, TGFB1, ACTA2, FAP, CD274, and PDCD1LG2) by RT-qPCR. Moreover, T cells or CD14-positive cells were co-cultured with culture supernatants from exosome-educated fibroblasts. T-cell proliferation and macrophage polarization were examined using flow cytometry. Then, RNA sequencing (RNA-seq) of exosome-educated fibroblasts and the corresponding control fibroblasts was performed. ResultsTumor-derived exosomes enhanced fibroblast proliferation and migration. Moreover, gene expression analysis revealed upregulation of the gene expression of proinflammatory cytokines and immunoregulatory genes, and activated fibroblast marker genes. The culture supernatants of tumor-derived exosome-educated fibroblasts suppressed T cell proliferation and the induction of protumoral macrophages compared with those of control fibroblasts. Next, comprehensive RNA-seq analysis data revealed the activation of 11 signaling pathways, including IL-6- and IL-17-related signaling. ConclusionThese results indicate that HNSCC tumor cells induce and/or differentiate into CAFs through exosome-based cell-to-cell communication to create an inflammatory tumor microenvironment.