PPP1R15A-expressing monocytic MDSCs promote immunosuppressive liver microenvironment in fibrosis-associated hepatocellular carcinoma.

Background & Aims: Recent studies demonstrated the importance of fibrosis in promoting an immunosuppressive liver microenvironment and thereby aggressive hepatocellular carcinoma (HCC) growth and resistance to immune checkpoint blockade (ICB), particularly via monocyte-to-monocytic myeloid-derived suppressor cell (M-MDSC) differentiation triggered by hepatic stellate cells (HSCs). We thus aimed to identify druggable targets in these immunosuppressive myeloid cells for HCC therapy. Methods: M-MDSC signature genes were identified by integrated transcriptomic analysis of a human HSC-monocyte culture system and tumor-surrounding fibrotic livers of patients with HCC. Mechanistic and functional studies were conducted using in vitro-generated and patient-derived M-MDSCs. The therapeutic efficacy of a M-MDSC targeting approach was determined in fibrosis-associated HCC mouse models. Results: We uncovered over-expression of protein phosphatase 1 regulatory subunit 15A (PPP1R15A), a myeloid cell-enriched endoplasmic reticulum stress modulator, in human M-MDSCs that correlated with poor prognosis and ICB non-responsiveness in patients with HCC. Blocking TGF-ß signaling reduced PPP1R15A expression in HSC-induced M-MDSCs, whereas treatment of monocytes by TGF-ß upregulated PPP1R15A, which in turn promoted ARG1 and S100A8/9 expression in M-MDSCs and reduced T-cell proliferation. Consistently, lentiviral-mediated knockdown of Ppp1r15a in vivo significantly reduced ARG1+S100A8/9+ M-MDSCs in fibrotic liver, leading to elevated intratumoral IFN-γ+GZMB+CD8+ T cells and enhanced anti-tumor efficacy of ICB. Notably, pharmacological inhibition of PPP1R15A by Sephin1 reduced the immunosuppressive potential but increased the maturation status of fibrotic HCC patient-derived M-MDSCs. Conclusions: PPP1R15A+ M-MDSC cells are involved in immunosuppression in HCC development and represent a novel potential target for therapies. Impact and implications: Our cross-species analysis has identified PPP1R15A as a therapeutic target governing the anti-T-cell activities of fibrosis-associated M-MDSCs (monocytic myeloid-derived suppressor cells). The results from the preclinical models show that specific inhibition of PPP1R15A can break the immunosuppressive barrier to restrict hepatocellular carcinoma growth and enhance the efficacy of immune checkpoint blockade. PPP1R15A may also function as a prognostic and/or predictive biomarker in patients with hepatocellular carcinoma.

PRAME expression in genital melanocytic lesions - Potential diagnostic pitfall of intermediate expression in atypical genital nevi.

INTRODUCTION: The Preferentially Expressed Antigen in Melanoma (PRAME) immunostain has seen significant diagnostic use in confirming malignancy for melanocytic lesions. However, the expression of PRAME in genital melanocytic lesions have not been reported. In this study, PRAME staining was performed on a cohort of genital melanocytic lesions, aiming to investigate the diagnostic role of PRAME in genital melanocytic lesions and its expression in atypical genital nevi. METHODOLOGY: A cohort including genital invasive melanoma, melanoma-in-situ, atypical genital nevus (AGN), compound nevus, intradermal nevus, blue nevus, lentigo and melanosis was retrieved with histology reviewed and PRAME immunostaining performed. RESULTS: A total of 66 cases were reviewed. The average proportion expression of PRAME were 56.75 % and 57.43 % for invasive melanoma and melanoma-in-situ, with average H-scores of 153.5/300 and 163.14/300 respectively, which were greater than AGN (3.25 %, 7.75/300, p<0.001), compound/intradermal nevi, lentigo/melanosis, and background junctional melanocytes (<1 %, <1/300, p<0.001). The different cutoffs of PRAME expression, the sensitivity and specificity were 65.22 % and 100 % (>100/300); 69.57 % and 95.83 % (>10/300); and 82.61 % and 93.75 % (≥1/300) respectively. Low level PRAME expression was seen in half of the cases of AGN (n=2/4, 50 %), and at low cutoffs (>10/300 and ≥1/300) unable to differentiate invasive melanoma from AGN (p>0.05). CONCLUSIONS: For genital melanocytic lesions, PRAME immunostain shows high specificity at strong and diffuse staining. AGN not uncommonly display low level expression. Focal and/or weak PRAME expression should not be considered as an absolute indication of malignancy, and comprehensive histological assessment remains the key to accurate diagnosis of melanocytic lesions.

The establishment of kidney cancer organoid line in drug testing.

INTRODUCTION: Kidney cancer is a common urological malignancy worldwide with an increasing incidence in recent years. Among all subtypes, renal cell carcinoma (RCC) represents the most predominant malignancy in kidney. Clinicians faced a major challenge to select the most effective and suitable treatment regime for patients from a wide range of modalities, despite improved understanding and diagnosis of RCC. OBJECTIVE: Recently, organoid culture gained more interest as the 3D model is shown to be highly patient specific which is hypothetically beneficial to the investigation of precision medicine. Nonetheless, the development and application of organotypic culture in RCC is still immature, therefore, the primary objective of this study was to establish an organoid model for RCC. MATERIALS AND METHODS: Patients diagnosed with renal tumor and underwent surgical intervention were recruited. RCC specimen was collected and derived into organoids. Derived organoids were validated by histological examminations, sequencing and xenograft. Drug response of organoids were compared with resistance cell line and patients' clinical outcomes. RESULTS: Our results demonstrated that organoids could be successfully derived from renal tumor and they exhibited high concordance in terms of immunoexpressional patterns. Sequencing results also depicted concordant mutations of driver genes in both organoids and parental tumor tissues. Critical and novel growth factors were discovered during the establishment of organoid model. Besides, organoids derived from renal tumor exhibited tumorigenic properties in vivo. In addition, organoids recapitulated patient's in vivo drug resistance and served as a platform to predict responsiveness of other therapeutic agents. CONCLUSION: Our RCC organoid model recaptiluated histological and genetic features observed in primary tumors. It also served as a potential platform in drug screening for RCC patients, though future studies are necessary before translating the outcomes into clinical practices.

Synthetic BZLF1-targeted transcriptional activator for efficient lytic induction therapy against EBV-associated epithelial cancers.

The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.

Lactococcus lactis HkyuLL 10 suppresses colorectal tumourigenesis and restores gut microbiota through its generated alpha-mannosidase.

OBJECTIVE: Probiotic Lactococcus lactis is known to confer health benefits to humans. Here, we aimed to investigate the role of L. lactis in colorectal cancer (CRC). DESIGN: L. lactis abundance was evaluated in patients with CRC (n=489) and healthy individuals (n=536). L. lactis was isolated from healthy human stools with verification by whole genome sequencing. The effect of L. lactis on CRC tumourigenesis was assessed in transgenic Apc Min/+ mice and carcinogen-induced CRC mice. Faecal microbiota was profiled by metagenomic sequencing. Candidate proteins were characterised by nano liquid chromatography-mass spectrometry. Biological function of L. lactis conditioned medium (HkyuLL 10-CM) and functional protein was studied in human CRC cells, patient-derived organoids and xenograft mice. RESULTS: Faecal L. lactis was depleted in patients with CRC. A new L. lactis strain was isolated from human stools and nomenclated as HkyuLL 10. HkyuLL 10 supplementation suppressed CRC tumourigenesis in Apc Min/+ mice, and this tumour-suppressing effect was confirmed in mice with carcinogen-induced CRC. Microbiota profiling revealed probiotic enrichment including Lactobacillus johnsonii in HkyuLL 10-treated mice. HkyuLL 10-CM significantly abrogated the growth of human CRC cells and patient-derived organoids. Such protective effect was attributed to HkyuLL 10-secreted proteins, and we identified that α-mannosidase was the functional protein. The antitumourigenic effect of α-mannosidase was demonstrated in human CRC cells and organoids, and its supplementation significantly reduced tumour growth in xenograft mice. CONCLUSION: HkyuLL 10 suppresses CRC tumourigenesis in mice through restoring gut microbiota and secreting functional protein α-mannosidase. HkyuLL 10 administration may serve as a prophylactic measure against CRC.

SMARCA4 deficiency and mutations are frequent in large cell lung carcinoma and are prognostically significant.

SMARCA4 mutation has emerged as a marker of poor prognosis in lung cancer and has potential predictive value in cancer treatment, but recommendations for which patients require its investigation are lacking. We comprehensively studied SMARCA4 alterations and the clinicopathological significance in a large cohort of immunohistochemically-subtyped non-small cell lung cancer (NSCLC). A total of 1416 patients was studied for the presence of SMARCA4 deficiency by immunohistochemistry (IHC). Thereafter, comprehensive sequencing of tumours was performed for 397 of these patients to study the mutational spectrum of SWI/SNF and SMARCA4 aberrations. IHC evidence of SMARCA4 deficiency was found in 2.9% of NSCLC. Of the sequenced tumours, 38.3% showed aberration in SWI/SNF complex, and 9.3% had SMARCA4 mutations. Strikingly, SMARCA4 aberrations were much more prevalent in large cell carcinoma (LCC) than other histological tumour subtypes. SMARCA4-deficient and SMARCA4-mutated tumours accounted for 40.5% and 51.4% of all LCC, respectively. Multivariable analyses confirmed SMARCA4 mutation was an independent prognostic factor in lung cancer. The immunophenotype of a subset of these tumours frequently showed TTF1 negativity and HepPAR1 positivity. SMARCA4 mutation or its deficiency was associated with positive smoking history and poor prognosis. It also demonstrated mutual exclusion with EGFR mutation. Taken together, the high incidence of SMARCA4 aberrations in LCC may indicate its diagnostic and prognostic value. Our study established the necessity of SMARCA4 IHC in the identification of SMARCA4-aberrant tumours, and this may be of particular importance in LCC and tumours without known driver events.

Protocol to study immunodynamics in the tumor microenvironment using a tyramide signal amplification-based immunofluorescent multiplex panel.

Immunodynamics in the tumor microenvironment can be precisely examined by using multiple antigen identification approaches. Here, we present a protocol for capturing expression levels of multiple target proteins in the same specimen at single-cell resolution using a tyramide signal amplification-based immunofluorescent multiplexing system. We describe steps for tumor tissue microarray preparation, multiplex immunohistochemistry staining, image acquisition, and quantification. This protocol can quantify immune cells in tissues from patients or experimental disease models at a protein level. For complete details on the use and execution of this protocol, please refer to Chung et al. (2023),1 Tang et al. (2022),2 and Tang et al. (2022).3.

Streptococcus anginosus promotes gastric inflammation, atrophy, and tumorigenesis in mice.

Streptococcus anginosus (S. anginosus) was enriched in the gastric mucosa of patients with gastric cancer (GC). Here, we show that S. anginosus colonized the mouse stomach and induced acute gastritis. S. anginosus infection spontaneously induced progressive chronic gastritis, parietal cell atrophy, mucinous metaplasia, and dysplasia in conventional mice, and the findings were confirmed in germ-free mice. In addition, S. anginosus accelerated GC progression in carcinogen-induced gastric tumorigenesis and YTN16 GC cell allografts. Consistently, S. anginosus disrupted gastric barrier function, promoted cell proliferation, and inhibited apoptosis. Mechanistically, we identified an S. anginosus surface protein, TMPC, that interacts with Annexin A2 (ANXA2) receptor on gastric epithelial cells. Interaction of TMPC with ANXA2 mediated attachment and colonization of S. anginosus and induced mitogen-activated protein kinase (MAPK) activation. ANXA2 knockout abrogated the induction of MAPK by S. anginosus. Thus, this study reveals S. anginosus as a pathogen that promotes gastric tumorigenesis via direct interactions with gastric epithelial cells in the TMPC-ANXA2-MAPK axis.

Cellular zinc metabolism and zinc signaling: from biological functions to diseases and therapeutic targets.

Zinc metabolism at the cellular level is critical for many biological processes in the body. A key observation is the disruption of cellular homeostasis, often coinciding with disease progression. As an essential factor in maintaining cellular equilibrium, cellular zinc has been increasingly spotlighted in the context of disease development. Extensive research suggests zinc's involvement in promoting malignancy and invasion in cancer cells, despite its low tissue concentration. This has led to a growing body of literature investigating zinc's cellular metabolism, particularly the functions of zinc transporters and storage mechanisms during cancer progression. Zinc transportation is under the control of two major transporter families: SLC30 (ZnT) for the excretion of zinc and SLC39 (ZIP) for the zinc intake. Additionally, the storage of this essential element is predominantly mediated by metallothioneins (MTs). This review consolidates knowledge on the critical functions of cellular zinc signaling and underscores potential molecular pathways linking zinc metabolism to disease progression, with a special focus on cancer. We also compile a summary of clinical trials involving zinc ions. Given the main localization of zinc transporters at the cell membrane, the potential for targeted therapies, including small molecules and monoclonal antibodies, offers promising avenues for future exploration.

Expanding the spectrum of tyrosine kinase fusions in calcified chondroid mesenchymal neoplasms: Identification of a novel PDGFRA::USP8 gene fusion.

Calcified chondroid mesenchymal neoplasms represent a distinct, and recently recognized, spectrum of tumors. To date most cases have been reported to be characterized by FN1 gene fusions involving multiple potential tyrosine kinase partners. Following incidental identification of a tumor morphologically corresponding to calcified chondroid mesenchymal neoplasm, but with a PDGFRA::USP8 gene fusion, we undertook a retrospective review to identify and characterize additional such cases. A total of four tumors were identified. Each was multilobulated and composed of polygonal-epithelioid-stellate cells with a background of chondroid matrix containing distinctive patterns of calcification. Targeted RNA sequencing revealed an identical PDGFRA (exon 22)::USP8 (exon 5) gene fusion in each case. Subsequent immunohistochemical staining confirmed the presence of PDGFRα overexpression. In summary, we report a series of four tumors within the morphologic spectrum of calcified chondroid mesenchymal neoplasms. In contrast to prior reports, these tumors harbored a novel PDGFRA::USP8 gene fusion, rather than FN1 rearrangement. Our findings expand the molecular diversity of these neoplasms, and suggest they are united through activation of protein kinases.

The nerve cells in gastrointestinal cancers: from molecular mechanisms to clinical intervention.

Gastrointestinal (GI) cancer is a formidable malignancy with significant morbidity and mortality rates. Recent studies have shed light on the complex interplay between the nervous system and the GI system, influencing various aspects of GI tumorigenesis, such as the malignance of cancer cells, the conformation of tumor microenvironment (TME), and the resistance to chemotherapies. The discussion in this review first focused on exploring the intricate details of the biological function of the nervous system in the development of the GI tract and the progression of tumors within it. Meanwhile, the cancer cell-originated feedback regulation on the nervous system is revealed to play a crucial role in the growth and development of nerve cells within tumor tissues. This interaction is vital for understanding the complex relationship between the nervous system and GI oncogenesis. Additionally, the study identified various components within the TME that possess a significant influence on the occurrence and progression of GI cancer, including microbiota, immune cells, and fibroblasts. Moreover, we highlighted the transformation relationship between non-neuronal cells and neuronal cells during GI cancer progression, inspiring the development of strategies for nervous system-guided anti-tumor drugs. By further elucidating the deep mechanism of various neuroregulatory signals and neuronal intervention, we underlined the potential of these targeted drugs translating into effective therapies for GI cancer treatment. In summary, this review provides an overview of the mechanisms of neuromodulation and explores potential therapeutic opportunities, providing insights into the understanding and management of GI cancers.

Hematopoietic Transcription Factor RUNX1 is Essential for Promoting Macrophage-Myofibroblast Transition in Non-Small-Cell Lung Carcinoma.

Macrophage-myofibroblast transition (MMT) is a newly discovered pathway for mass production of pro-tumoral cancer-associated fibroblasts (CAFs) in non-small cell lung carcinoma (NSCLC) in a TGF-ß1/Smad3 dependent manner. Better understanding its regulatory signaling in tumor microenvironment (TME) may identify druggable target for the development of precision medicine. Here, by dissecting the transcriptome dynamics of tumor-associated macrophage at single-cell resolution, a crucial role of a hematopoietic transcription factor Runx1 in MMT formation is revealed. Surprisingly, integrative bioinformatic analysis uncovers Runx1 as a key regulator in the downstream of MMT-specific TGF-ß1/Smad3 signaling. Stromal Runx1 level positively correlates with the MMT-derived CAF abundance and mortality in NSCLC patients. Mechanistically, macrophage-specific Runx1 promotes the transcription of genes related to CAF signatures in MMT cells at genomic level. Importantly, macrophage-specific genetic deletion and systemic pharmacological inhibition of TGF-ß1/Smad3/Runx1 signaling effectively prevent MMT-driven CAF and tumor formation in vitro and in vivo, representing a potential therapeutic target for clinical NSCLC.

The molecular classification of cancer-associated fibroblasts on a pan-cancer single-cell transcriptional atlas.

BACKGROUND: Cancer-associated fibroblasts (CAFs), integral to the tumour microenvironment, are pivotal in cancer progression, exhibiting either pro-tumourigenic or anti-tumourigenic functions. Their inherent phenotypic and functional diversity allows for the subdivision of CAFs into various subpopulations. While several classification systems have been suggested for different cancer types, a unified molecular classification of CAFs on a single-cell pan-cancer scale has yet to be established. METHODS: We employed a comprehensive single-cell transcriptomic atlas encompassing 12 solid tumour types. Our objective was to establish a novel molecular classification and to elucidate the evolutionary trajectories of CAFs. We investigated the functional profiles of each CAF subtype using Single-Cell Regulatory Network Inference and Clustering and single-cell gene set enrichment analysis. The clinical relevance of these subtypes was assessed through survival curve analysis. Concurrently, we employed multiplex immunofluorescence staining on tumour tissues to determine the dynamic changes of CAF subtypes across different tumour stages. Additionally, we identified the small molecule procyanidin C1 (PCC1) as a target for matrix-producing CAF (matCAF) using molecular docking techniques and further validated these findings through in vitro and in vivo experiments. RESULTS: In our investigation of solid tumours, we identified four molecular clusters of CAFs: progenitor CAF (proCAF), inflammatory CAF (iCAF), myofibroblastic CAF (myCAF) and matCAF, each characterised by distinct molecular traits. This classification was consistently applicable across all nine studied solid tumour types. These CAF subtypes displayed unique evolutionary pathways, functional roles and clinical relevance in various solid tumours. Notably, the matCAF subtype was associated with poorer prognoses in several cancer types. The targeting of matCAF using the identified small molecule, PCC1, demonstrated promising antitumour activity. CONCLUSIONS: Collectively, the various subtypes of CAFs, particularly matCAF, are crucial in the initiation and progression of cancer. Focusing therapeutic strategies on targeting matCAF in solid tumours holds significant potential for cancer treatment.

Tumour-associated macrophages: versatile players in the tumour microenvironment.

Tumour-Associated Macrophages (TAMs) are one of the pivotal components of the tumour microenvironment. Their roles in the cancer immunity are complicated, both pro-tumour and anti-cancer activities are reported, including not only angiogenesis, extracellular matrix remodeling, immunosuppression, drug resistance but also phagocytosis and tumour regression. Interestingly, TAMs are highly dynamic and versatile in solid tumours. They show anti-cancer or pro-tumour activities, and interplay between the tumour microenvironment and cancer stem cells and under specific conditions. In addition to the classic M1/M2 phenotypes, a number of novel dedifferentiation phenomena of TAMs are discovered due to the advanced single-cell technology, e.g., macrophage-myofibroblast transition (MMT) and macrophage-neuron transition (MNT). More importantly, emerging information demonstrated the potential of TAMs on cancer immunotherapy, suggesting by the therapeutic efficiency of the checkpoint inhibitors and chimeric antigen receptor engineered cells based on macrophages. Here, we summarized the latest discoveries of TAMs from basic and translational research and discussed their clinical relevance and therapeutic potential for solid cancers.

H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling.

BACKGROUND: Gastric cancer (GC) is one of the most common tumours in East Asia countries and is associated with Helicobacter pylori infection. H. pylori utilizes virulence factors, CagA and VacA, to up-regulate pro-inflammatory cytokines and activate NF-κB signaling. Meanwhile, the PIEZO1 upregulation and cancer-associated fibroblast (CAF) enrichment were found in GC progression. However, the mechanisms of PIEZO1 upregulation and its involvement in GC progression have not been fully elucidated. METHODS: The CAF enrichment and clinical significance were investigated in animal models and primary samples. The expression of NF-κB and PIEZO1 in GC was confirmed by immunohistochemistry staining, and expression correlation was analysed in multiple GC datasets. GSEA and Western blot analysis revealed the YAP1-CTGF axis regulation by PIEZO1. The stimulatory effects of CTGF on CAFs were validated by the co-culture system and animal studies. Patient-derived organoid and peritoneal dissemination models were employed to confirm the role of the PIEZO1-YAP1-CTGF cascade in GC. RESULTS: Both CAF signature and PIEZO1 were positively correlated with H. pylori infection. PIEZO1, a mechanosensor, was confirmed as a direct downstream of NF-κB to promote the transformation from intestinal metaplasia to GC. Mechanistic studies revealed that PIEZO1 transduced the oncogenic signal from NF-κB into YAP1 signaling, a well-documented oncogenic pathway in GC progression. PIEZO1 expression was positively correlated with the YAP1 signature (CTGF, CYR61, and c-Myc, etc.) in primary samples. The secreted CTGF by cancer cells stimulated the CAF infiltration to form a stiffened collagen-enrichment microenvironment, thus activating PIEZO1 to form a positive feedback loop. Both PIEZO1 depletion by shRNA and CTGF inhibition by Procyanidin C1 enhanced the efficacy of 5-FU in suppressing the GC cell peritoneal metastasis. CONCLUSION: This study elucidates a novel driving PIEZO1-YAP1-CTGF force, which opens a novel therapeutic avenue to block the transformation from precancerous lesions to GC. H. pylori-NF-κB activates the PIEZO1-YAP1-CTGF axis to remodel the GC microenvironment by promoting CAF infiltration. Targeting PIEZO1-YAP1-CTGF plus chemotherapy might serve as a potential therapeutic option to block GC progression and peritoneal metastasis.

Exploring the Microbiome in Gastric Cancer: Assessing Potential Implications and Contextualizing Microorganisms beyond H. pylori and Epstein-Barr Virus.

While previous research has primarily focused on the impact of H. pylori and Epstein-Barr virus (EBV), emerging evidence suggests that other microbial influences, including viral and fungal infections, may also contribute to gastric cancer (GC) development. The intricate interactions between these microbes and the host's immune response provide a more comprehensive understanding of gastric cancer pathogenesis, diagnosis, and treatment. The review highlights the roles of established players such as H. pylori and EBV and the potential impacts of gut bacteria, mainly Lactobacillus, Streptococcus, hepatitis B virus, hepatitis C virus, and fungi such as Candida albicans. Advanced sequencing technologies offer unprecedented insights into the complexities of the gastric microbiome, from microbial diversity to potential diagnostic applications. Furthermore, the review highlights the potential for advanced GC diagnosis and therapies through a better understanding of the gut microbiome.

Identification of GRIN2D as a novel therapeutic target in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a dismal prognosis, and despite significant advances in our understanding of its genetic drivers, like KRAS, TP53, CDKN2A, and SMAD4, effective therapies remain limited. Here, we identified a new therapeutic target GRIN2D and then explored its functions and mechanisms in PDAC progression. METHODS: We performed a genome-wide RNAi screen in a PDAC xenograft model and identified GRIN2D, which encodes the GluN2D subunit of N-methyl-D-aspartate receptors (NMDARs), as a potential oncogene. Western blot, immunohistochemistry, and analysis on Gene Expression Omnibus were used for detecting the expression of GRIN2D in PDAC. Cellular experiments were conducted for exploring the functions of GRIN2D in vitro while subcutaneous and orthotopic injections were used in in vivo study. To clarify the mechanism, we used RNA sequencing and cellular experiments to identify the related signaling pathway. Cellular assays, RT-qPCR, and western blot helped identify the impacts of the NMDAR antagonist memantine. RESULTS: We demonstrated that GRIN2D was highly expressed in PDAC cells, and further promoted oncogenic functions. Mechanistically, transcriptome profiling identified GRIN2D-regulated genes in PDAC cells. We found that GRIN2D promoted PDAC progression by activating the p38 MAPK signaling pathway and transcription factor CREB, which in turn promoted the expression of HMGA2 and IL20RB. The upregulated GRIN2D could effectively promote tumor growth and liver metastasis in PDAC. We also investigated the therapeutic potential of NMDAR antagonism in PDAC and found that memantine reduced the expression of GRIN2D and inhibited PDAC progression. CONCLUSION: Our results suggested that NMDA receptor GRIN2D plays important oncogenic roles in PDAC and represents a novel therapeutic target.

MLK4 promotes glucose metabolism in lung adenocarcinoma through CREB-mediated activation of phosphoenolpyruvate carboxykinase and is regulated by KLF5.

MLK4, a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, has been implicated in cancer progression. However, its role in lung adenocarcinoma has not been characterized. Here, we showed that MLK4 was overexpressed in a significant subset of lung adenocarcinoma, associated with a worse prognosis, and exerted an oncogenic function in vitro and in vivo. Bioinformatics analyses of clinical datasets identified phosphoenolpyruvate carboxykinase 1 (PCK1) as a novel target of MLK4. We validated that MLK4 regulated PCK1 expression at transcriptional level, by phosphorylating the transcription factor CREB, which in turn mediated PCK1 expression. We further demonstrated that PCK1 is an oncogenic factor in lung adenocarcinoma. Given the importance of PCK1 in the regulation of cellular metabolism, we next deciphered the metabolic effects of MLK4. Metabolic and mass spectrometry analyses showed that MLK4 knockdown led to significant reduction of glycolysis and decreased levels of glycolytic pathway metabolites including phosphoenolpyruvate and lactate. Finally, the promoter analysis of MLK4 unravelled a binding site of transcription factor KLF5, which in turn, positively regulated MLK4 expression in lung adenocarcinoma. In summary, we have revealed a KLF5-MLK4-PCK1 signalling pathway involved in lung tumorigenesis and established an unusual link between MAP3K signalling and cancer metabolism.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.

Gut microbiome dysbiosis across early Parkinson's disease, REM sleep behavior disorder and their first-degree relatives.

The microbiota-gut-brain axis has been suggested to play an important role in Parkinson's disease (PD). Here we performed a cross-sectional study to profile gut microbiota across early PD, REM sleep behavior disorder (RBD), first-degree relatives of RBD (RBD-FDR), and healthy controls, which could reflect the gut-brain staging model of PD. We show gut microbiota compositions are significantly altered in early PD and RBD compared with control and RBD-FDR. Depletion of butyrate-producing bacteria and enrichment of pro-inflammatory Collinsella have already emerged in RBD and RBD-FDR after controlling potential confounders including antidepressants, osmotic laxatives, and bowel movement frequency. Random forest modelling identifies 12 microbial markers that are effective to distinguish RBD from control. These findings suggest that PD-like gut dysbiosis occurs at the prodromal stages of PD when RBD develops and starts to emerge in the younger RBD-FDR subjects. The study will have etiological and diagnostic implications.