Cancer Burden Is Controlled by Mural Cell-ß3-Integrin Regulated Crosstalk with Tumor Cells.

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-ß3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-ß3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell ß3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by ß3-integrin, providing a previously unrecognized mechanism of cancer growth control.

Immunization with a multi-antigen targeted DNA vaccine eliminates chemoresistant pancreatic cancer by disrupting tumor-stromal cell crosstalk.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterised by limited responses to chemoimmunotherapy attributed to highly desmoplastic tumor microenvironment. Disrupting the tumor-stromal cell crosstalk is considered as an improved PDAC treatment strategy, whereas little progress has been made due to poor understanding of its underlying mechanism. Here, we examined the cellular role of melanoma associated antigen A isoforms (MAGEA) in regulating tumor-stromal crosstalk mediated chemoresistance. METHODS: We used clinical samples to explore the correlation between MAGEA expression and patient prognosis in multiple cancers. We utilized cancer cell lines, patient derived organoids and orthotopic PDAC model to examine the function of MAGEA in chemoresistance. We performed biochemical, proteome profiler array and transcriptional analysis to uncover a mechanism that governs tumor-stromal crosstalk. We developed a multi-MAGEA antigen targeted DNA vaccine and tested its effect on PDAC tumor growth. RESULTS: We establish MAGEA as a regulator of the tumor-stromal crosstalk in PDAC. We provide strong clinical evidence indicating that high MAGEA expression, including MAGEA2, MAGEA3 and MAGEA10, correlates with worse chemotherapeutic response and poor prognosis in multiple cancers, while their expression is up-regulated in chemoresistant PDAC patient derived organoids and cancer cell lines. Mechanistically, MAGEA2 prohibits gemcitabine-induced JNK-c-Jun-p53 mediated cancer cell apoptosis, while gemcitabine stimulated pancreatic stellate cells secretes GDF15 to further enhance the gemcitabine resistance of MAGEA2 expressing cells by activating GFRAL-RET mediated Akt and ERK1/2 dependent survival pathway. Strikingly, immunization with a DNA vaccine that targeting multiple MAGEA antigens, including MAGEA2, MAGEA3 and MAGEA10, elicits robust immune responses against the growth of gemcitabine resistant tumors. CONCLUSIONS: These findings suggest that targeting MAGEA-mediated paracrine regulation of chemoresistance by immunotherapy can be an improved pancreatic cancer treatment strategy.

Ceramide kinase confers tamoxifen resistance in estrogen receptor-positive breast cancer by altering sphingolipid metabolism.

Dysregulated sphingolipid metabolism contributes to ER+ breast cancer progression and therapeutic response, whereas its underlying mechanism and contribution to tamoxifen resistance (TAMR) is unknown. Here, we establish sphingolipid metabolic enzyme CERK as a regulator of TAMR in breast cancer. Multi-omics analysis reveals an elevated CERK driven sphingolipid metabolic reprogramming in TAMR cells, while high CERK expression associates with worse patient prognosis in ER+ breast cancer. CERK overexpression confers tamoxifen resistance and promotes tumorigenicity in ER+ breast cancer cells. Knocking out CERK inhibits the orthotopic breast tumor growth of TAMR cells while rescuing their tamoxifen sensitivity. Mechanistically, the elevated EHF expression transcriptionally up-regulates CERK expression to prohibit tamoxifen-induced sphingolipid ceramide accumulation, which then inhibits tamoxifen-mediated repression on PI3K/AKT dependent cell proliferation and its driven p53/caspase-3 mediated apoptosis in TAMR cells. This work provides insight into the regulation of sphingolipid metabolism in tamoxifen resistance and identifies a potential therapeutic target for this disease.

Systematic analysis to identify transcriptome-wide dysregulation of Alzheimer's disease in genes and isoforms.

Alzheimer's disease (AD) is one of the most common neurodegeneration diseases caused by multiple factors. The mechanistic insight of AD remains limited. To disclose molecular mechanisms of AD, many studies have been proposed from transcriptome analyses. However, no analysis across multiple levels of transcription has been conducted to discover co-expression networks of AD. We performed gene-level and isoform-level analyses of RNA sequencing (RNA-seq) data from 544 brain tissues of AD patients, mild cognitive impaired (MCI) patients, and healthy controls. Gene and isoform levels of co-expression modules were constructed by RNA-seq data. The associations of modules with AD were evaluated by integrating cognitive scores of patients, Genome-wide association studies (GWAS), alternative splicing analysis, and dementia-related genes expressed in brain tissues. Totally, 29 co-expression modules were found with expressions significantly correlated with the cognitive scores. Among them, two isoform modules were enriched with AD-associated SNPs and genes whose mRNA splicing displayed significant alteration in relation to AD disease. These two modules were further found enriched with dementia-related genes expressed in four brain regions of 125 AD patients. Analyzing expressions of these two modules revealed expressions of 39 isoforms (corresponding to 35 genes) significantly correlated with cognitive scores of AD patients, in which 38 isoforms were significantly up-regulated in AD patients comparing to controls, and 33 isoforms (corresponding to 29 genes) were not reported as AD-related previously. Employing the co-expression modules and the drug-induced gene expression data from Connectivity Map (CMAP), 12 drugs were predicted as significant in restoring the gene expression of AD patients towards health, which include nine drugs reported for relieving AD. In comparison, four of the top 12 significant drugs were known for relieving AD if the drug prediction was performed by the genes expressed significantly different in AD and healthy controls. Analysis of multiple levels of the transcriptomic organization is useful in suggesting AD-related co-expression networks and discovering drugs.

DStruBTarget: Integrating Binding Affinity with Structure Similarity for Ligand-Binding Protein Prediction.

Motivation: Identification of ligand-binding proteins is an important issue for drug development. Most of the current computational approach is developed only utilizing ligand structure similarity. However, the ligand structure similarity has failed to reflect the binding quality between the ligand and the target protein, which limited the performance of current methods. Results: The present study integrated two-dimensional (2D) and three-dimensional (3D) ligand structure similarity between query ligand and template with known ligand-protein binding affinity (BA) to identify proteins binding with the query ligand. This method is named as DStruBTarget. The performance of DStruBTarget was evaluated by 10-fold cross-validation in a dataset containing 9197 ligands and 1111 ligand-binding proteins (DBD dataset). This dataset was constructed by excluding the ligands with similar structures and the proteins with high sequence identity. The DStruBTarget achieved a hit rate of 77% in top 1 prediction, which is 4.80 and 3.00% better than the methods only using 2D structure similarity, and the method integrating 2D and 3D structure similarity (2D + 3D), respectively. An independent test of DStruBTarget was performed in a publicly available dataset constructed by SwissTargetPrediction. In this dataset, the top 1 hit rate of DStruBTarget reached 44.02%, which was better than the SwissTargetPrediction, and also outstands other methods, such as 2D, 3D, 2D + 3D, 2D integrating binding affinity (2D + BA), and 3D integrating binding affinity (3D + BA). DStruBTarget was compared to another newly published method HitPickV2 and achieved 52.17% hit rate of the top 1 prediction, which was significantly better than the result of HitpickV2 (30.43%). Finally, DStruBTarget was integrated with protein BLAST to predict the ligand-binding proteins not limited in a certain database. DStruBTarget with BLAST was tested in the DBD dataset. Its top 1 hit rate was 51.15%, which is lower than DStruBTarget without BLAST. Further comparison was on the ligands that bind to multiple numbers of proteins, which illustrated that DStruBTarget with BLAST performed better than without BLAST when the number of binding proteins of the query ligands is larger than six. Meanwhile, the prediction power of the DStruBTarget with BLAST in top 1 prediction was found to be positively correlated with the number of proteins binding with the query ligands, while the top 1 prediction power of DStruBTarget without BLAST was negatively correlated with the number of binding proteins for query ligands. Thus, DStruBTarget with BLAST is a potentially useful approach for predicting novel proteins for ligands that bind to multiple proteins.

Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy.

Chemoresistance is a serious limitation of cancer treatment. Until recently, almost all the work done to study this limitation has been restricted to tumour cells. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.

Cancer stem cell mimicry for immune evasion and therapeutic resistance.

Cancer stem cells (CSCs) are heterogeneous, possess self-renewal attributes, and orchestrate important crosstalk in tumors. We propose that the CSC state represents "mimicry" by cancer cells that leads to phenotypic plasticity. CSC mimicry is suggested as CSCs can impersonate immune cells, vasculo-endothelia, or lymphangiogenic cells to support cancer growth. CSCs facilitate both paracrine and juxtracrine signaling to prime tumor-associated immune and stromal cells to adopt pro-tumoral phenotypes, driving therapeutic resistance. Here, we outline the ingenuity of CSCs' mimicry in their quest to evade immune detection, which leads to immunotherapeutic resistance, and highlight CSC-mimicry-targeted therapeutic strategies for robust immunotherapy.

Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers.

Low migratory dendritic cell (DC) levels pose a challenge in cancer immune surveillance, yet their impact on tumor immune status and immunotherapy responses remains unclear. We present clinical evidence linking reduced migratory DC levels to immune-cold tumor status, resulting in poor patient outcomes. To address this, we develop an autologous DC-based nanovaccination strategy using patient-derived organoid or cancer cell lysate-pulsed cationic nanoparticles (cNPs) to load immunogenic DC-derived microvesicles (cNPcancer cell@MVDC). This approach transforms immune-cold tumors, increases migratory DCs, activates T cells and natural killer cells, reduces tumor growth, and enhances survival in orthotopic pancreatic and lung cancer models, surpassing conventional methods. In vivo imaging reveals superior cNPcancer cell@MVDC accumulation in tumors and lymph nodes, promoting immune cell infiltration. Mechanistically, cNPs enrich mitochondrial DNA, enhancing cGAS-STING-mediated DC activation and migration. Our strategy shifts cold tumors to a hot state, enhancing antitumor immunity for potential personalized cancer treatments.

Cancer associated fibroblast derived gene signature determines cancer subtypes and prognostic model construction in head and neck squamous cell carcinomas.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCC) are the most common type of head and neck cancer with an unimproved prognosis over the past decades. Although the role of cancer-associated-fibroblast (CAF) has been demonstrated in HNSCC, the correlation between CAF-derived gene expression and patient prognosis remains unknown. METHODS: A total of 528 patients from TCGA database and 270 patients from GSE65858 database were contained in this study. After extracting 66 CAF-related gene expression data from TCGA database, consensus clustering was performed to identify different HNSCC subtypes. Limma package was used to distinguish the differentially expression genes (DEGs) between these subtypes, followed by Lasso regression analysis to construct a prognostic model. The model was validated by performing Kaplan-Meier survival, ROC and risk curve, univariate and multivariate COX regression analysis. GO, KEGG, GSEA, ESTIMATE and ssGSEA analyses was performed to explort the potential mechanism leading to different prognosis. RESULTS: Based on the 66 CAF-related gene expression pattern we stratitied HNSCC patients into two previously unreported subtypes with different clinical outcomes. A prognostic model composed of 15 DEGs was constructed and validated. In addition, bioinformatics analysis showed that the prognostic risk of HNSCC patients was also negatively correlated to immune infiltration, implying the role of tumor immune escape in HNSCC prognosis and treatment option. CONCLUSIONS: The study develops a reliable prognostic prediction tool and provides a theoretical treatment guidance for HNSCC patients.

Identification of a distinct tumor endothelial cell-related gene expression signature associated with patient prognosis and immunotherapy response in multiple cancers.

BACKGROUND: Tumor endothelial cells (TECs) play a significant role in regulating the tumor microenvironment, drug response, and immune cell activities in various cancers. However, the association between TEC gene expression signature and patient prognosis or therapeutic response remains poorly understood. METHODS: We analyzed transcriptomics data of normal and tumor endothelial cells obtained from the GEO database to identify differentially expressed genes (DEGs) associated with TECs. We then compared these DEGs with those commonly found across five different tumor types from the TCGA database to determine their prognostic relevance. Using these genes, we constructed a prognostic risk model integrated with clinical features to develop a nomogram model, which we validated through biological experiments. RESULTS: We identified 12 TEC-related prognostic genes across multiple tumor types, of which five genes were sufficient to construct a prognostic risk model with an AUC of 0.682. The risk scores effectively predicted patient prognosis and immunotherapeutic response. Our newly developed nomogram model provided more accurate prognostic estimates of cancer patients than the TNM staging method (AUC = 0.735) and was validated using external patient cohorts. Finally, RT-PCR and immunohistochemical analyses indicated that the expression of these 5 TEC-related prognostic genes was up-regulated in both patient-derived tumors and cancer cell lines, while depletion of the hub genes reduced cancer cell growth, migration and invasion, and enhanced their sensitivity to gemcitabine or cytarabine. CONCLUSIONS: Our study discovered the first TEC-related gene expression signature that can be used to construct a prognostic risk model for guiding treatment options in multiple cancers.

Targeting Src reactivates pyroptosis to reverse chemoresistance in lung and pancreatic cancer models.

Pancreatic and lung cancers frequently develop resistance to chemotherapy-induced cell apoptosis during the treatment, indicating that targeting nonapoptotic-related pathways, such as pyroptosis, can be an alternative cancer treatment strategy. Pyroptosis is a gasdermin-driven lytic programmed cell death triggered by inflammatory caspases when initiated by canonical or noncanonical pathways that has been recently seen as a potential therapeutic target in cancer treatment. However, overcoming chemoresistance in cancers by modulating pyroptosis has not been explored. Here, we demonstrate that ß5-integrin represses chemotherapy-induced canonical pyroptosis to confer cancer chemoresistance through ASAH2-driven sphingolipid metabolic reprogramming. Clinically, high ß5-integrin expression associates with poor patient prognosis and chemotherapeutic responses in cancers. In addition, chemoresistant cells in vitro fail to undergo chemotherapy-induced pyroptosis, which is controlled by ß5-integrin. Mechanistically, proteomic and lipidomic analyses indicate that ß5-integrin up-regulates sphingolipid metabolic enzyme ceramidase (ASAH2) expression through Src-signal transducer and activator of transcription 3 (STAT3) signaling, which then reduces the metabolite ceramide concentration and subsequent ROS production to prohibit chemotherapy-induced canonical pyroptosis. Using cancer cell lines, patient-derived tumor organoids, and orthotopic lung and pancreatic animal models, we show that administration of a Src or ceramidase inhibitor rescues the response of chemoresistant pancreatic and lung cancer cells to chemotherapy by reactivating pyroptosis in vitro and in vivo. Overall, our results suggest that pyroptosis-based therapy is a means to improve cancer treatment and warrants further investigation.

Cathepsin-facilitated invasion of BMI1-high hepatocellular carcinoma cells drives bile duct tumor thrombi formation.

Bile duct tumor thrombosis (BDTT) is a complication mostly observed in patients with advanced hepatocellular carcinoma (HCC), causing jaundice and associated with poor clinical outcome. However, its underlying molecular mechanism is unclear. Here, we develop spontaneous preclinical HCC animal models with BDTT to identify the role of BMI1 expressing tumor initiating cells (BMI1high TICs) in inducing BDTT. BMI1 overexpression transforms liver progenitor cells into BMI1high TICs, which possess strong tumorigenicity and increased trans-intrahepatic biliary epithelial migration ability by secreting lysosomal cathepsin B (CTSB). Orthotopic liver implantation of BMI1high TICs into mice generates tumors and triggers CTSB mediated bile duct invasion to form tumor thrombus, while CTSB inhibitor treatment prohibits BDTT and extends mouse survival. Clinically, the elevated serum CTSB level determines BDTT incidence in HCC patients. Mechanistically, BMI1 epigenetically up-regulates CTSB secretion in TICs by repressing miR-218-1-3p expression. These findings identify a potential diagnostic and therapeutic target for HCC patients with BDTT.

Chemoresistant fibroblasts dictate neoadjuvant chemotherapeutic response of head and neck cancer via TGFα-EGFR paracrine signaling.

Conventional chemotherapy targets malignant cells without evaluating counter protection from the tumor microenvironment that often causes treatment failure. Herein, we establish chemoresistant fibroblasts (rCAFs) as regulators of neoadjuvant chemotherapeutic (NACT) response in head and neck squamous cell carcinoma (HNSCC). Clinically, high expression of CAF-related gene signature correlates with worse prognosis and chemotherapeutic response in multiple cancers, while the population of CAFs in the residual tumors of chemoresistant HNSCC patients remains unchanged after NACT treatment, compared to chemosensitive patients. Using a murine cancer model or patient-derived organoid, and primary CAFs isolated from chemo-sensitive (sCAFs) or -resistant patients, we show that rCAFs, but not sCAFs, are resistant to chemotherapy-induced apoptosis while reducing HNSCC cell chemosensitivity via paracrine signals. Combined multi-omics and biochemical analyses indicate an elevated PI3K/AKT/p65 driven cell survival and cytokine production in rCAFs, while rCAF-secreted TGFα promotes cancer cell chemoresistance by activating EGFR/Src/STAT3 survival signaling axis. Treatment with anti-EGFR cetuximab restores the chemosensitivity of tumors derived from co-injection of cancer cells and rCAFs in vivo, while the serum level of TGFα determines NACT response in HNSCC patients. Overall, our findings uncover a novel insight whereby the crosstalk between tumor cell and rCAF determines chemotherapeutic response and prognosis in cancer patients.

Acetylation of Rb by PCAF is required for nuclear localization and keratinocyte differentiation.

Although the retinoblastoma protein (Rb) functions as a checkpoint in the cell cycle, it also regulates differentiation. It has recently been shown that Rb is acetylated during differentiation; however, the role of this modification has not been identified. Depletion of Rb levels with short hairpin RNA resulted in inhibition of human keratinocyte differentiation, delayed cell cycle exit and allowed cell cycle re-entry. Restoration of Rb levels rescued defects in differentiation and cell cycle exit and re-entry; however, re-expression of Rb with the major acetylation sites mutated did not. During keratinocyte differentiation, acetylation of Rb is mediated by PCAF and it is further shown that PCAF acetyltransferase activity is also required for normal differentiation. The major acetylation sites in Rb are located within the nuclear localization sequence and, although mutation did not alter Rb localization in cycling cells, the mutant is mislocalized to the cytoplasm during differentiation. Studies indicate that acetylation is a mechanism for controlling Rb localization in human keratinocytes, with either reduction of the PCAF or exogenous expression of the deacetylase SIRT1, resulting in mislocalization of Rb. These findings identify PCAF-mediated acetylation of Rb as an event required to retain Rb within the nucleus during keratinocyte differentiation.

Activation of the Notch1-c-myc-VCAM1 signalling axis initiates liver progenitor cell-driven hepatocarcinogenesis and pulmonary metastasis.

The cellular origin of hepatocellular carcinomas (HCC) and the role of Notch1 signalling in HCC initiation are controversial. Herein, we establish Notch1 as a regulator of HCC development and progression. Clinically, high Notch1 expression correlates with enhanced cancer progression, elevated lung metastasis, increased cancer stem cell (CSC)-like cells' gene signature expression, and poor overall survival in HCC patients. Notch1 intracellular domain (N1ICD) overexpression spontaneously transforms rat liver progenitor cells (LPC) into CSC-like cells (WBN1ICD C5) under a selective growth environment, while orthotopic injection of these cells generates liver tumors and spontaneous pulmonary metastasis in an isogenic rat model. Mechanistically, the elevated Notch1 activity increases c-myc expression, which then transcriptionally upregulates VCAM1 expression to activate macrophage dependent HCC transendothelial migration. In vivo, silencing c-myc prohibits the tumorigenicity of WBN1ICD C5 cells, while depletion of VCAM1 reduces spontaneous lung metastasis without affecting primary WBN1ICD C5 orthotopic liver tumor growth. Importantly, depletion of macrophage or blockade of macrophage VCAM1 binding receptor α4ß1-integrin reduces the number of WBN1ICD C5 lung nodules in an experimental metastasis model. Overall, our work discovers that the Notch1-c-myc-VCAM1 signaling axis initiates LPC-driven hepatocarcinogenesis and metastasis, providing a preclinical model for HCC study and therapeutic targets for an improved HCC treatment.

Suppression of Endothelial Cell FAK Expression Reduces Pancreatic Ductal Adenocarcinoma Metastasis after Gemcitabine Treatment.

Despite substantial advances in the treatment of solid cancers, resistance to therapy remains a major obstacle to prolonged progression-free survival. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with a high level of liver metastasis. Primary PDAC is highly hypoxic, and metastases are resistant to first-line treatment, including gemcitabine. Recent studies have indicated that endothelial cell (EC) focal adhesion kinase (FAK) regulates DNA-damaging therapy-induced angiocrine factors and chemosensitivity in primary tumor models. Here, we show that inducible loss of EC-FAK in both orthotopic and spontaneous mouse models of PDAC is not sufficient to affect primary tumor growth but reduces liver and lung metastasis load and improves survival rates in gemcitabine-treated, but not untreated, mice. EC-FAK loss did not affect primary tumor angiogenesis, tumor blood vessel leakage, or early events in metastasis, including the numbers of circulating tumor cells, tumor cell homing, or metastatic seeding. Phosphoproteomics analysis showed a downregulation of the MAPK, RAF, and PAK signaling pathways in gemcitabine-treated FAK-depleted ECs compared with gemcitabine-treated wild-type ECs. Moreover, low levels of EC-FAK correlated with increased survival and reduced relapse in gemcitabine-treated patients with PDAC, supporting the clinical relevance of these findings. Altogether, we have identified a new role of EC-FAK in regulating PDAC metastasis upon gemcitabine treatment that impacts outcome. SIGNIFICANCE: These findings establish the potential utility of combinatorial endothelial cell FAK targeting together with gemcitabine in future clinical applications to control metastasis in patients with pancreatic ductal adenocarcinoma.

The Emerging Roles of Pericytes in Modulating Tumor Microenvironment.

Pericytes (PCs), known as mural cells, play an important blood vessel (BV) supporting role in regulating vascular stabilization, permeability and blood flow in microcirculation as well as blood brain barrier. In carcinogenesis, defective interaction between PCs and endothelial cells (ECs) contributes to the formation of leaky, chaotic and dysfunctional vasculature in tumors. However, recent works from other laboratories and our own demonstrate that the direct interaction between PCs and other stromal cells/cancer cells can modulate tumor microenvironment (TME) to favor cancer growth and progression, independent of its BV supporting role. Furthermore, accumulating evidence suggests that PCs have an immunomodulatory role. In the current review, we focus on recent advancement in understanding PC's regulatory role in the TME by communicating with ECs, immune cells, and tumor cells, and discuss how we can target PC's functions to re-model TME for an improved cancer treatment strategy.

Hexokinase 2-driven glycolysis in pericytes activates their contractility leading to tumor blood vessel abnormalities.

Defective pericyte-endothelial cell interaction in tumors leads to a chaotic, poorly organized and dysfunctional vasculature. However, the underlying mechanism behind this is poorly studied. Herein, we develop a method that combines magnetic beads and flow cytometry cell sorting to isolate pericytes from tumors and normal adjacent tissues from patients with non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). Pericytes from tumors show defective blood vessel supporting functions when comparing to those obtained from normal tissues. Mechanistically, combined proteomics and metabolic flux analysis reveals elevated hexokinase 2(HK2)-driven glycolysis in tumor pericytes, which up-regulates their ROCK2-MLC2 mediated contractility leading to impaired blood vessel supporting function. Clinically, high percentage of HK2 positive pericytes in blood vessels correlates with poor patient overall survival in NSCLC and HCC. Administration of a HK2 inhibitor induces pericyte-MLC2 driven tumor vasculature remodeling leading to enhanced drug delivery and efficacy against tumor growth. Overall, these data suggest that glycolysis in tumor pericytes regulates their blood vessel supporting role.

A circular network of coregulated sphingolipids dictates lung cancer growth and progression.

BACKGROUND: Sphingolipid metabolism is among the top dysregulated pathways in non-small cell lung carcinomas (NSCLC). However, the molecular control of sphingolipid metabolic reprogramming in cancer progression remains unclear. METHODS: We first determined the correlation between sphingolipid metabolic gene expression and patient prognosis. We then carried out sphingolipidomics analysis of health individual and NSCLC patient sera as well as B3GNT5 and GAL3ST1 genetically perturbed NSCLC cell lines. We used these cell lines to perform tumorigenesis study to determine the cellular role of B3GNT5 and GAL3ST1 in cancer growth and progression. FINDINGS: The expression of B3GNT5 and GAL3ST1 among sphingolipid metabolic enzymes is most significantly associated with patient prognosis, whilst sphingolipidomics analysis of healthy individual and NSCLC patient sera identifies their metabolites, lacto/neolacto-series glycosphingolipid and sulfatide species, as potential biomarkers that were more effective than current clinical biomarkers for staging patients. Further network analysis of the sphingolipidomes reveals a circular network of coregulated sphingolipids, indicating that the lacto/neolacto-series glycosphingolipid/sulfatide balance functions as a checkpoint to determine sphingolipid metabolic reprograming during patient progression. Sphingolipidomics analysis of B3GNT5/GAL3ST1 genetically perturbed NSCLC cell lines confirms their key regulatory role in sphingolipid metabolism, while B3GNT5 and GAL3ST1 expression has an opposite role on tumorigenesis. INTERPRETATION: Our results provide new insights whereby B3GNT5 and GAL3ST1 differentially regulate sphingolipid metabolism in lung cancer growth and progression. FUNDING: This work was supported by the Natural Science Foundation of China (81872142, 81920108028); Guangzhou Science and Technology Program (201904020008); Guangdong Science and Technology Department (2020A0505100029, 2019A1515011802, 2020A1515011280, 2020B1212060018, 2020B1212030004); China Postdoctoral Science Foundation (2019M650226, 2019M650227).

Galactosyltransferase B4GALT1 confers chemoresistance in pancreatic ductal adenocarcinomas by upregulating N-linked glycosylation of CDK11p110.

Aberrant glycosylation in pancreatic cancer has been linked to cancer development, progression and chemoresistance. However, the role of glycogene, such as galactosyltransferase, in pancreatic cancer remains unknown. Herein, we establish beta-1.4-galactosyltransferase 1 (B4GALT1) as a clinical marker and regulator of chemoresistance. Clinically, high B4GALT1 expression correlates with poor survival, enhanced tumor size, increased lymph node metastasis, elevated cancer progression and enhanced incidence of relapse in PDAC patients. Expression of B4GALT1 is up-regulated in gemcitabine resistant patient derived organoids as well as chemoresistant cancer cell lines, while genetic perturbation of its expression in PDAC cell lines regulates cancer progression and chemoresistance. Mechanistically, we show that elevated p65 activity transcriptionally up-regulates B4GALT1 expression, which then interacts with and stabilizes cyclin dependent kinase 11 isomer CDK11p110 protein via N-linked glycosylation, in order to promote cancer progression and chemoresistance. Finally, depletion of B4GALT1 rescues the response of chemoresistant cells to gemcitabine in an orthotopic PDAC model. Overall, our data uncovers a mechanism by which p65-B4GALT1-CDK11p110 signalling axis determines cancer progression and chemoresistance, providing a new therapeutic target for an improved pancreatic cancer treatment.