Neuron-derived neurotensin promotes pancreatic cancer invasiveness and gemcitabine resistance via the NTSR1/Akt pathway.

Perineural invasion and neurogenesis are frequently observed in pancreatic ductal adenocarcinoma (PDAC) and link to poor outcome. However, how neural factors affect PDAC prognosis and the underlying mechanism as well as counteracting therapeutic are still unclear. In silico systematic analysis was performed with PROGgene to identify potential neural factor and its receptor in pancreatic cancer. In vitro assays including migration, invasion, 3D recruitment, and gemcitabine resistance were performed to study the effect of neuron-derived neurotensin (NTS) on pancreatic cancer behavior. Orthotopic animal study was used to validate the in vitro findings. Gene set enrichment analysis (GSEA) was performed to confirm the results from in silico to in vivo. Expression of NTS and its receptor 1 (NTSR1) predicted poor prognosis in PDAC. NTS synthetic peptide or neuron-derived condition medium promoted pancreatic cancer invasiveness and recruitment in 2D and 3D assays. NTS-induced effects depended on NTSR1 and PI3K activation. GDC-0941, a clinically approved PI3K inhibitor, counteracted NTS-induced effects in vitro. Inhibition of NTSR1 in pancreatic cancer cells resulted in decreased tumor dissemination and diminished PI3K activation in vivo. NTS boosted gemcitabine resistance via NTSR1 in pancreatic cancer. Our results suggest that neural cell-secreted NTS plays an important role in promoting PDAC.

RNF43 Inactivation Enhances the B-RAF/MEK Signaling and Creates a Combinatory Therapeutic Target in Cancer Cells.

RING finger 43 (RNF43), a RING-type E3 ubiquitin ligase, is a key regulator of WNT signaling and is mutated in 6-10% of pancreatic tumors. However, RNF43-mediated effects remain unclear, as only a few in vivo substrates of RNF43 are identified. Here, it is found that RNF43-mutated pancreatic cancer cells exhibit elevated B-RAF/MEK activity and are highly sensitive to MEK inhibitors. The depletion of RNF43 in normal pancreatic ductal cells also enhances MEK activation, suggesting that it is a physiologically regulated process. It is confirmed that RNF43 ubiquitinates B-RAF at K499 to promote proteasome-dependent degradation, resulting in reduced MEK activity and proliferative ability in cancer cells. In addition, phosphorylation of B-RAF at T491 suppresses B-RAF ubiquitination by decreasing the interaction between RNF43 and B-RAF. Mutations at K499 in B-RAF are identified in various cancer types. MEK and WNT inhibitors synergistically suppress the growth of RNF43-mutated pancreatic cancer cells in vitro and in vivo. Collectively, the research reveals a novel mechanism by which RNF43 inhibits B-RAF/MEK signaling to suppress tumor growth and provide a new strategy for the treatment of RNF43-inactivated pancreatic cancer.

Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination.

Perineural invasion and neurogenesis are frequently observed in pancreatic ductal adenocarcinoma (PDAC), and they are associated with a poor prognosis. Axon guidance factor semaphorin 3A (SEMA3A) is upregulated in PDAC. However, it remains unclear whether cancer-derived SEMA3A influences nerve innervation and pancreatic tumorigenesis. In silico analyses were performed using PROGgene and NetworkAnalyst to clarify the importance of SEMA3A and its receptors, plexin A1 (PLXNA1) and neuropilin 2 (NRP2), in pancreatic cancer. In vitro assays, including migration, neurite outgrowth, and 3D recruitment, were performed to study the effects of SEMA3A on neuronal behaviors. Additionally, an orthotopic animal study using C57BL/6 mice was performed to validate the in vitro findings. Expression of SEMA3A and its receptors predicted worse prognosis for PDAC. Cancer-derived SEMA3A promoted neural migration, neurite outgrowth, and neural recruitment. Furthermore, SEMA3A-induced effects depended on PLXNA1, NRP2, and MAPK activation. Trametinib, an approved MAPK kinase (MEK) inhibitor, counteracted SEMA3A-enhanced neuronal activity in vitro. Inhibition of SEMA3A by shRNA in pancreatic cancer cells resulted in decreased neural recruitment, tumor growth, and dissemination in vivo. Our results suggested that cancer-secreted SEMA3A plays an important role in promoting neo-neurogenesis and progression of PDAC.

ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance.

ARID1A is a tumor suppressor gene mutated in 7-10% of pancreatic cancer patients. However, its function in pancreas development and endocrine regulation is unclear. We generated mice that lack Arid1a expression in the pancreas. Our results showed that deletion of the Arid1a gene in mice caused a reduction in islet numbers and insulin production, both of which are associated with diabetes mellitus (DM) phenotype. RNA sequencing of isolated islets confirmed DM gene signature and decrease of developmental lineage genes. We identified neurogenin3, a transcription factor that controls endocrine fate specification, is a direct target of Aird1a. Gene set enrichment analysis indicated the enhancement of histone deacetylase (HDAC) pathway after Arid1a depletion and a clinically approved HDAC inhibitor showed therapeutic benefit by suppressing disease onset. Our data suggest that Arid1a is required for the development of pancreatic islets by regulating Ngn3+-mediated transcriptional program and is important in maintaining endocrine function.

Protein arginine methyltransferase 3: A crucial regulator in metabolic reprogramming and gene expression in cancers.

Post-translational modification (PTM) of proteins increases proteome diversity, which is critical for maintaining cellular homeostasis. The importance of protein methylation in the regulation of diverse biological processes has been highlighted in the past decades. Methylation of the arginine residue on proteins is catalyzed by members of the protein arginine methyltransferase (PRMT) family. PRMTs play indispensable roles in various pathways that regulate cancer development, progression, and drug response. In this review, we discuss the role of PRMT3, a member of the PRMT family, in controlling oncogenic processes. Additionally, the effects of PRMT3 on the methylation of regulatory proteins involved in transcription, post-transcriptional control, ribosomal maturation, translation, biological synthesis, and metabolic signaling are summarized. Moreover, recent progresses in the development of PRMT3 inhibitors are introduced. Overall, this review highlights the importance of PRMT3 in tumorigenesis and discusses the underlying mechanisms by which PRMT3 modulates cellular metabolism and gene expression. These results also provide a molecular basis for therapeutic modalities by targeting PRMT3.

Mitochondrial phosphoenolpyruvate carboxykinase promotes tumor growth in estrogen receptor-positive breast cancer via regulation of the mTOR pathway.

BACKGROUND: Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer. METHODS: We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer. RESULTS: PEPCK-M was highly expressed in estrogen receptor-positive (ER+ ) breast cancers. Decreased cell proliferation and G0 /G1 arrest were induced in ER+ breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER+ breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER+ breast cancer were attenuated by PCK2 knockdown. CONCLUSION: PEPCK-M promotes proliferation and cell cycle progression in ER+ breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER+ breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER+ breast cancer.

Skp2-mediated Zeb1 expression facilitates cancer migration by a ubiquitination-independent pathway.

AIMS: Pancreatic ductal adenocarcinoma (PDAC) constitutes one of the most dismal malignancies worldwide. Despite multidisciplinary involvement in interventions involving surgery, radiotherapy, and chemotherapy, most pancreatic cancer patients eventually develop distant metastasis. S-phase kinase-associated protein 2 (Skp2) plays an important role in cell-cycle regulation in pancreatic cancer. However, the role of Skp2 in individualized PDAC treatment is largely unknown. MAIN METHODS: Immunoblotting, quantitative reverse transcription polymerase chain reaction, cell viability test, chromatin immunoprecipitation assay, and xenograft in vivo assay were performed in parental and Skp2-depleted cells. The immunohistochemistry of Skp2 was analyzed on the tissue microarrays of 45 PDAC cases and mice tissues. KEY FINDINGS: In this study, we observed that Skp2 is a marker for poor prognosis in PDAC patients. Upregulation of the inhibitor of κB (IκB)-inducing kinase-nuclear factor kappa B (NF-κB) signal cascade mediated Skp2 expression thereby promoting epithelial-mesenchymal transition (EMT). Depletion of NF-κB-associated signaling effectively prevented Skp2-mediated pancreatic cancer cell migration. As a functional consequence, Skp2 orchestrated with Myc to induce zinc finger E-box binding homeobox 1 (Zeb1) transcription by recruiting p300 to the Zeb1 promoter independent of Skp2 E3-ligase activity. Therefore, blockade of Skp2 could significantly reduce the expression of Zeb1 and inhibit cancer cell migration. In conclusion, Skp2 regulated Zeb1 activity to control the migration and invasion abilities of pancreatic cancer cells. Skp2 expression in PDAC may affect cell vulnerability to standard chemotherapy regimens. SIGNIFICANCE: Therefore, in patients with PDAC, modulation of Skp2 expression could be a novel strategy for preventing cancer cell metastasis.

PTEN regulates invasiveness in pancreatic neuroendocrine tumors through DUSP19-mediated VEGFR3 dephosphorylation.

BACKGROUND: Phosphatase and tensin homolog (PTEN) is a tumor suppressor. Low PTEN expression has been observed in pancreatic neuroendocrine tumors (pNETs) and is associated with increased liver metastasis and poor survival. Vascular endothelial growth factor receptor 3 (VEGFR3) is a receptor tyrosine kinase and is usually activated by binding with vascular endothelial growth factor C (VEGFC). VEGFR3 has been demonstrated with lymphangiogenesis and cancer invasiveness. PTEN is also a phosphatase to dephosphorylate both lipid and protein substrates and VEGFR3 is hypothesized to be a substrate of PTEN. Dual-specificity phosphatase 19 (DUSP19) is an atypical DUSP and can interact with VEGFR3. In this study, we investigated the function of PTEN on regulation of pNET invasiveness and its association with VEGFR3 and DUSP19. METHODS: PTEN was knocked down or overexpressed in pNET cells to evaluate its effect on invasiveness and its association with VEGFR3 phosphorylation. In vitro phosphatase assay was performed to identify the regulatory molecule on the regulation of VEGFR3 phosphorylation. In addition, immunoprecipitation, and immunofluorescence staining were performed to evaluate the molecule with direct interaction on VEGFR3 phosphorylation. The animal study was performed to validate the results of the in vitro study. RESULTS: The invasion and migration capabilities of pNETs were enhanced by PTEN knockdown accompanied with increased VEGFR3 phosphorylation, ERK phosphorylation, and increased expression of epithelial-mesenchymal transition molecules in the cells. The enhanced invasion and migration abilities of pNET cells with PTEN knockdown were suppressed by addition of the VEGFR3 inhibitor MAZ51, but not by the VEGFR3-Fc chimeric protein to neutralize VEGFC. VEGFR3 phosphorylation is responsible for pNET cell invasiveness and is VEGFC-independent. However, an in vitro phosphatase assay failed to show VEGFR3 as a substrate of PTEN. In contrast, DUSP19 was transcriptionally upregulated by PTEN and was shown to dephosphorylate VEGFR3 via direct interaction with VEGFR3 by an in vitro phosphatase assay, immunoprecipitation, and immunofluorescence staining. Increased tumor invasion into peripheral tissues was validated in xenograft mouse model. Tumor invasion was suppressed by treatment with VEGFR3 or MEK inhibitors. CONCLUSIONS: PTEN regulates pNET invasiveness via DUSP19-mediated VEGFR3 dephosphorylation. VEGFR3 and DUSP19 are potential therapeutic targets for pNET treatment.

Reprogramming of sentinel lymph node microenvironment during tumor metastasis.

Metastasis is a major cause of death in patients with cancer. The two main routes for cancer cell dissemination are the blood and lymphatic systems. The underlying mechanism of hematogenous metastasis has been well characterized in the past few decades. However, our understanding of the molecular basis of lymphatic metastasis remains at a premature stage. Conceptually, cancer cells invade into lymphatic capillary, passively move to collecting lymphatic vessels, migrate into sentinel lymph node (SLN;, the first lymph node to which cancer cells spread from the primary tumor), and enter the blood circulatory system via the subclavian vein. Before arriving, cancer cells release specific soluble factors to modulate the microenvironment in SLN to establish a beachhead for successful colonization. After colonization, cancer cells inhibit anti-tumor immunity by inducing the recruitment of regulatory T cell and myeloid-derived suppressor cells, suppressing the function of dendritic cell and CD8+ T cell, and promoting the release of immunosuppressive cytokines. The development of novel strategies to reverse cancer cell-triggered SLN remodeling may re-activate immunity to reduce beachhead buildup and distant metastasis. In addition to being a microanatomic location for metastasis, the SLN is also an important site for immune modulation. Nanotechnology-based approaches to deliver lymph node-tropic antibodies or drug-conjugated nanoparticles to kill cancer cells on site are a new direction for cancer treatment. Conversely, the induction of stronger immunity by promoting antigen presentation in lymph nodes provides an alternate way to enhance the efficacy of immune checkpoint therapy and cancer vaccine. In this review article, we summarize recent findings on the reprogramming of SLN during lymphatic invasion and discuss the possibility of inhibiting tumor metastasis and eliciting anti-tumor immunity by targeting SLN.

The Trinity: Interplay among Cancer Cells, Fibroblasts, and Immune Cells in Pancreatic Cancer and Implication of CD8+ T Cell-Orientated Therapy.

The microenvironment in tumors is complicated and is constituted by different cell types and stromal proteins. Among the cell types, the abundance of cancer cells, fibroblasts, and immune cells is high and these cells work as the "Trinity" in promoting tumorigenesis. Although unidirectional or bidirectional crosstalk between two independent cell types has been well characterized, the multi-directional interplays between cancer cells, fibroblasts, and immune cells in vitro and in vivo are still unclear. We summarize recent studies in addressing the interaction of the "Trinity" members in the tumor microenvironment and propose a functional network for how these members communicate with each other. In addition, we discuss the underlying mechanisms mediating the interplay. Moreover, correlations of the alterations in the distribution and functionality of cancer cells, fibroblasts, and immune cells under different circumstances are reviewed. Finally, we point out the future application of CD8+ T cell-oriented therapy in the treatment of pancreatic cancer.

Semaphorin 4C promotes motility and immunosuppressive activity of cancer cells via CRMP3 and PD-L1.

Semaphorins (SEMAs) are membrane-bound or soluble proteins that participate in organ development and cancer progression, however, the detailed role of SEMAs in carcinogenesis is not fully elucidated yet. Our in silico analysis showed among the differentially expressed SEMAs in colon cancer tissues, patients with higher SEMA4C expression tumors had worse survival. The migration and invasion of the HCT116 and CT26 colon cancer cells were significantly suppressed by SEMA4C neutralizing antibody treatment; while enhanced by ectopic expression of SEMA4C. Subsequently, RNA sequencing study revealed microtubule polymerization- and nucleation-related genes are highly enriched in SEMA4C overexpression HCT116 cells. Western blotting showed the negative correlation between the levels of SEMA4C expression and tubulin acetylation. Mechanistic study showed SEMA4C interacted with and stabilized collapsin response mediator protein 3 (CRMP3), a novel deacetylase, to increase α-tubulin deacetylation and cell motility, which could be effectively attenuated after HDAC inhibitors treatment. We also found that a tumor-suppressive miRNA let-7b can target SEMA4C and act synergistically with SEMA4C neutralizing antibody to suppress the motility of colon cancer cells. In addition, blockade of SEMA4C could attenuate the expression of program death ligand 1 (PD-L1). Collectively, our results highlight that SEMA4C may promote colon cancer progression through modulating CRMP3-mediated tubulin deacetylation and PD-L1-mediated immunosuppression.

Semaphorin 6C Suppresses Proliferation of Pancreatic Cancer Cells via Inhibition of the AKT/GSK3/ß-Catenin/Cyclin D1 Pathway.

Semaphorins (SEMAs) are axon guidance factors that participate in axonal connections and nerve system development. However, the functional roles of SEMAs in tumorigenesis are still largely uncovered. By using in silico data analysis, we found that SEMA6C was downregulated in pancreatic cancer, and its reduction was correlated with worse survival rates. RNA sequencing revealed that cell cycle-related genes, especially cyclin D1, were significantly altered after blockage of SEMA6C by neutralizing antibodies or ectopic expressions of SEMA6C. Mechanistic investigation demonstrated that SEMA6C acts as a tumor suppressor in pancreatic cancer by inhibiting the AKT/GSK3 signaling axis, resulting in a decrease in cyclin D1 expression and cellular proliferation. The enhancement of cyclin D1 expression and cyclin-dependent kinase activation in SEMA6C-low cancer created a druggable target of CDK4/6 inhibitors. We also elucidated the mechanism underlying SEMA6C downregulation in pancreatic cancer and demonstrated a novel regulatory role of miR-124-3p in suppressing SEMA6C. This study provides new insights of SEMA6C-mediated anti-cancer action and suggests the treatment of SEMA6C-downregulated cancer by CDK4/6 inhibitors.

c-Myc promotes lymphatic metastasis of pancreatic neuroendocrine tumor through VEGFC upregulation.

Pancreatic neuroendocrine tumor (pNET) is a pancreatic neoplasm with neuroendocrine differentiation. pNET in early stage can be treated with surgical resection with long-term survival, whereas the prognosis of pNET with locoregional or distant metastasis is relatively poor. Lymphangiogenesis is essential for tumor metastasis via the lymphatic system and may overhead distant metastasis. c-Myc overexpression is involved in tumorigenesis. The role of c-Myc in lymphangiogenesis is unclear. In this study, we evaluated the mechanism and effect of c-Myc on lymphangiogenesis of pNET via interaction of lymphatic endothelial cells (LECs) and pNET cells. Lymph node metastasis was evaluated in pNET xenograft mice. Potential target agents to inhibit lymph node metastasis were evaluated in an animal model. We found that vascular endothelial growth factor C (VEGFC) expression and secretion was increased in pNET cell lines with c-Myc overexpression. c-Myc transcriptionally upregulates VEGFC expression and the secretion of pNET cells by directly binding to the E-box of the VEGFC promoter and enhances VEGF receptor 3 phosphorylation and the tube formation of LECs. c-Myc overexpression is associated with lymph node metastasis in pNET xenograft mice. Combinational treatment with an mTOR inhibitor and c-Myc inhibitor or VEGFC-neutralizing chimera protein reduced lymph node metastasis in the mice with c-Myc overexpression. The mTOR inhibitor acts on lymphangiogenesis by reducing VEGFC expression in pNET cells and inhibiting the tube formation of LECs. In conclusion, mTOR and c-Myc are important for lymphangiogenesis of pNET and are potential therapeutic targets for prevention and treatment of lymph node metastasis in pNET.

Lysine demethylase 2A expression in cancer-associated fibroblasts promotes breast tumour growth.

BACKGROUND: Our previous study demonstrated that lysine demethylase 2A (KDM2A) enhances stemness in breast cancer cells. This demethylase is also highly expressed in cancer-associated fibroblasts (CAFs). However, its clinical significance is unclear. METHODS: The expression of KDM2A in CAFs was studied using immunohistochemical staining and its association with clinicopathological features and patient's survival was tested. Overexpression and knockdown strategies were used to investigate KDM2A-regulated genes in fibroblasts. Senescent cells were detected by using ß-galactosidase staining. The in vivo tumour-promoting activity of stromal KDM2A was confirmed by animal study. RESULTS: Increase of stromal KDM2A is associated with advanced tumour stage and poor clinical outcome in breast cancer patients. Cancer-derived cytokines stimulated KDM2A expression in normal fibroblasts and transformed them into CAFs. Upregulation of KDM2A induced p53-dependent senescence in fibroblasts and enhanced the release of cytokines, which reciprocally promoted cancer cell proliferation. Additionally, KDM2A upregulated programmed death-ligand 1 (PD-L1) expression via transcriptional activation in fibroblasts. Knockdown of KDM2A completely abolished the tumour-promoting activity of CAFs on breast tumour growth in vivo and diminished PD-L1 expression in the stroma of tumour tissues. CONCLUSIONS: Stromal KDM2A plays an oncogenic role in breast cancer and inhibition of KDM2A reduces fibroblast senescence and suppresses tumour growth.

Comprehensive Transcriptomic Analysis Identifies ST8SIA1 as a Survival-Related Sialyltransferase Gene in Breast Cancer.

Hypersialylation caused by the overexpression of sialyltransferases (STs) is a common feature in cancer that is associated with several characteristics of tumorigenesis. Thus, identifying cancer-associated STs is critical for cancer therapy. However, ST screening has been frequently conducted in cell line models. In this study, we conducted a comprehensive analysis of STs in the clinical database and identified the STs related with the survival of breast cancer patients. RNA sequencing (RNA-Seq) data of 496 patients were obtained from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA). Of the eight mapped STs, ST3GAL5, and ST8SIA1 met the acceptable area under the curve (AUC) criteria for overall survival (OS). Using Kaplan-Meier methods, we determined that high expression of ST8SIA1 was associated with poor 10-year OS in all patients, triple-negative breast cancer (TNBC), and non-TNBC patients, and poor disease-free survival (DFS) rates particularly in TNBC. ST8SIA1 also had superior AUC values in terms of OS/DFS. High ST8SIA1 levels showed a higher risk for poor OS in different groups of patients and a higher risk for poor DFS particularly in TNBC. In summary, we conducted a comprehensive analysis of STs from the clinical database and identified ST8SIA1 as a crucial survival-related ST, which might be a potential therapeutic target for breast cancer and TNBC patients.

Single-cell analysis reveals immune modulation and metabolic switch in tumor-draining lymph nodes.

Lymph-node metastasis is a prognosis factor for poor clinical outcome of breast cancer patients. Currently, how breast cancer cells establish pre-metastatic niche in the tumor-draining lymph nodes (TDLNs) is still unclear. To address this question, we isolated heterogeneous cells including immune and stromal cells from naive lymph nodes (LNs) of the FVB/NJ mice and TDLNs of the MMTV-PyMT mice. Single-cell RNA sequencing was performed to investigate the transcriptome of the cells and various bioinformatics analyses were used to identify the altered pathways. Our results revealed several significant changes between naïve LNs and TDLNs. First, according to immunologic signature and pathway analysis, CD4+ and CD8 + T cells showed upregulated angiogenesis pathway genes and higher regulatory T (Treg)-associated genes while they demonstrated downregulation of interferon response and inflammatory response gene signatures, concurrently suggesting an immunosuppressive microenvironment in the TDLNs. Second, profiling of B cells showed down-regulation of marginal zone B lymphocytes in the TDLNs, which was validated by flow cytometric analysis. Third, we found the enhancement of oxidative phosphorylation pathway in the fibroblastic reticular cells (FRCs) of the MMTV-PyMT mice and the elevation of related genes including Prdx3, Ndufa4 and Uqcrb, suggesting massive ATP consumption and TCA cycle metabolism in the FRCs. Collectively, our results reveal the reprogramming of TDLNs during breast cancer progression at single-cell level in a spontaneous breast cancer model and suggest the changes in immune modulation and metabolic switch are key alterations in the preparation of pre-metastatic niche by breast cancer cells.

Prognostic role of RECK in pathological outcome-dependent buccal mucosa squamous cell carcinoma.

BACKGROUND: Buccal mucosal squamous cell carcinoma (BMSCC) is an aggressive oral cancer. Moreover, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a well-known tumor suppressor in many cancers. Our aim was to investigate the association of RECK expression with prognosis in BMSCC patients with different clinicopathological features. MATERIALS AND METHODS: The expression level of RECK was determined by immunohistochemistry using tissue microarrays containing specimens from 193 BMSCC patients. The association of RECK expression with outcomes in BMSCC patients stratified by different clinicopathological features was analyzed by Cox proportional hazards models. RESULTS: The low expression level of RECK was associated with shorter disease-specific survival, especially in patients with age >40 years, moderate or poor cell differentiation, advanced pathological stage, and history of postoperative radiotherapy. However, the low expression level of RECK was not associated with poor disease-free survival, except in BMSCC patients with age ≦40 years, advanced pathological stage and lymph node metastasis. Furthermore, RECK-knockdowned cells showed higher cell viability and abilities of invasion/migration, indicating that RECK might be a tumor suppressor for tumor progression in oral cancer. CONCLUSION: The low expression of RECK might be a potential prognostic biomarker for pathological outcome-dependent BMSCC patients.

Cancer-Derived VEGF-C Increases Chemokine Production in Lymphatic Endothelial Cells to Promote CXCR2-Dependent Cancer Invasion and MDSC Recruitment.

Breast cancer-derived vascular endothelial growth factor-C (VEGF-C) has been shown to enhance lymphangiogenesis in lymph nodes to accelerate cancer metastasis. However, the remodeling of lymph node microenvironments by VEGF-C remains elusive. By in vivo selection, we established a subline (named as "LC") with strong lymphatic tropism and high VEGF-C expression from the human MDA-MB-231 breast cancer cell line. Co-culture with LC cells or treatment with LC-conditioned medium upregulated the expression of CXC chemokines in lymphatic endothelial cells (LECs), which could be inhibited by pre-incubation with VEGF-C-neutralizing antibodies and VEGFR3 inhibitors. The chemokines produced by LECs enhanced recruitment of myeloid-derived suppressor cells (MDSCs) to tumor-draining and distant lymph nodes in tumor-bearing mice. Treatment with a CXCR2 inhibitor after tumor cell inoculation dramatically decreased the number of MDSCs in lymph nodes, suggesting the importance of the chemokine/CXCR2 signaling axis in MDSC recruitment. In addition, LEC-released chemokines also stimulated the expression of serum amyloid A1 (SAA1) in cancer cells, enhancing their lymphatic invasion by increasing VE-cadherin phosphorylation, junction disruption, and vascular permeability of LECs. Clinical sample validation confirmed that SAA1 expression was associated with increased lymph node metastasis. Collectively, we reveal a novel mechanism by which cancer cell-derived VEGF-C remodels lymphovascular microenvironments by regulating chemokine production in LECs to promote cancer invasion and MDSC recruitment. Our results also suggest that inhibition of CXCR2 is effective in treating lymphatic metastasis.

Protein arginine methyltransferase 3-induced metabolic reprogramming is a vulnerable target of pancreatic cancer.

BACKGROUND: The biological function of protein arginine methyltransferase 3 (PRMT3) is not well known because very few physiological substrates of this methyltransferase have been identified to date. METHODS: The clinical significance of PRMT3 in pancreatic cancer was studied by database analysis. The PRMT3 protein level of human pancreatic tumors was detected by immunoblotting and immunohistochemical staining. PRMT3-associated proteins and the methylation sites on the proteins were investigated using mass spectrometry. Seahorse Bioscience analyzed the metabolic reprogramming. Combination index analysis and xenograft animal model were conducted to explore the effects of combination of inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidative phosphorylation on tumor growth. RESULTS: We found that the expression of PRMT3 is upregulated in pancreatic cancer, and its expression is associated with poor survival. We identified GAPDH as a PRMT3-binding protein and demonstrated that GAPDH is methylated at R248 by PRMT3 in vivo. The methylation of GAPDH by PRMT3 enhanced its catalytic activity while the mutation of R248 abolished the effect. In cells, PRMT3 overexpression triggered metabolic reprogramming and enhanced glycolysis and mitochondrial respiration simultaneously in a GAPDH-dependent manner. PRMT3-overexpressing cancer cells were addicted to GAPDH-mediated metabolism and sensitive to the inhibition of GAPDH and mitochondrial respiration. The combination of inhibitors of GAPDH and oxidative phosphorylation induced a synergistic inhibition on cellular growth in vitro and in vivo. CONCLUSION: Our results suggest that PRMT3 mediates metabolic reprogramming and cellular proliferation through methylating R248 of GAPDH, and double blockade of GAPDH and mitochondrial respiration could be a novel strategy for the treatment of PRMT3-overexpressing pancreatic cancer.

Alteration of Epigenetic Modifiers in Pancreatic Cancer and Its Clinical Implication.

The incidence of pancreatic cancer has considerably increased in the past decade. Pancreatic cancer has the worst prognosis among the cancers of the digestive tract because the pancreas is located in the posterior abdominal cavity, and most patients do not show clinical symptoms for early detection. Approximately 55% of all patients are diagnosed with pancreatic cancer only after the tumors metastasize. Therefore, identifying useful biomarkers for early diagnosis and screening high-risk groups are important to improve pancreatic cancer therapy. Recent emerging evidence has suggested that genetic and epigenetic alterations play a crucial role in the molecular aspects of pancreatic tumorigenesis. Here, we summarize recent progress in our understanding of the epigenetic alterations in pancreatic cancer and propose potential synthetic lethal strategies to target these genetic defects to treat this deadly disease.