A GAPDH serotonylation system couples CD8+ T cell glycolytic metabolism to antitumor immunity.

Apart from the canonical serotonin (5-hydroxytryptamine [5-HT])-receptor signaling transduction pattern, 5-HT-involved post-translational serotonylation has recently been noted. Here, we report a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serotonylation system that promotes the glycolytic metabolism and antitumor immune activity of CD8+ T cells. Tissue transglutaminase 2 (TGM2) transfers 5-HT to GAPDH glutamine 262 and catalyzes the serotonylation reaction. Serotonylation supports the cytoplasmic localization of GAPDH, which induces a glycolytic metabolic shift in CD8+ T cells and contributes to antitumor immunity. CD8+ T cells accumulate intracellular 5-HT for serotonylation through both synthesis by tryptophan hydroxylase 1 (TPH1) and uptake from the extracellular compartment via serotonin transporter (SERT). Monoamine oxidase A (MAOA) degrades 5-HT and acts as an intrinsic negative regulator of CD8+ T cells. The adoptive transfer of 5-HT-producing TPH1-overexpressing chimeric antigen receptor T (CAR-T) cells induced a robust antitumor response. Our findings expand the known range of neuroimmune interaction patterns by providing evidence of receptor-independent serotonylation post-translational modification.

Nucleolar HEAT Repeat Containing 1 Up-regulated by the Mechanistic Target of Rapamycin Complex 1 Signaling Promotes Hepatocellular Carcinoma Growth by Dominating Ribosome Biogenesis and Proteome Homeostasis.

BACKGROUND & AIMS: Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression. METHODS: HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells. RESULTS: HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth in vitro and in vivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1-mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis. CONCLUSIONS: Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1-mammalian target of rapamycin complex 1-specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.

CCBE1 promotes mitochondrial fusion by inhibiting the TGFß-DRP1 axis to prevent the progression of hepatocellular carcinoma.

The extracellular matrix (ECM), as an important component of the tumor microenvironment, exerts various roles in tumor formation. Mitochondrial dynamic disorder is closely implicated in tumorigenesis, including hyperfission in HCC. We aimed to determine the influence of the ECM-related protein CCBE1 on mitochondrial dynamics in HCC. Here, we found that CCBE1 was capable of promoting mitochondrial fusion in HCC. Initially, CCBE1 expression was found to be significantly downregulated in tumors compared with nontumor tissues, which resulted from hypermethylation of the CCBE1 promoter in HCC. Furthermore, CCBE1 overexpression or treatment with recombinant CCBE1 protein dramatically inhibited HCC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, CCBE1 functioned as an inhibitor of mitochondrial fission by preventing the location of DRP1 on mitochondria through inhibiting its phosphorylation at Ser616 by directly binding with TGFßR2 to inhibit TGFß signaling activity. In addition, a higher percentage of specimens with higher DRP1 phosphorylation was present in patients with lower CCBE1 expression than in patients with higher CCBE1 expression, which further confirmed the inhibitory effect of CCBE1 on DRP1 phosphorylation at Ser616. Collectively, our study highlights the crucial roles of CCBE1 in mitochondrial homeostasis, suggesting strong evidence for this process as a potential therapeutic strategy for HCC.

ACE2 negatively regulates the Warburg effect and suppresses hepatocellular carcinoma progression via reducing ROS-HIF1α activity.

Aerobic glycolysis has pleiotropic roles in the pathogenesis of hepatocellular carcinoma (HCC). Emerging studies revealed key promoters of aerobic glycolysis, however, little is known about its negative regulators in HCC. In this study, an integrative analysis identifies a repertoire of differentially expressed genes (DNASE1L3, SLC22A1, ACE2, CES3, CCL14, GYS2, ADH4, and CFHR3) that are inversely associated with the glycolytic phenotype in HCC. ACE2, a member of the rennin-angiotensin system, is revealed to be downregulated in HCC and predicts a poor prognosis. ACE2 overexpression significantly inhibits the glycolytic flux as evidenced by reduced glucose uptake, lactate release, extracellular acidification rate, and the expression of glycolytic genes. Opposite results are noticed in loss-of-function studies. Mechanistically, ACE2 metabolizes Ang II to Ang-(1-7), which activates Mas receptor and leads to the phosphorylation of Src homology 2-containing inositol phosphatase 2 (SHP-2). SHP2 activation further blocks reactive oxygen species (ROS)-HIF1α signaling. Addition of Ang-(1-7) or the antioxidant N-acetylcysteine compromises in vivo additive tumor growth and aerobic glycolysis induced by ACE2 knockdown. Moreover, growth advantages afforded by ACE2 knockdown are largely glycolysis-dependent. In clinical settings, a close link between ACE2 expression and HIF1α or the phosphorated level of SHP2 is found. Overexpression of ACE2 significantly retards tumor growth in patient-derived xenograft model. Collectively, our findings suggest that ACE2 is a negative glycolytic regulator, and targeting the ACE2/Ang-(1-7)/Mas receptor/ROS/HIF1α axis may be a promising therapeutic strategy for HCC treatment.

Dipyridamole enhances the anti-cancer ability of aspirin against colorectal cancer by inducing apoptosis in an unfolded protein response-dependent manner.

PURPOSE: Available evidence indicates that dipyridamole enhances the anti-thrombotic effects of aspirin for the prevention of secondary strokes. Aspirin is a well-known non-steroid anti-inflammatory drug. This anti-inflammatory property has turned aspirin into a potential drug for inflammation-related cancers such as colorectal cancer (CRC). Here, we aimed to explore whether the anti-cancer effect of aspirin against CRC could be improved by combined administration with dipyridamole. METHODS: Population-based clinical data analysis was conducted to assess a possible therapeutic effect of combined dipyridamole and aspirin treatment in inhibiting CRC compared with either monotherapy. This therapeutic effect was further verified in different CRC mouse models, i.e. an orthotopic xenograft mouse model, an AOM/DSS mouse model, an Apcmin/+ mouse model and a patient derived xenograft (PDX) mouse model. The in vitro effects of the drugs on CRC cells were tested using CCK8 and flow cytometry assays. RNA-Seq, Western blotting, qRT-PCR and flow cytometry were used to identify the underlying molecular mechanisms. RESULTS: We found that dipyridamole combined with aspirin had a better inhibitory effect on CRC than either monotherapy alone. The enhanced anti-cancer effect of the combined use of dipyridamole with aspirin was found to rely on the induction of an overwhelmed endoplasmic reticulum (ER) stress and subsequent pro-apoptotic unfolded protein response (UPR), which was different from the anti-platelet effect. CONCLUSIONS: Our data indicate that the anti-cancer effect of aspirin against CRC may be enhanced by combined administration with dipyridamole. In case further clinical studies confirm our findings, these may be repurposed as adjuvant agents.

The SLITRK4-CNPY3 axis promotes liver metastasis of gastric cancer by enhancing the endocytosis and recycling of TrkB in tumour cells.

PURPOSE: Gastric cancer (GC) is a malignant tumour with high mortality, and liver metastasis is one of the main causes of poor prognosis. SLIT- and NTRK-like family member 4 (SLITRK4) plays an important role in the nervous system, such as synapse formation. Our study aimed to explore the functional role of SLITRK4 in GC and liver metastasis. METHODS: The mRNA level of SLITRK4 was evaluated using publicly available transcriptome GEO datasets and Renji cohort. The protein level of SLITRK4 in the tissue microarray of GC was observed using immunohistochemistry. Cell Counting Kit-8, colony formation, transwell migration assays in vitro and mouse model of liver metastasis in vivo was performed to investigate the functional roles of SLITRK4 in GC. Bioinformatics predictions and Co-IP experiments were applied to screen and identify SLITRK4-binding proteins. Western blot was performed to detect Tyrosine Kinase receptor B (TrkB)-related signaling molecules. RESULTS: By comparing primary and liver metastases from GC, SLITRK4 was found to be upregulated in tissues of GC with liver metastasis and to be closely related to poor clinical prognosis. SLITRK4 knockdown significantly abrogated the growth, invasion, and metastasis of GC in vitro and in vivo. Further study revealed that SLITRK4 could interact with Canopy FGF Signalling Regulator 3 (CNPY3), thus enhancing TrkB- related signaling by promoting the endocytosis and recycling of the TrkB receptor. CONCLUSION: In conclusion, the CNPY3-SLITRK4 axis contributes to liver metastasis of GC according to the TrkB-related signaling pathway. which may be a therapeutic target for the treatment of GC with liver metastasis.

A low amino acid environment promotes cell macropinocytosis through the YY1-FGD6 axis in Ras-mutant pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), cancer with a high mortality rate and the highest rate of KRAS mutation, reportedly internalizes proteins via macropinocytosis to adapt to low amino acid levels in the tumor microenvironment. Here, we aimed to identify a key regulator of macropinocytosis for the survival of tumor cells in a low amino acid environment in PDAC. FYVE, RhoGEF, and PH domain-containing protein 6 (FGD6) were identified as key regulators of macropinocytosis. FGD6 promoted PDAC cell proliferation, macropinocytosis, and tumor growth both in vitro and in vivo. The macropinocytosis level was decreased with FGD6 knockdown in PDAC cell lines. Moreover, FGD6 promoted macropinocytosis by participating in the trans-Golgi network and enhancing the membrane localization of growth factor receptors, especially the TGF-beta receptor. TGF-beta enhanced macropinocytosis in PDAC cells. Additionally, YAP nuclear translocation induced by a low amino acid tumor environment initiated FGD6 expression by coactivation with YY1. Clinical data analysis based on TCGA and GEO datasets showed that FGD6 expression was upregulated in PDAC tissue, and high FGD6 expression was correlated with poor prognosis in patients with PDAC. In tumor tissue from KrasG12D/+/Trp53R172H/-/Pdx1-Cre (KPC) mice, FGD6 expression escalated during PDAC development. Our results uncover a previously unappreciated mechanism of macropinocytosis in PDAC. Strategies to target FGD6 and growth factors membrane localization might be developed for the treatment of PDAC.

Increased Nuclear Transporter KPNA2 Contributes to Tumor Immune Evasion by Enhancing PD-L1 Expression in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies and is known for its high resistance and low response to treatment. Tumor immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies. Karyopherin alpha 2 (KPNA2), a member of the nuclear transporter family, is elevated in multiple human cancers and accelerates carcinogenesis. However, the specific role of KPNA2 in PDAC remains unclear. In this study, we found that expression of KPNA2 was significantly upregulated in PDAC compared to adjacent nontumor tissue and its high expression was correlated with poor survival outcome by analyzing the GEO datasets. Similar KPNA2 expression pattern was also found in both human patient samples and KPC mouse models through IHC staining. Although KPNA2 knockdown failed to impair the vitality and migration ability of PDAC cells in vitro, the in vivo tumor growth was significantly impeded and the expression of immune checkpoint ligand PD-L1 was reduced by silencing KPNA2. Furthermore, we uncovered that KPNA2 modulated the expression of PD-L1 by mediating nuclear translocation of STAT3. Collectively, our data suggested that KPNA2 has the potential to serve as a promising biomarker for diagnosis in PDAC.

Single-cell RNA sequencing reveals that targeting HSP90 suppresses PDAC progression by restraining mitochondrial bioenergetics.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, which lacks effective treatment strategies. There is an urgent need for the development of new strategies for PDAC therapy. The genetic and phenotypic heterogeneity of PDAC cancer cell populations poses further challenges in the clinical management of PDAC. In this study, we performed single-cell RNA sequencing to characterize PDAC tumors from KPC mice. Functional studies and clinical analysis showed that PDAC cluster 2 cells with highly Hsp90 expression is much more aggressive than the other clusters. Genetic and pharmacologic inhibition of Hsp90 impaired tumor cell growth both in vitro and in vivo. Further mechanistic study revealed that HSP90 inhibition disrupted the interaction between HSP90 and OPA1, leading to a reduction in mitochondrial cristae amount and mitochondrial energy production. Collectively, our study reveals that HSP90 might be a potential therapeutic target for PDAC.

CTHRC1 promotes liver metastasis by reshaping infiltrated macrophages through physical interactions with TGF-ß receptors in colorectal cancer.

Metastasis is a major cause of cancer-related deaths. Tumor-intrinsic properties can determine whether tumor metastasis occurs or not. Here, by comparing the gene expression patterns in primary colorectal cancer (CRC) patients with or without metastasis, we found that Collagen Triple Helix Repeat Containing 1 (CTHRC1) in primary CRC served as a metastasis-associated gene. Animal experiments verified that CTHRC1 secreted by CRC cells promoted hepatic metastasis, which was closely correlated with macrophage infiltration. Depletion of macrophages by liposomal clodronate largely abolished the promoting effect of CTHRC1 on CRC liver metastasis. Furthermore, we demonstrated that CTHRC1 modulated macrophage polarization to M2 phenotypes through TGF-ß signaling. A mechanistic study revealed that CTHRC1 bound directly to TGF-ß receptor II and TGF-ß receptor III, stabilized the TGF-ß receptor complex, and activated TGF-ß signaling. The combination treatment of CTHRC1 monoclonal antibody and anti-PD-1 blocking antibody effectively suppressed CRC hepatic metastasis. Taken together, our data demonstrated that CTHRC1 is an intrinsic marker of CRC metastasis and further revealed that CTHRC1 promoted CRC liver metastasis by reshaping infiltrated macrophages through TGF-ß signaling, suggesting that CTHRC1 could be a potential biomarker for the early prediction of and a therapeutic target of CRC hepatic metastasis.

Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis.

The immunosuppressive microenvironment that is shaped by hepatic metastatic pancreatic ductal adenocarcinoma (PDAC) is essential for tumor cell evasion of immune destruction. Neutrophils are important components of the metastatic tumor microenvironment and exhibit heterogeneity. However, the specific phenotypes, functions and regulatory mechanisms of neutrophils in PDAC liver metastases remain unknown. Here, we show that a subset of P2RX1-negative neutrophils accumulate in clinical and murine PDAC liver metastases. RNA sequencing of murine PDAC liver metastasis-infiltrated neutrophils show that P2RX1-deficient neutrophils express increased levels of immunosuppressive molecules, including PD-L1, and have enhanced mitochondrial metabolism. Mechanistically, the transcription factor Nrf2 is upregulated in P2RX1-deficient neutrophils and associated with PD-L1 expression and metabolic reprogramming. An anti-PD-1 neutralizing antibody is sufficient to compromise the immunosuppressive effects of P2RX1-deficient neutrophils on OVA-activated OT1 CD8+ T cells. Therefore, our study uncovers a mechanism by which metastatic PDAC tumors evade antitumor immunity by accumulating a subset of immunosuppressive P2RX1-negative neutrophils.

Dual Convergence of Facial Nerve Branches Innervating Whisker Pad in Rats.

The precise anatomy of the facial nerve branches innervating rat whisker pad and the distribution of their corresponding motor neurons in facial nucleus area were investigated. The extratemporal facial nerves of 6 rats were anatomically observed under a surgical microscope, and then the nerve specimens of facial nerve branches at 7 anatomical sites were taken and examined for the axons and myelin sheath using Luxol fast blue staining. The distribution of facial motor neurons innervating the facial branches was observed in 12 rats by retrograde labelling. The distal pes, a fusing architecture of the buccal and marginal mandibular branches, was found to furcate into superior, middle and inferior branches to innervate whisker pad. Histologically, the myelin sheath of each branch was morphologically consistent, and the nerve fiber bundles of facial nerve branches became increasingly thinner and scattered, particularly after crossing the distal pes site and innervating the whisker pad. The facial motor neurons innervating the buccal and marginal mandibular branches were clearly distributed in similar regions in facial nucleus. This study confirmed the highly spatial synergy between the buccal and marginal mandibular branches innervating the whisker pad from extratemporal anatomy and distribution of facial motor neurons.

Microglia--friend or foe.

Microglia, as the immune effectors in the central nervous system, respond to pathological conditions and participate in the initiation and progression of neurological disorders such as inflammation and brain tumor by releasing potential neurotrophic or cytotoxic molecules, presenting the antigen to T cell and interacting with brain tumor. Evidences also suggest that microglia are capable of promoting or inhibiting the proliferation and differentiation of neural stem cells by secreting series of biologically active molecules. In this review, we focus on three aspects-inflammation, neurogensis and brain tumor to illustrate the multi-faceted activities of microglia in the normal and pathologic brain.