The GFPT2-O-GlcNAcylation-YBX1 axis promotes IL-18 secretion to regulate the tumor immune microenvironment in pancreatic cancer.

The immunosuppressive microenvironment caused by several intrinsic and extrinsic mechanism has brought great challenges to the immunotherapy of pancreatic cancer. We identified GFPT2, the key enzyme in hexosamine biosynthesis pathway (HBP), as an immune-related prognostic gene in pancreatic cancer using transcriptome sequencing and further confirmed that GFPT2 promoted macrophage M2 polarization and malignant phenotype of pancreatic cancer. HBP is a glucose metabolism pathway leading to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is further utilized for protein O-GlcNAcylation. We confirmed GFPT2-mediated O-GlcNAcylation played an important role in regulating immune microenvironment. Through cellular proteomics, we identified IL-18 as a key downstream of GFPT2 in regulating the immune microenvironment. Through CO-IP and protein mass spectrum, we confirmed that YBX1 was O-GlcNAcylated and nuclear translocated by GFPT2-mediated O-GlcNAcylation. Then, YBX1 functioned as a transcription factor to promote IL-18 transcription. Our study elucidated the relationship between the metabolic pathway of HBP in cancer cells and the immune microenvironment, which might provide some insights into the combination therapy of HBP vulnerability and immunotherapy in pancreatic cancer.

Deubiquitinating PABPC1 by USP10 upregulates CLK2 translation to promote tumor progression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant with limited treatment options. Deubiquitinases (DUBs), which cleave ubiquitin on substrates, can regulate tumor progression and are appealing therapeutic targets, but there are few related studies in PDAC. In our study, we screened the expression levels and prognostic value of USP family members based on published databases and selected USP10 as the potential interventional target in PDAC. IHC staining of the PDAC microarray revealed that USP10 expression was an adverse clinical feature of PDAC. USP10 promoted tumor growth both in vivo and in vitro in PDAC. Co-IP experiments revealed that USP10 directly interacts with PABPC1. Deubiquitination assays revealed that USP10 decreased the K27/29-linked ubiquitination level of the RRM2 domain of PABPC1. Deubiquitinated PABPC1 was able to couple more CLK2 mRNA and eIF4G1, which increased the translation efficiency. Replacing PABPC1 with a mutant that could not be ubiquitinated impaired USP10 knock-down-mediated tumor suppression in PDAC. Targeting USP10 significantly delayed the growth of cell-derived xenograft and patient-derived xenograft tumors. Collectively, our study first identified USP10 as the DUB of PABPC1 and provided a rationale for potential therapeutic options for PDAC with high USP10 expression.

Inhibition of epithelial-to-mesenchymal transition augments antitumor efficacy of nanotherapeutics in pancreatic ductal adenocarcinoma.

Intrinsic drug resistance mechanisms of tumor cells often reduce intracellular drug concentration to suboptimal levels. Epithelial-to-mesenchymal transition (EMT) is a pivotal process in tumor progression and metastasis that confers an aggressive phenotype as well as resistance to chemotherapeutics. Therefore, it is imperative to develop novel strategies and identify new targets to improve the overall efficacy of cancer treatment. We developed SN38 (active metabolite of irinotecan)-assembled glycol chitosan nanoparticles (cSN38) for the treatment of pancreatic ductal adenocarcinoma (PDAC). Furthermore, cSN38 and the TGF-ß1 inhibitor LY364947 formed composite nanoparticles upon self-assembly (cSN38 + LY), which obviated the poor aqueous solubility of LY364947 and enhanced drug sensitivity. The therapeutic efficacy of cSN38 + LY nanotherapeutics was studied in vitro and in vivo using suitable models. The cSN38 nanoparticles exhibited an antitumor effect that was significantly attenuated by TGF-ß-induced EMT. The cellular uptake of SN38 was impeded during EMT, which affected the therapeutic efficacy. The combination of LY364947 and cSN38 markedly enhanced the cellular uptake of SN38, increased cytotoxic effects, and inhibited EMT in PDAC cells in vitro. Furthermore, cSN38 + LY significantly inhibited PDAC xenograft growth in vivo. The cSN38 + LY nanoparticles increased the therapeutic efficacy of cSN38 via repressing the EMT of PDAC cells. Our findings provide a rationale for designing nanoscale therapeutics to combat PDAC.

SIGLEC15 amplifies immunosuppressive properties of tumor-associated macrophages in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) usually presents infrequent infiltration of T lymphocytes. The known immune-checkpoint inhibitors to date focus on activating T cells and manifest limited effectiveness in PDAC. SIGLEC15 was identified as a novel tumor-associated macrophage (TAM)-related immune-checkpoint in other cancer types, while its immunosuppressive role and clinical significance remained unclear in PDAC. In our study, SIGLEC15 presented immunosuppressive relevance in PDAC via bioinformatic analysis and expressed on TAM and PDAC cells. SIGLEC15+ TAM, rather than SIGLEC15+ PDAC cells or SIGLEC15- TAM, correlated with poor prognosis and immunosuppressive microenvironment in the PDAC microarray cohort. Compared with SIGLEC15- TAM, SIGLEC15+ TAM presented an M2-like phenotype that could be modulated by SIGLEC15 in a tumor cell-dependent manner. In mechanism, SIGLEC15 interacted with PDAC-expressed sialic acid, preferentially α-2, 3 sialic acids, to stimulate SYK phosphorylation in TAM, which further promoted its immunoregulatory cytokines and chemokines production. In vivo, SIGLEC15+ TAM also presented an M2-like phenotype, accelerated tumor growth, and facilitated immunosuppressive microenvironment, which was greatly abolished by SYK inhibitor. Our study highlighted a novel M2-promoting function of SIGLEC15 and strongly suggested SIGLEC15 as a potential immunotherapeutic target for PDAC.

Mutation and Expression of Gene YY1 in Pancreatic Neuroendocrine Tumors and Its Clinical Significance.

OBJECTIVE: The clinical significance of the YY1 gene mutation and expression in pancreatic neuroendocrine tumors (PNETs) remains unknown. Therefore, this study aimed to comprehensively analyze the somatic mutation of YY1 in the different subtypes of PNETs. METHODS: A total of 143 PNETs were assessed by Sanger sequencing to identify the somatic mutation of YY1 gene in various subtypes of PNETs. YY1 protein expression was examined in 103 PNETs by immunohistochemical staining and western blot. Gene mutation and its protein expression were correlated with clinicopathologic features. RESULTS: A recurrent mutation (chr14:100743807C>G) in the YY1 gene was identified in 15 of 83 insulinomas (18%) and in only 1 of 60 noninsulinoma PNETs (1.7%) (P = .0045). The YY1 mutation was not found in MEN1-associated insulinomas. The YY1 mutation in insulinomas was correlated with older age and lower serum glucose levels (age, 57 vs 42.5 years, P = .006; blood glucose, 25.2 vs 33.6 mg/dL, P = .008). YY1 protein expression was found in 100 of 103 PNETs, although expression was weaker in metastases than in localized tumors (P = .036). The stronger expression of YY1 protein was associated with favorable disease-free survival of patients with PNETs (log-rank, P = .011; n = 70). Multivariable statistical analysis showed that YY1 protein expression could be an independent predictor of prognosis. CONCLUSION: The hotspot YY1 mutation mostly occurred in insulinomas and rarely in noninsulinoma PNETs. The stronger YY1 protein expression was correlated with the better prognosis of PNETs patients.

A Ki-67 Index to Predict Treatment Response to the Capecitabine/Temozolomide Regimen in Neuroendocrine Neoplasms: A Retrospective Multicenter Study.

OBJECTIVE: The efficacy of the capecitabine/temozolomide (CAPTEM) regimen has been demonstrated in metastatic neuroendocrine neoplasms (NENs), but because of varying response rates among the patients, biomarkers to predict its response are greatly needed. Here, we investigated the clinical utility of a Ki-67 index to predict the CAPTEM regimen objective responses and select patients who could benefit from this regimen. METHODS: Metastatic NENs patients treated with the CAPTEM regimen from 4 high-volume medical centers were selected and grouped in a training and validation cohort. The classification and regression tree (CART) was generated to identify the optimal threshold of Ki-67 for stratifying the patients into different Ki-67 range groups based on their response to the CAPTEM regimen. RESULTS AND CONCLUSIONS: The overall response rate (ORR) and disease control rate of the entire cohort (N = 151) were 26.5 and 76.2%, respectively, with a median progression-free survival (PFS) of 12.0 months. CART analysis showed that patients in the Ki-67 range group 10-40% demonstrated a significantly higher ORR than those in Ki-67 >40 and <10% groups (p < 0.001 in the training cohort and p = 0.036 in the validation cohort). Response to the CAPTEM regimen was not influenced by the expression of O6-methylguanine-DNA methyltransferase or primary tumor location. Multivariate analysis identified the Ki-67 index as the only independent prognostic factor for overall survival (p = 0.031) and PFS (p = 0.006). The proposed Ki-67 index was externally validated and could be used to clinically identify suitable metastatic NENs patients who could achieve an optimal cytoreduction using the CAPTEM regimen.

Clinicopathological features and prognostic validity of the European Neuroendocrine Tumor Society (ENETS) and American Joint Committee on Cancer (AJCC) 8th staging systems in colonic neuroendocrine neoplasms.

PURPOSE: This study aimed to investigate the characteristics of colonic neuroendocrine neoplasms (NENs) and to validate the prognostic value of the European Neuroendocrine Tumor Society (ENETS) and American Joint Committee on Cancer (AJCC) 8th staging systems. METHODS: A total of 167 and 1248 patients with colonic NENs from 12 medical centers across China and from the Surveillance, Epidemiology, and End Results (SEER) cancer registry in the United States, respectively, were reviewed. Patients were staged according to the ENETS and AJCC 8th staging systems. RESULTS: Clinicopathological features of colonic NENs in the Chinese cohort and SEER cohort were significantly distinct. In both the Chinese cohort and the SEER cohort, colonic neuroendocrine carcinoma (NEC) and mixed adeno-neuroendocrine carcinoma (MANEC) were more frequent in the midgut than in the hindgut. Tumors originating from the midgut tended to be larger and at a more advanced stage than those from the hindgut. The AJCC 8th staging system and the ENETS system appeared to have similar prognostic ability for colonic NEC/MANEC. CONCLUSIONS: Our study revealed that tumors originating from the midgut and the hindgut shared different clinicopathological features. The AJCC 8th staging system and the ENETS system appeared to have similar prognostic ability for colonic NEC/MANEC.

Annexin A4-nuclear factor-κB feedback circuit regulates cell malignant behavior and tumor growth in gallbladder cancer.

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system. However, the mechanisms underlying its tumor initiation, progression, and metastasis are not yet fully understood. The annexin A4 (ANXA4) gene is highly expressed in GBC tissues and may play an important role in the initiation and progression of this disease. In this study, we examined the up-regulation of ANXA4 in human GBC tissues and cell lines. Elevated ANXA4 correlated well with invasion depth in GBC patients and predicted a poor prognosis. In vitro, GBC-SD and NOZ cells with ANXA4 knockdown demonstrated increased apoptosis and inhibited cell growth, migration, and invasion. Interactions between ANXA4 and nuclear factor-κB (NF-κB) p65 proteins were detected. In vivo, ANXA4 knockdown inhibited tumor growth of GBC cells in nude mice and down-regulated the expression of downstream factors in the NF-κB signaling pathway. Taken together, these data indicate that up-regulation of ANXA4 leads to activation of the NF-κB pathway and its target genes in a feedback regulatory mechanism via the p65 subunit, resulting in tumor growth in GBC.

Hepatocyte nuclear factor 4α suppresses the aggravation of colon carcinoma.

Hepatocyte nuclear factor 4-α (HNF4α), a nuclear receptor, is expressed at lower levels in colon carcinoma tissues than in adjacent normal tissues. However, the relation between HNF4α and colon cancer progression and the underlying molecular mechanisms remain unclear. Here, we investigated the role of HNF4α in the progression of colon carcinoma. We showed that HNF4α mRNA and protein were downregulated in colon carcinoma specimens. HNF4α expression was related to pT classification (P < 0.001), lymph node metastasis (P = 0.002), distant metastasis (P < 0.001) and clinical stage (P < 0.001) in colon carcinoma patients. Patients with low or negative HNF4α expression had worse 3-year progression-free survival (PFS, P = 0.006) and overall survival (OS, P = 0.005) than patients with high HNF4α expression. Low HNF4α expression was an independent prognostic factor for 3-year PFS (hazard ratio 2.94; 95% confidence interval 1.047-8.250; P = 0.041). Ectopic expression of HNF4α inhibited colon carcinoma cell (HT29, LoVo, and SW480) proliferation, migration, and invasion, induced G2/M phase arrest and promoted apoptosis. Ectopic expression of HNF4α upregulated E-cadherin and downregulated vimentin in vitro, and suppressed SW480 xenograft tumor growth and liver metastasis in vivo. Furthermore, HNF4α overexpression downregulated the expression of snail, slug and twist. HNF4α inhibited EMT through its effect on the Wnt/ß-catenin signaling pathway, and HNF4α downregulation may be mediated by promoter methylation in cancer tissues. Our results suggest that downregulation of HNF4α plays a critical role in the aggravation of colon carcinoma possibly by promoting EMT via the Wnt/ß-catenin signaling pathway and by affecting apoptosis and cell cycle progression.

Proteomic identification of tumor biomarkers associated with primary gallbladder cancer.

AIM: To identify potential biomarkers of primary gallbladder cancer (PGC). METHODS: Fresh PGC, cholecystitis and normal gallbladder tissue specimens collected from 10 patients, respectively, were subjected to comparative proteomic analysis. The proteomic patterns of PGC were compared with those of cholecystitis and normal gallbladder tissues using two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were then identified using a MALDI-TOF mass spectrometer (MS) and database searches. To further validate these proteins, 20 samples of PGC tissues and normal tumor-adjacent tissues were collected for Western blot, quantitative real-time PCR, and immunohistochemical staining assay. RESULTS: Seven differentially expressed protein spots were detected by 2-ED analysis by comparing the average maps of PGC, cholecystitis and normal gallbladder tissues. Six of the seven differentially expressed proteins were identified using MALDI-TOF MS, with three overexpressed and three underexpressed in PGC tissue. Protein levels of annexin A4 (ANXA4) were significantly elevated, and heat shock protein 90-beta (Hsp90ß) and dynein cytoplasmic 1 heavy chain 1 (Dync1h1) were decreased in PGC tissues relative to the normal tumor-adjacent tissues as shown by Western blot analysis. However, levels of actin, aortic smooth muscle and gamma-actin were unchanged. In addition, the mRNA levels of all 5 proteins showed similar changes to those of the protein levels (P < 0.01). Further validation by immunohistochemical analysis showed the upregulated expression of ANXA4 and decreased expression of Hsp90ß and Dync1h1 in the cytoplasm of PGC tissues relative to the normal tumor-adjacent tissues. CONCLUSION: Three proteins are identified as potential biomarkers of PGC using proteomic analysis. The functions of these proteins in the carcinogenesis of PGC remain to be studied.

Impact of intra-subunit interactions on the dimeric arginine kinase activity and structural stability.

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. In this research, six conserved residues located on the intra-subunit domain-domain interfaces were mutated to explore their roles in the activity and structural stability of dimer AK. The mutations D69A, E70A, E71A and F80A led to pronounced loss of AK activity and structural stability. Although the mutations V75A and F76A had little effect on AK activity and structure, they caused gradually decreased the stability and reactivation of dimer AK. Our results suggested that the mutations might affect the correct positioning of the N-loop and C-loop thus disrupted the efficient recognition and interactions between the N-terminal domain and C-terminal domain which may influence the compact dimer structure, and result in decreased activity and structural stability.