Blocking EGR1/TGF-ß1 and CD44s/STAT3 Crosstalk Inhibits Peritoneal Metastasis of Gastric Cancer.

Peritoneal metastasis (PM) continues to limit the clinical efficacy of gastric cancer (GC). Early growth response 1 (EGR1) plays an important role in tumor cell proliferation, angiogenesis and invasion. However, the role of EGR1 derived from the tumor microenvironment in reshaping the phenotypes of GC cells and its specific molecular mechanisms in increasing the potential for PM are still unclear. In this study, we reported that EGR1 was significantly up-regulated in mesothelial cells from GC peritoneal metastases, leading to enhanced epithelial-mesenchymal transformation (EMT) and stemness phenotypes of GC cells under co-culture conditions. These phenotypes were achieved through the transcription and secretion of TGF-ß1 by EGR1 in mesothelial cells, which could regulate the expression and internalization of CD44s. After being internalized into the cytoplasm, CD44s interacted with STAT3 to promote STAT3 phosphorylation and activation, and induced EMT and stemness gene transcription, thus positively regulating the metastasis of GC cells. Moreover, TGF-ß1 secretion in the PM microenvironment was significantly increased compared with the matched primary tumor. The blocking effect of SHR-1701 on TGF-ß1 was verified by inhibiting peritoneal metastases in xenografts. Collectively, the interplay of EGR1/TGF-ß1/CD44s/STAT3 signaling between mesothelial cells and GC cells induces EMT and stemness phenotypes, offering potential as a therapeutic target for PM of GC.

Tumor-mesothelium HOXA11-PDGF BB/TGF ß1-miR-181a-5p-Egr1 feedforward amplifier circuity propels mesothelial fibrosis and peritoneal metastasis of gastric cancer.

A proportion of gastric cancer (GC) patients suffer from peritoneal metastasis (PM) in the late stage of tumor and these patients have a poor prognosis. To provide more care for GC patient with PM, a deeper exploration of the molecular characteristics of GC-PM is needed. Here we performed the in vitro and in vivo study to illustrate the effect of HOXA11 over-expressed GC cells on peritoneal mesothelial cells (HMrSV5), transcriptomics analyses of HMrSV5 cells co-cultured with HOXA11 over-expressed GC cells, counterparts or alone, cytokine array analyses of serum-free culture medium of HOXA11 over-expressed GC cells, we validated our findings through genetic manipulation of HMrSV5 cells and neutralizing antibodies targeting cytokines secreted by HOXA11 over-expressed GC cells in vitro, as well as utilized human peritoneal metastatic lesions to validate expression of potential targets. We identified that HOXA11 over-expressed GC cells strongly propelled mesothelial fibrosis in vivo and in vitro, and HOXA11 regulated paracrine and autocrine of PDGF BB and TGF ß1 in GC cells to propel mesothelial fibrosis. Meanwhile, HOXA11 over-expressed GC cells drove PDGF BB and TGF ß1 secretion to activate developmental-process related genes in HMrSV5 cells, including Egr1, which processes dependent on miR-181a-5p. Then, Egr1 could mediate peritoneal mesothelial fibrosis. Correspondingly, Egr1 over-expressed HMrSV5 cells supported migration and peritoneal dissemination of GC cells. Together our results suggest that a feedforward amplifier circuity governing GC cells and mesothelial cells in peritoneum contribute to peritoneal metastasis of GC cells.

VPS35 promotes gastric cancer progression through integrin/FAK/SRC signalling-mediated IL-6/STAT3 pathway activation in a YAP-dependent manner.

VPS35 is a key subunit of the retromer complex responsible for recognising cytosolic retrieval signals in cargo and is involved in neurodegenerative disease and tumour progression. However, the function and molecular mechanism of VPS35 in gastric cancer (GC) remains largely unknown. Here, we demonstrated that VPS35 was significantly upregulated in GC, which was associated with poor survival. VPS35 promoted GC cell proliferation and metastasis both in vitro and in vivo. Mechanistically, VPS35 activated FAK-SRC kinases through integrin-mediated outside-in signalling, leading to the activation of YAP and subsequent IL-6 expression induction in tumour cells. What's more, combined mass spectrometry analysis of MGC-803 cell and bioinformatic analysis, we found that phosphorylation of VPS35 was enhanced in GC cells, and phosphorylated VPS35 has enhanced interaction with ITGB3. VPS35 interacted with ITGB3 and affected the recycling of ITGB3 in GC cells. Gain- and loss-of-function experiments revealed that VPS35 promoted tumour proliferation and metastasis via the IL-6/STAT3 pathway. Interestingly, we also found that STAT3 directly bound to the VPS35 promoter and increased VPS35 transcription, thereby establishing a positive regulatory feedback loop. In addition, we demonstrated that VPS35 knockdown sensitised GC cells to 5-FU and cisplatin. These findings provide evidence that VPS35 promotes tumour proliferation and metastasis, and highlight the potential of targeting VPS35- and IL-6/STAT3-mediated tumour interactions as promising therapeutic strategies for GC.

Paracrine activin B-NF-κB signaling shapes an inflammatory tumor microenvironment in gastric cancer via fibroblast reprogramming.

BACKGROUND: Important roles of INHBB in various malignancies are increasingly identified. The underlying mechanisms in gastric cancer (GC) microenvironment are still greatly unexplored. METHODS: The clinical significance of INHBB and the correlation between INHBB and p-p65 in GC were assessed through analyzing publicly available databases and human paraffin embedded GC tissues. The biological crosstalk of INHBB between GC cells and fibroblasts was explored both in vitro and in vivo. RNA-seq analyses were performed to determine the mechanisms which regulating fibroblasts reprogramming. Luciferase reporter assay and chromatin immunoprecipitation (CHIP) assay were used to verify the binding relationship of p65 and INHBB in GC cells. RESULTS: Our study showed that INHBB level was significantly higher in GC, and that increased INHBB was associated with poor survival. INHBB positively regulates the proliferation, migration, and invasion of GC cells in vitro. Also, activin B promotes the occurrence of GC by reprogramming fibroblasts into cancer-associated fibroblasts (CAFs). The high expression of INHBB in GC cells activates the NF-κB pathway of normal gastric fibroblasts by secreting activin B, and promotes fibroblasts proliferation, migration, and invasion. In addition, activin B activates NF-κB pathway by controlling TRAF6 autoubiquitination to induce TAK1 phosphorylation in fibroblasts. Fibroblasts activated by activin B can induce the activation of p65 phosphorylation of GC cells by releasing pro-inflammatory factors IL-1ß. p65 can directly bind to the INHBB promoter and increase the INHBB transcription of GC cells, thus establishing a positive regulatory feedback loop to promote the progression of GC. CONCLUSIONS: GC cells p65/INHBB/activin B and fibroblasts p65/IL-1ß signal loop led to the formation of a whole tumor-promoting inflammatory microenvironment, which might be a promising therapeutic target for GC.

HNF4A-BAP31-VDAC1 axis synchronously regulates cell proliferation and ferroptosis in gastric cancer.

B cell receptor associated protein 31 (BAP31) is closely associated with tumor progression, while the role and mechanism of BAP31 in gastric cancer (GC) remains unknown. This study explored that BAP31 was upregulated in GC tissues and high expression indicated poor survival of GC patients. BAP31 knockdown inhibited cell growth and induced G1/S arrest. Moreover, BAP31 attenuation increased the lipid peroxidation level of the membrane and facilitated cellular ferroptosis. Mechanistically, BAP31 regulated cell proliferation and ferroptosis by directly binding to VDAC1 and affected VDAC1 oligomerization and polyubiquitination. HNF4A was bound to BAP31 at the promoter and increased its transcription. Furthermore, knockdown of BAP31 inclined to make GC cells vulnerable to 5-FU and ferroptosis inducer, erastin, in vivo and in vitro. Our work suggests that BAP31 may serve as prognostic factor for gastric cancer and act as potential therapeutic strategy for gastric cancer.

Targeting KRASG12V mutations with HLA class II-restricted TCR for the immunotherapy in solid tumors.

KRAS mutation is a significant driving factor of tumor, and KRASG12V mutation has the highest incidence in solid tumors such as pancreatic cancer and colorectal cancer. Thus, KRASG12V neoantigen-specific TCR-engineered T cells could be a promising cancer treatment approach for pancreatic cancer. Previous studies had reported that KRASG12V-reactive TCRs originated from patients' TILs could recognized KRASG12V neoantigen presented by specific HLA subtypes and remove tumor persistently in vitro and in vivo. However, TCR drugs are different from antibody drugs in that they are HLA-restricted. The different ethnic distribution of HLA greatly limits the applicability of TCR drugs in Chinese population. In this study, we have identified a KRASG12V-specific TCR which recognized classII MHC from a colorectal cancer patient. Interestingly, we observed that KRASG12V-specific TCR-engineered CD4+ T cells, not CD8+ T cells, demonstrated significant efficacy in vitro and in xenograft mouse model, exhibiting stable expression and targeting specificity of TCR when co-cultured with APCs presenting KRASG12V peptides. TCR-engineered CD4+ T cells were co-cultured with APCs loaded with neoantigen, and then HLA subtypes were identified by the secretion of IFN-γ. Collectively, our data suggest that TCR-engineered CD4+ T cells can be used to target KRASG12V mutation presented by HLA-DPB1*03:01 and DPB1*14:01, which provide a high population coverage and are more suitable for the clinical transformation for Chinese, and mediate tumor killing effect like CD8+ T cells. This TCR hold promise for precision therapy in immunotherapy of solid tumors as an attractive candidate.

Specific human leukocyte antigen class I genotypes predict prognosis in resected pancreatic adenocarcinoma: a retrospective cohort study.

BACKGROUND: Patients with resected pancreatic adenocarcinoma (PAAD) often experience short-term relapse and dismal survival, suggesting an urgent need to develop predictive and/or prognostic biomarkers for these populations. Given the potential associations of the human leukocyte antigen class I ( HLA -I) genotype with oncogenic mutational profile and immunotherapy efficacy, we aimed to assess whether differential HLA -I genotype could predict the postoperative outcomes in resected PAAD patients. MATERIALS AND METHODS: HLA -I ( A , B , and C ) genotyping and somatic variants of 608 Chinese PAAD patients were determined by targeted next-generation sequencing of matched blood cells and tumor tissues. HLA - A / B alleles were classified with the available definition of 12 supertypes. The Kaplan-Meier curves of disease-free survival (DFS) and multivariable Cox proportional-hazards regression analyses were performed to determine the survival difference in 226 selected patients with radical resection. Early-stage (I-II) patients constituted the majority (82%, 185/226) and some stage I-II individuals with high-quality tumor samples were analyzed by RNA-sequencing to examine immunophenotypes. RESULTS: Patients with HLA-A02 + B62 + B44 - had significantly shorter DFS (median, 239 vs. 410 days; hazard ratio=1.65, P =0.0189) than patients without this genotype. Notably, stage I-II patients carrying HLA-A02 + B62 + B44 - had sharply shorter DFS than those without HLA-A02 + B62 + B44 - (median, 237 vs. 427 days; hazard ratio=1.85, P =0.007). Multivariate analysis revealed that HLA-A02 + B62 + B44 - was associated with significantly inferior DFS ( P =0.014) in stage I-II patients but not in stage III patients. Mechanistically, HLA-A02 + B62 + B44 - patients were associated with a high rate of KRAS G12D and TP53 mutations, lower HLA-A expression, and less inflamed T-cell infiltration. CONCLUSION: The current results suggest that a specific combination of germline HLA-A02/B62/B44 supertype, HLA-A02 + B62 + B44 - , was a potential predictor for DFS in early-stage PAAD patients after surgery.

Identification of copy number variation-driven molecular subtypes informative for prognosis and treatment in pancreatic adenocarcinoma of a Chinese cohort.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is one of the most lethal carcinomas, and the current histopathological classifications are of limited use in clinical decision-making. There is an unmet need to identify new biomarkers for prognosis-informative molecular subtyping and ultimately for precision medicine. METHODS: We profiled genomic alterations for 608 PAAD patients in a Chinese cohort, including somatic mutations, pathogenic germline variants and copy number variations (CNV). Using the CNV information, we performed unsupervised consensus clustering of these patients, differential CNV analysis and functional/pathway enrichment analysis. Cox regression was conducted for progression-free survival analysis, the elastic net algorithm used for prognostic model construction, and rank-based gene set enrichment analysis for exploring tumor microenvironments. FINDINGS: Our data did not support prognostic value of point mutations in either highly mutated genes (such as KRAS, TP53, CDKN2A and SMAD4) or homologous recombination repair genes. Instead, associated with worse prognosis were amplified genes involved in DNA repair and receptor tyrosine kinase (RTK) related signalings. Motivated by this observation, we categorized patients into four molecular subtypes (namely repair-deficient, proliferation-active, repair-proficient and repair-enhanced) that differed in prognosis, and also constructed a prognostic model that can stratify patients with low or high risk of relapse. Finally, we analyzed publicly available datasets, not only reinforcing the prognostic value of our identified genes in DNA repair and RTK related signalings, but also identifying tumor microenvironment correlates with prognostic risks. INTERPRETATION: Together with the evidence from genomic footprint analysis, we suggest that repair-deficient and proliferation-active subtypes are better suited for DNA damage therapies, while immunotherapy is highly recommended for repair-proficient and repair-enhanced subtypes. Our results represent a significant step in molecular subtyping, diagnosis and management for PAAD patients. FUNDING: This work was supported by the National Natural Science Foundation of China (grant numbers 81470894, 81502695, 81672325, 81871906, 82073326, 82103482 and 32170663), the Shanghai Sailing Program (grant number 20YF1426900), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (awarded to H.F.).

A Novel c-MET-Targeting Antibody-Drug Conjugate for Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-associated death in the United States and has a 5-year survival rate of <4%. Although much effort has been invested in the research and development of pancreatic cancer drugs over the past 30 years, due to the lack of effective targetable carcinogenic drivers, no new targeted therapies that can improve patient prognosis have been approved for clinical use. SHR-A1403 is a new c-mesenchymal-epithelial transition factor (c-MET) antibody-drug conjugate that can be used for the targeted treatment of PDAC with high c-MET expression. This study reports for the first time the application prospects of SHR-A1403 in preclinical models of PDAC. SHR-A1403 significantly inhibited the proliferation, migration, and invasion of pancreatic cancer cells and induced cell cycle arrest and apoptosis. These changes were caused by inhibition of intracellular cholesterol biosynthesis by SHR-A1403. Therefore, targeting c-MET through SHR-A1403 showed strong preclinical anti-tumour efficacy in pancreatic cancer. Our work suggests the potential application of c-MET-targeted antibody-drug conjugate treatment for PDAC in clinical practise.

miR-934 as a Prognostic Marker Facilitates Cell Proliferation and Migration of Pancreatic Tumor by Targeting PROX1.

BACKGROUND: Pancreatic cancer is an extremely lethal digestive cancer with late diagnosis and poor prognosis. miR-934 has been reported to serve as an oncogene in multiple cancers, such as ovarian cancer and bladder cancer. However, its role in pancreatic cancer remains undiscovered. MATERIALS AND METHODS: The expression data of miR-934 were obtained from the Gene Expression Omnibus database and from our own patient samples. The clinicopathological data and corresponding follow-up data were retrieved from The Cancer Genome Atlas database. CCK8 and colony formation assays were conducted to measure cell proliferation capacity in vitro. Wound healing and transwell assays were performed to detect the migration ability of pancreatic cancer cell. RESULTS: We found that miR-934 was significantly upregulated in pancreatic tumor samples and cell lines. The expression of miR-934 was related to pathological stages. Upregulated miR-934 was associated with poor prognosis in patients with pancreatic cancer. Mir-934 inhibition reduced, while overexpression promoted, cell proliferation and migration. Mechanically, we found  miR-934 could directly bind to 3'-UTR of PROX1 leading to mRNA derogation. Furthermore, increased cell proliferation and migration caused by miR-934 overexpression could be reversed by forced PROX1 expression. CONCLUSION: miR-934 is an oncogene in pancreatic cancer and could serve as a prognosis indicator for patients with pancreatic cancer, suggesting that miR-934 is a promising therapeutic target for pancreatic cancer.