Background: Cancer-associated fibroblasts (CAFs) are a vital constituent of the tumor microenvironment (TME) and have several activities, but the effect of CAF heterogeneity on the molecular features and clinical outcomes of pancreatic ductal adenocarcinoma (PDAC) remains unknown. Methods: An algorithm “scFrac” based on single-cell sequencing data from the Gene Expression Omnibus was introduced to emulate the enrichment of CAF subtypes in a TCGA-PDAC cohort and their prognostic influence, and confirmed by an external validation group (66 patients with PDAC) with multiplex immunohistochemistry staining. A comprehensive analysis including metabolic profile and transcription factor regulon activity was carried out among CAF subtypes. Results: Three distinct CAF populations were confirmed: myofibroblast (myCAF), inflammatory CAF (iCAF), and antigen-presenting CAF (apCAF). These subtypes expressed distinct metabolic profiles and transcriptional regulon activity. KEGG pathway annotation demonstrated that complement and coagulation cascades, as well as cytokine-cytokine receptor interaction were dominant in iCAFs, and pathways related to focal adhesion, and ECM-receptor interaction showed dominance in myCAFs, while antigen processing and presentation were the top enriched pathways in apCAFs. iCAFs trended to glycolysis with CREB3L1, EGR2 and SOX4 activation, whereas myCAFs depend on the tricarboxylic acid cycle and its derivatives with NRF2, CEBPD and YBX1 activation. iCAF is a protective factor associated with an inflammatory phenotype, but myCAF is an important factor in the poor prognosis of PDAC. Conclusions: We identified distinct molecular characteristics of 3 CAF subtypes in PDAC and plotted their metabolism profile. We introduced a novel algorism, scFrac, for exploring how CAF subgroups dysregulate cancer biology, and also shed a new therapeutic light on targeting the CAF subtype in TME.

Pancreatic cancer is a deadly disease that is largely resistant to immunotherapy, in part because of the accumulation of immunosuppressive cells in the tumor microenvironment (TME). Much evidence suggests that tumor-derived exosomes (TDE) contribute to the immunosuppressive activity mediated by myeloid-derived suppressor cells (MDSC) within the pancreatic cancer TME. However, the underlying mechanisms remain elusive. Herein, we report that macrophage migration inhibitory factor (MIF) in TDEs has a key role in inducing MDSC formation in pancreatic cancer. We identified MIF in both human and murine pancreatic cancer–derived exosomes. Upon specific shRNA-mediated knockdown of MIF, the ability of pancreatic cancer–derived exosomes to promote MDSC differentiation was abrogated. This phenotype was rescued by reexpression of the wild-type form of MIF rather than a tautomerase-null mutant or a thiol-protein oxidoreductase-null mutant, indicating that both MIF enzyme activity sites play a role in exosome-induced MDSC formation in pancreatic cancer. RNA sequencing data indicated that MIF tautomerase regulated the expression of genes required for MDSC differentiation, recruitment, and activation. We therefore developed a MIF tautomerase inhibitor, IPG1576. The inhibitor effectively inhibited exosome-induced MDSC differentiation in vitro and reduced tumor growth in an orthotopic pancreatic cancer model, which was associated with decreased numbers of MDSCs and increased infiltration of CD8+ T cells in the TME. Collectively, our findings highlight a pivotal role for MIF in exosome-induced MDSC differentiation in pancreatic cancer and underscore the potential of MIF tautomerase inhibitors to reverse the immunosuppressive pancreatic cancer microenvironment, thereby augmenting anticancer immune responses.

Driver mutations can contribute to the initial processes of cancer, and their identification is crucial for understanding tumorigenesis as well as for molecular drug discovery and development. Allostery regulates protein function away from the functional regions at an allosteric site. In addition to the known effects of mutations around functional sites, mutations at allosteric sites have been associated with protein structure, dynamics, and energy communication. As a result, identifying driver mutations at allosteric sites will be beneficial for deciphering the mechanisms of cancer and developing allosteric drugs. In this study, we provided a platform called DeepAlloDriver to predict driver mutations using a deep learning method that exhibited >93% accuracy and precision. Using this server, we found that a missense mutation in RRAS2 (Gln72 to Leu) might serve as an allosteric driver of tumorigenesis, revealing the mechanism of the mutation in knock-in mice and cancer patients. Overall, DeepAlloDriver would facilitate the elucidation of the mechanisms underlying cancer progression and help prioritize cancer therapeutic targets. The web server is freely available at: https://mdl.shsmu.edu.cn/DeepAlloDriver.

Pancreatic Adenocarcinoma (PDAC) is one of the most deadly malignant tumors worldwide. A variety of mechanisms are involved in PDAC biological behaviors, of which, the mechanisms of immune escape may be a pivotal hallmark. HLA-G is a tolerant molecule implicated in tumor escape and serves as a prognostic biomarker in tumors. Our study evaluated the expression of HLA-G in PDAC and explored its clinical significance. In a cohort of 122 PDAC patients, 78 patents (63.9%) exhibited high level of HLA-G tumor tissues. Multivariate analysis suggested that HLA-G level was an independent predictor for OS (HR = 3.894, 95% CI = 2.380-6.370, p <0.001). High level of HLA-G significantly correlated with PDAC aggressive features, such as more advanced stage (TNM Stage II) (p<0.001), extrapancreatic infiltration (T3 stage) (p<0.001), lymph node involvement (p=0.019) and poor differentiation (p=0.010). In western blot analysis, almost all of the tumor cell lines (5/6) expressed high levels of HLA-G. In ELISA analysis, the level of plasma sHLA-G in PDAC patients were significantly increased than in healthy control (P=0.0037). Further analysis revealed the level of sHLA-G inversely related to numbers of peripheral activated T cells (CD8+CD28+ T cells), which may indicate that sHLA-G inactivates T cell responses resulting in tumor immune escape. In conclusion, tumor-derived HLA-G may indicate the mechanism of immune escape and impaired PDAC clinical outcome, especially in early-stage patients, which may also be a potential therapeutic target.

N-myc downstream-regulated gene 1 (NDRG1) is known as tumor/metastasis suppressor in a variety of cancers including pancreas, being involved in angiogenesis, cancer growth and metastasis. However, the precise molecular mechanism how NDRG1 exerts its inhibitory function in pancreatic cancer remains unclear. In this investigation, we demonstrated that K-Ras plays a vital role in modulating NDRG1 protein level in PDAC cancer cells in vitro, which is mediated through ERK signaling. Noteworthy, K-Ras downstream Akt/mTOR signaling is inhibited upon NDRG1 overexpression, resulting in decease of HIF1α level. Moreover, NDRG1 has a unique role in modulating cancer metabolism of pancreatic ductal adenocarcinoma (PDAC). The mechanism accounting for NDRG1 in modulating aerobic glycolysis, at least partly, relied on its regulation of glycolysis genes including GLUT1, HK2, LDHA and PDK1. Additionally, NDRG1 is shown to suppress the activity of HIF1α, which is responsible for regulation of glycolysis enzymes. The current study is the first to elucidate a unique facet of the potent tumor/metastasis suppressor NDRG1 in the regulation of PDAC glycolysis, leading to important insights into the mechanism by which NDRG1 exert inhibitory function in PDAC.

Background: Cell adhesion molecule cluster of differentiation 44 (CD44) plays a significant role in cancer cell local invasion, intravasation, migration, and the establishment of metastatic lesions. However, little is known about the underlying mechanism of how CD44 regulates hepatocellular carcinoma (HCC) extrahepatic metastasis (EHM). Methods: The expression of CD44 in HCC tissues and cell lines was detected through western blot and immunohistochemistry (IHC). Through gain- and loss-of-function assays, we examined the oncogenic roles of CD44 in regulating HCC cell growth and metastasis in vitro and in vivo. To identify the potential mechanism, we employed quantitative real-time polymerase chain reaction, and western blot. Results: In this study, CD44 was highly expressed in HCC cells and HCC-patient specimens that exhibited high malignancy potential. The overall survival (OS) was worse and the cumulative recurrence rate was higher in HCC patients with CD44 overexpression than those with low levels of CD44 expression. Our in-vitro and in-vivo experiments showed that CD44 downregulation reduced HCC cell colony formation, migration, and invasion, and HCC tumor growth and metastasis, and that the pro-metastasis effect of CD44 was mediated by the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling-chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) axis. The reported capacity of CD44 to induce CXCR4 expression and increase the propensity of tumors to invade and metastasize to distant organs is consistent with the aggressive clinical characteristics of HCCs. Conclusions: CD44 could represent a future therapeutic target for EHM.