SMAD4 endows TGF-ß1-induced highly invasive tumor cells with ferroptosis vulnerability in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy prone to recurrence and metastasis. Studies show that tumor cells with increased invasive and metastatic potential are more likely to undergo ferroptosis. SMAD4 is a critical molecule in the transforming growth factor ß (TGF-ß) pathway, which affects the TGF-ß-induced epithelial-mesenchymal transition (EMT) status. SMAD4 loss is observed in more than half of patients with PDAC. In this study, we investigated whether SMAD4-positive PDAC cells were prone to ferroptosis because of their high invasiveness. We showed that SMAD4 status almost determined the orientation of transforming growth factor ß1 (TGF-ß1)-induced EMT via the SMAD4-dependent canonical pathway in PDAC, which altered ferroptosis vulnerability. We identified glutathione peroxidase 4 (GPX4), which inhibited ferroptosis, as a SMAD4 down-regulated gene by RNA sequencing. We found that SMAD4 bound to the promoter of GPX4 and decreased GPX4 transcription in PDAC. Furthermore, TGF-ß1-induced high invasiveness enhanced sensitivity of SMAD4-positive organoids and pancreas xenograft models to the ferroptosis inducer RAS-selective lethal 3 (RSL3). Moreover, SMAD4 enhanced the cytotoxic effect of gemcitabine combined with RSL3 in highly invasive PDAC cells. This study provides new ideas for the treatment of PDAC, especially SMAD4-positive PDAC.

NMI promotes tumor progression and gemcitabine resistance in pancreatic cancer via STAT3-IFIT3 axis.

N-myc and STAT interactor (NMI) has been reported to interact with several transcription factors, including STATs family, c-Myc, N-Myc, and BRCA1, to indirectly affect transcription events and participate in multiple cellular processes. However, its function in pancreatic ductal adenocarcinoma (PDAC) has seldom been studied. In this study, we investigated the regulation of NMI on PDAC progression and uncovered the underlying molecular mechanisms. We found that NMI expression was significantly upregulated in PDAC and high NMI expression was related to a worse patient survival. Cell proliferation and migration assay, including cell viability, transwell assay, wound healing, and subcutaneous mouse model were utilized to confirm the function of NMI in PDAC progression. Downregulation of NMI abrogates tumor progression of PDAC both in vitro and in vivo. RNA sequencing was utilized to identify the downstream molecules of NMI and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) was confirmed to be regulated by NMI in both mRNA and protein level. The binding function of NMI to STAT3 was essential in regulating the IFIT3 expression. Moreover, the NMI/STAT3-IFIT3 axis was identified to markedly facilitate the gemcitabine resistance in PDAC cells.

Reconsidering the role of prophylactic pancreaticojejunostomy in pancreatic enucleation: balancing the benefits and risks.

BACKGROUND: The incidence of postoperative pancreatic fistula (POPF) following enucleation is high, and prophylactic pancreaticojejunostomy (PPJ) is frequently performed. Minimally invasive enucleation (MEN) has been demonstrated to be safe and feasible, leaving most enucleation wounds exposed. METHODS: The clinical data of 40 patients who underwent open enucleation with PPJ at our center between 2012 and 2021 were compared with those of 80 patients who underwent MEN. RESULTS: The MEN group had better outcomes than the PPJ group in terms of intraoperative bleeding (50.0 versus 100.0 mL), postoperative semi-liquid diet recovery (2.0 versus 5.0 days), and postoperative length of stay (7.7 versus 12.5 days). While the MEN group had higher rates of complex enucleation (60.0% versus 40.0%), main pancreatic duct repair (32.5% versus 10.0%), discharge with drains (48.8% versus 25.0%), and grade B POPFs (47.5% versus 17.5%). Both surgical methods effectively preserved pancreatic function; however, two patients in the PPJ group experienced severe haemorrhaging and died. Additionally, during the follow-up period, gastrointestinal bleeding was found and discomfort in the surgical area was reported. CONCLUSION: Pancreatic enucleation combined with PPJ should be avoided, and although a biochemical or grade B POPF may develop after MEN, it can be compensated for by preserving pancreatic function and ensuring a good long-term quality of life in the patients.

Development and validation of CT-based radiomics deep learning signatures to predict lymph node metastasis in non-functional pancreatic neuroendocrine tumors: a multicohort study.

Background: Lymph node status is an important factor for the patients with non-functional pancreatic neuroendocrine tumors (NF-PanNETs) with respect to the surgical methods, prognosis, recurrence. Our aim is to develop and validate a combination model based on contrast-enhanced CT images to predict the lymph node metastasis (LNM) in NF-PanNETs. Methods: Retrospective data were gathered for 320 patients with NF-PanNETs who underwent curative pancreatic resection and CT imaging at two institutions (Center 1, n = 236 and Center 2, n = 84) between January 2010 and March 2022. RDPs (Radiomics deep learning signature) were developed based on ten machine-learning techniques. These signatures were integrated with the clinicopathological factors into a nomogram for clinical applications. The evaluation of the model's performance was conducted through the metrics of the area under the curve (AUC). Findings: The RDPs showed excellent performance in both centers with a high AUC for predicting LNM and disease-free survival (DFS) in Center 1 (AUC, 0.88; 95% CI: 0.84-0.92; DFS, p < 0.05) and Center 2 (AUC, 0.91; 95% CI: 0.85-0.97; DFS, p < 0.05). The clinical factors of vascular invasion, perineural invasion, and tumor grade were associated with LNM (p < 0.05). The combination nomogram showed better prediction capability for LNM (AUC, 0.93; 95% CI: 0.89-0.96). Notably, our model maintained a satisfactory predictive ability for tumors at the 2-cm threshold, demonstrating its effectiveness across different tumor sizes in Center 1 (≤2 cm: AUC, 0.90 and >2 cm: AUC, 0.86) and Center 2 (≤2 cm: AUC, 0.93 and >2 cm: AUC, 0.91). Interpretation: Our RDPs may have the potential to preoperatively predict LNM in NF-PanNETs, address the insufficiency of clinical guidelines concerning the 2-cm threshold for tumor lymph node dissection, and provide precise therapeutic strategies. Funding: This work was supported by JSPS KAKENHI Grant Number JP22K20814; the Rare Tumor Research Special Project of the National Natural Science Foundation of China (82141104) and Clinical Research Special Project of Shanghai Municipal Health Commission (202340123).

Minimally invasive enucleation of pancreatic tumors: The main pancreatic duct is no longer a restricted area.

Background: Tumors involving the main pancreatic duct (MPD) used to be a contraindication for enucleation. Methods: Clinical data of consecutive patients with pancreatic tumors who received laparoscopic or robotic enucleation (LEN or REN) between January 2019 and December 2021 at Fudan University Shanghai Cancer Center were analyzed. Results: Ninety-six patients were included in the analysis, with 55 in the LEN group and 41 in the REN group, and no conversion to laparotomy. Most tumors were located in the head of pancreas (71.9 %). The tumor diameter (3.1 vs. 1.9 cm) was larger, and more cystic tumors (92.7 % vs. 56.4 %) and more tumors involving the MPD (34.1 % vs. 3.6 %) were observed in the REN group. MPD support tube insertion was performed in 15 cases, with 11 in the REN group and 4 in the LEN group. The incidence of biochemical and grade B postoperative pancreatic fistula (POPF) was both 46.9 %, and no grade C POPF occurred. Among the 45 patients with grade B POPF, 28 cases (62.2 %) were due to carrying drainage tube >3 weeks without additional treatment, and only 4 cases required invasive treatment. For patients with MPD support tube implantation (n = 15), support tube fall-offs were observed in 12 cases, 2 patients had MPD dilatation, and no MPD stricture, stone formation or pancreatic atrophy was observed during follow-up. Conclusions: The incidence of POPF was high but still controllable without serious complications after minimally invasive enucleation. The MPD is no longer a restricted area, and the robotic system has advantages in handling complex enucleations.

Hernandezine induces autophagic cell death in human pancreatic cancer cells via activation of the ROS/AMPK signaling pathway.

Hernandezine (Her) is a bisbenzylisoquinoline alkaloid extracted from the traditional Chinese herbal medicine Thalictrum glandulosissimum. Evidence shows that Her is a natural agonist of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and induces apoptosis and autophagy in tumor cells. In this study, we investigated the role of autophagy in Her-induced cell death in human pancreatic cancer cell lines. We showed that Her dose-dependently suppressed cell proliferation, promoted autophagy and induced autophagic death in pancreatic ductal adenocarcinoma (PDAC) cell lines Capan-1 and SW1990. The IC50 values of Her in inhibition of Capan-1 and SW1990 cells were 47.7 µM and 40.1 µM, respectively. Immunoblotting showed that Her (1-40 µM) promoted the conversion of LC3-I to LC3-II, and Her exerted concentration-dependent and time-dependent effects on autophagy activation in PDAC cells. In transmission electron microscopy and fluorescence image analysis, we found that autophagic vacuoles were significantly increased in Her-treated cells. Knockdown of ATG5, a key gene in the autophagy pathway, alleviated the activation of autophagy by Her. These results demonstrated that Her induced autophagy in PDAC cells. Intensely activated autophagy could promote cell death. The autophagy inhibitors, BafA1 and HCQ significantly inhibited Her-induced cell death, implying that Her induced autophagic cell death in PDAC cells. Moreover, we showed that Her activated autophagy by increasing the phosphorylation of AMPK and decreasing the phosphorylation of mTOR/p70S6K. Knockdown of AMPKα relieves the autophagic cell death induced by Her. Furthermore, Her concentration-dependently enhanced reactive oxygen species (ROS) generation in PDAC cells. Antioxidants could reduce the phosphorylation of AMPK and suppress autophagic cell death induced by Her. Our study provides evidence for the development of Her as a therapeutic agent for the treatment of pancreatic cancer.

Maximum Value on Arterial Phase Computed Tomography Predicts Prognosis and Treatment Efficacy of Sunitinib for Pancreatic Neuroendocrine Tumours.

PURPOSE: This study was designed to assess the computed tomography maximum (CTmax) value on pretherapeutic arterial phase computed tomography (APCT) images to predict pancreatic neuroendocrine tumours (pNETs) recurrence and clarify its role in predicting the outcome of tumour therapy. METHODS: This retrospective study enrolled 250 surgical patients and 24 nonsurgical patients with sunitinib-based treatment in our hospital from 2008 to 2019. CT images were assessed, the maximum value was defined as "CTmax," and recurrence-free survival (RFS) or progression-free survival (PFS) was compared between a high-CTmax group and a low-CTmax group among patients who underwent surgical resection or nonsurgical, sunitinib-based treatment according to the CTmax cutoff value. RESULTS: In ROC curve analysis, a CTmax of 108 Hounsfield units, as the cutoff value, achieved an AUC of 0.796 in predicting recurrence. Compared with the low-CTmax group, the high-CTmax group had a longer RFS (p < 0.001). Low CTmax was identified as an independent factor for RFS (p < 0.001) in multivariate analysis; these results were confirmed using the internal validation set. The CTmax value was significantly correlated with the microvascular density (MVD) value (p < 0.001) and the vascular endothelial growth factor receptor 2 (VEGFR2) score (p < 0.001). Furthermore, the high-CTmax group had a better PFS than the low-CTmax group among the sunitinib treatment group (p = 0.007). CONCLUSIONS: The tumour CTmax on APCT might be a potential and independent indicator for predicting recurrence in patients who have undergone surgical resection and assessing the efficacy of sunitinib for patients with advanced metastatic pNETs.

The Deep Ocean's Carbon Exhaust.

The deep ocean releases large amounts of old, pre-industrial carbon dioxide (CO2) to the atmosphere through upwelling in the Southern Ocean, which counters the marine carbon uptake occurring elsewhere. This Southern Ocean CO2 release is relevant to the global climate because its changes could alter atmospheric CO2 levels on long time scales, and also affects the present-day potential of the Southern Ocean to take up anthropogenic CO2. Here, year-round profiling float measurements show that this CO2 release arises from a zonal band of upwelling waters between the Subantarctic Front and wintertime sea-ice edge. This band of high CO2 subsurface water coincides with the outcropping of the 27.8 kg m-3 isoneutral density surface that characterizes Indo-Pacific Deep Water (IPDW). It has a potential partial pressure of CO2 exceeding current atmospheric CO2 levels (∆PCO2) by 175 ± 32 µatm. Ship-based measurements reveal that IPDW exhibits a distinct ∆PCO2 maximum in the ocean, which is set by remineralization of organic carbon and originates from the northern Pacific and Indian Ocean basins. Below this IPDW layer, the carbon content increases downwards, whereas ∆PCO2 decreases. Most of this vertical ∆PCO2 decline results from decreasing temperatures and increasing alkalinity due to an increased fraction of calcium carbonate dissolution. These two factors limit the CO2 outgassing from the high-carbon content deep waters on more southerly surface outcrops. Our results imply that the response of Southern Ocean CO2 fluxes to possible future changes in upwelling are sensitive to the subsurface carbon chemistry set by the vertical remineralization and dissolution profiles.

The vertebrate- and testis- specific transmembrane protein C11ORF94 plays a critical role in sperm-oocyte membrane binding.

Sperm-oocyte membrane fusion is necessary for mammalian fertilization. The factors that determine the fusion of sperm with oocytes are largely unknown. So far, spermatozoon factor IZUMO1 and the IZUMO1 counter-receptor JUNO on the oocyte membrane has been identified as a protein requiring fusion. Some sperm membrane proteins such as FIMP, SPACA6 and TEME95, have been proved not to directly regulate fusion, but their knockout will affect the fusion process of sperm and oocytes. Here, we identified a novel gene C11orf94 encoding a testicular-specific small transmembrane protein that emerges in vertebrates likely acquired via horizontal gene transfer from bacteria and plays an indispensable role in sperm-oocyte binding. We demonstrated that the deletion of C11orf94 dramatically decreased male fertility in mice. Sperm from C11orf94-deficient mice could pass through the zona pellucida, but failed to bind to the oocyte membrane, thus accumulating in the perivitelline space. In consistence, when the sperm of C11orf94-deficient mice were microinjected into the oocyte cytoplasm, fertilized oocytes were obtained and developed normally to blastocysts. Proteomics analysis revealed that C11orf94 influenced the expression of multiple gene products known to be indispensable for sperm-oocyte binding and fusion, including IZUMO1, EQTN and CRISP1. Thus, our study indicated that C11ORF94 is a vertebrate- and testis-specific small transmembrane protein that plays a critical role in sperm binding to the oolemma.

Differential MC5R loss in whales and manatees reveals convergent evolution to the marine environment.

Melanocortin 5 receptor (MC5R), which is expressed in the terminally differentiated sebaceous gland, is a G protein-coupled receptor (GPCR). MC5R exists mostly in mammals but is completely lost in whales; only the relic of MC5R can be detected in manatees, and phenotypically, they have lost sebaceous glands. Interestingly, whales and manatees are both aquatic mammals but have no immediate common ancestors. The loss of MC5R and sebaceous glands in whales and manatees is likely to be a result of convergent evolution. Here, we find that MC5R in whales and manatees are lost by two different mechanisms. Homologous recombination of MC5R in manatees and the insertion of reverse transcriptase in whales lead to the gene loss, respectively. On one hand, in manatees, there are two "TTATC" sequences flanking MC5R, and homologous recombination of the segments between the two "TTATC" sequences resulted in the partial loss of the sequence of MC5R. On the other hand, in whales, reverse transcriptase inserts between MC2R and RNMT on the chromosome led to the loss of MC5R. Based on these two different mechanisms for gene loss in whales and manatees, we finally concluded that MC5R loss might be the result of convergent evolution to the marine environment, and we explored the impact on biological function that is significant to environmental adaptation.

Whole-genome sequencing identifies rare missense variants of WNT16 and ERVW-1 causing the systemic lupus erythematosus.

Systemic lupus erythematosus (SLE, OMIM 152700) is a rare autoimmune disease with high heritability that affects ~0.1% of the population. Previous studies have revealed several common variants with small effects in European and East Asian SLE patients. However, there is still no rare variant study on Chinese SLE patients using the whole-genome sequencing technology (WGS). Here, we designed a family based WGS study to identify novel rare variants with large effects. Based on large-scale allele frequency data from the gnomAD database, we identified rare protein-coding gene variants with disruptive and sequence-altering impacts in SLE patients. We found that the burden of rare variants was significantly higher than that of common variants in patients, suggesting a larger effect of rare variants on the SLE pathogenesis. We identified the pathogenic risk of rare missense variants with significant odds ratios (p < 0.05) in two genes, including WNT16 (NC_000007.14:g.121329757G > C, NP_057171.2:p.(Ala86Pro) and 7 g.121329760G > C, NP_057171.2:p.(Ala87Pro)), which explains five out of seven patients covering all three families but are absent from all controls, and ERVW-1 (NC_000007.14:g.92469882A > G, NP_001124397.1:p.(Leu167Pro), rs74545114; NC_000007.14:g.92469907G > A, NP_001124397.1:p.(Arg159Cys), rs201142302; NC_000007.14:g.92469919G > A, NP_001124397.1:p.(His155Tyr), rs199552228), which explains the other two patients. None of these variants were identified in any of the controls. These associations are supported by known gene expression studies in SLE patients based on literature review. We further tested the wild and mutant types using the luciferase assays and qPCR in cells. We found that WNT16 can activate the canonical Wnt/ß-catenin pathway while the mutant cannot. Additionally, the wild ERVW-1 expression can be significantly up-regulated by cAMP while the mutant cannot. Our study provides the first direct genetic and in vitro evidence for the pathogenic risk of mutant WNT16 and ERVW-1, which may facilitate the design of precision therapy for SLE.

High-speed rail model reveals the gene tandem amplification mediated by short repeated sequence in eukaryote.

The occurrence of gene duplication/amplification (GDA) provide potential material for adaptive evolution with environmental stress. Several molecular models have been proposed to explain GDA, recombination via short stretches of sequence similarity plays a crucial role. By screening genomes for such events, we propose a "SRS (short repeated sequence) *N + unit + SRS*N" amplified unit under USCE (unequal sister-chromatid exchange) for tandem amplification mediated by SRS with different repeat numbers in eukaryotes. The amplified units identified from 2131 well-organized amplification events that generate multi gene/element copy amplified with subsequent adaptive evolution in the respective species. Genomic data we analyzed showed dynamic changes among related species or subspecies or plants from different ecotypes/strains. This study clarifies the characteristics of variable copy number SRS on both sides of amplified unit under USCE mechanism, to explain well-organized gene tandem amplification under environmental stress mediated by SRS in all eukaryotes.

MEN1 promotes ferroptosis by inhibiting mTOR-SCD1 axis in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumor (pNET) is the second most common malignant tumors of the pancreas. Multiple endocrine neoplasia 1 ( MEN1) is the most frequently mutated gene in pNETs and MEN1-encoded protein, menin, is a scaffold protein that interacts with transcription factors and chromatin-modifying proteins to regulate various signaling pathways. However, the role of MEN1 in lipid metabolism has not been studied in pNETs. In this study, we perform targeted metabolomics analysis and find that MEN1 promotes the generation and oxidation of polyunsaturated fat acids (PUFAs). Meanwhile lipid peroxidation is a hallmark of ferroptosis, and we confirm that MEN1 promotes ferroptosis by inhibiting the activation of mTOR signaling which is the central hub of metabolism. We show that stearoyl-coA desaturase (SCD1) is the downstream of MEN1-mTOR signaling and oleic acid (OA), a metabolite of SCD1, recues the lipid peroxidation caused by MEN1 overexpression. The negative correlation between MEN1 and SCD1 is further verified in clinical specimens. Furthermore, we find that BON-1 and QGP-1 cells with MEN1 overexpression are more sensitive to everolimus, a widely used drug in pNETs that targets mTOR signaling. In addition, combined use everolimus with ferroptosis inducer, RSL3, possesses a more powerful ability to kill cells, which may provide a new strategy for the comprehensive therapy of pNETs.

ALDOA inhibits cell cycle arrest induced by DNA damage via the ATM-PLK1 pathway in pancreatic cancer cells.

BACKGROUND: ALDOA is a glycolytic enzyme found mainly in developing embryos, adult muscle and various malignant tumours, including pancreatic tumours. Our previous study revealed that ALDOA, an oncogene, can promote the proliferation and metastasis of pancreatic tumours. Furthermore, ALDOA could predict poor prognosis in patients with pancreatic tumours. METHODS: IHC analysis of PDAC tissues was conducted. Western blotting, PCR, cellular IF experiments and cell cycle assessment were conducted utilizing cell lines. GSEA and KEGG pathway analysis were used to identify potential downstream pathways. RESULTS: To explore the effects of ALDOA on the occurrence and development of pancreatic tumours, we analysed the RNA sequencing results and found that ALDOA could inhibit the DDR. Under normal circumstances, when DNA is damaged, initiation of the DDR causes cell cycle arrest, DNA repair or cell apoptosis. Further experiments showed that ALDOA could inhibit DNA repair and reverse cell cycle arrest induced by DNA damage so that DNA damage persisted to promote the occurrence and progression of cancer. CONCLUSIONS: Regarding the molecular mechanism, we found that ALDOA inhibited the DDR and improved activation of the cell cycle checkpoint PLK1 by suppressing ATM, which promotes tumour cell progression. Consequently, ALDOA has a profound effect on pancreatic cancer development.

Organoid model: A new hope for pancreatic cancer treatment?

Pancreatic cancer is a rapidly progressing disease with a poor prognosis. We still have many questions about the pathogenesis, early diagnosis and precise treatment of this disease. Organoids, a rapidly emerging technology, can simulate the characteristics of pancreatic tumors. Using the organoid model of pancreatic cancer, we can study and explore the characteristics of pancreatic cancer, thereby effectively guiding clinical practice and improving patient prognosis. This review introduces the development of organoids, comparisons of organoids with other preclinical models and the status of organoids in basic research and clinical applications for pancreatic cancer.

Evolution of a splice variant that acts as an endogenous antagonist of the original INSL3 in primates.

Alu sequences are the most abundant repetitive elements in the human genome, and have proliferated to more than one million copies in the human genome. Primate-specific Alu sequences account for ~10% of the human genome, and their spread within the genome has the potential to generate new exons. The new exons produced by Alu elements appear in various primate genes, and their functions have been elucidated. Here, we identified a new exon in the insulin-like 3 gene (INSL3), which evolved ~50 million years ago, and led to a splicing variant with 31 extra amino acid residues in addition to the original 95 nucleotides (NTs) of INSL3. The Alu-INSL3 isoform underwent diverse changes during primate evolution; we identified that human Alu-INSL3 might be on its way to functionality and has potential to antagonize LGR8-INSL3 function. Therefore, the present study is designed to provide an example of the evolutionary trajectory of a variant peptide hormone antagonist that caused by the insertion of an Alu element in primates.

Constitutively active BRS3 is a genuinely orphan GPCR in placental mammals.

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent the most productive drug targets. Orphan GPCRs, with their endogenous ligands unknown, were considered a source of drug targets and consequently attract great interest to identify their endogenous cognate ligands for deorphanization. However, a contrary view to the ubiquitous existence of endogenous ligands for every GPCR is that there might be a significant overlooked fraction of orphan GPCRs that function constitutively in a ligand-independent manner only. Here, we investigated the evolution of the bombesin receptor-ligand family in vertebrates in which one member-bombesin receptor subtype-3 (BRS3)-is a potential orphan GPCR. With analysis of 17 vertebrate BRS3 structures and 10 vertebrate BRS3 functional data, our results demonstrated that nonplacental vertebrate BRS3 still connects to the original ligands-neuromedin B (NMB) and gastrin-releasing peptide (GRP)-because of adaptive evolution, with significantly changed protein structure, especially in three altered key residues (Q127R, P205S, and R294H) originally involved in ligand binding/activation, whereas the placental mammalian BRS3 lost the binding affinity to NMB/GRP and constitutively activates Gs/Gq/G12 signaling in a ligand-independent manner. Moreover, the N terminus of placental mammalian BRS3 underwent positive selection, exhibiting significant structural differences compared to nonplacental vertebrate BRS3, and this domain plays an important role in constitutive activity of placental mammalian BRS3. In conclusion, constitutively active BRS3 is a genuinely orphan GPCR in placental mammals, including human. To our knowledge, this study identified the first example that might represent a new group of genuinely orphan GPCRs that will never be deorphanized by the discovery of a natural ligand and provided new perspectives in addition to the current ligand-driven GPCR deorphanization.