Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

Chemical Proteomic Approach for In-Depth Glycosylation Profiling of Plasma Carcinoembryonic Antigen in Cancer Patients.

Carcinoembryonic antigen (CEA) of human plasma is a biomarker of many cancer diseases, and its N-glycosylation accounts for 60% of molecular mass. It is highly desirable to characterize its glycoforms for providing additional dimension of features to increase its performance in prognosis and diagnosis of cancers. However, to systematically characterize its site-specific glycosylation is challenging because of its low abundance. Here, we developed a highly sensitive strategy for in-depth glycosylation profiling of plasma CEA through chemical proteomics combined with multienzymatic digestion. A trifunctional probe was utilized to generate covalent bond of plasma CEA and its antibody upon UV irradiation. As low as 1 ng/ml CEA in plasma could be captured and digested with trypsin and chymotrypsin for intact glycopeptide characterization. Twenty six of 28 potential N-glycosylation sites were well identified, which were the most comprehensive N-glycosylation site characterization of CEA on intact glycopeptide level as far as we known. Importantly, this strategy was applied to the glycosylation analysis of plasma CEA in cancer patients. Differential site-specific glycoforms of plasma CEA were observed in patients with colorectal cancers (CRCs) and lung cancer. The distributions of site-specific glycoforms were different as the progression of CRC, and most site-specific glycoforms were overexpressed in stage II of CRC. Overall, we established a highly sensitive chemical proteomic method to profile site-specific glycosylation of plasma CEA, which should generally applicable to other well-established cancer glycoprotein biomarkers for improving their cancer diagnosis and monitoring performance.

A real-world observation on thrombopoietic agents for patients with cancer treatment-induced thrombocytopenia in China: A multicenter, cross-sectional study.

BACKGROUND: Studies on various thrombopoietic agents for cancer treatment-induced thrombocytopenia (CTIT) in China are lacking. This study aimed to provide detailed clinical profiles to understand the outcomes and safety of different CTIT treatment regimens. METHODS: In this retrospective, cross-sectional study, 1664 questionnaires were collected from 33 hospitals between March 1 and July 1, 2021. Patients aged >18 years were enrolled who were diagnosed with CTIT and treated with recombinant interleukin 11 (rhIL-11), recombinant thrombopoietin (rhTPO), or a thrombopoietin receptor agonist (TPO-RA). The outcomes, compliance, and safety of different treatments were analyzed. RESULTS: Among the 1437 analyzable cases, most patients were treated with either rhTPO alone (49.3%) or rhIL-11 alone (27.0%). The most common combination regimen used was rhTPO and rhIL-11 (10.9%). Platelet transfusions were received by 117 cases (8.1%). In multivariate analysis, rhTPO was associated with a significantly lower proportion of platelet recovery, platelet transfusion, and hospitalization due to chemotherapy-induced thrombocytopenia (CIT) than rhIL-11 alone. No significant difference was observed in the time taken to achieve a platelet count of >100 × 109/L and chemotherapy dose reduction due to CIT among the different thrombopoietic agents. The outcomes of thrombocytopenia in 170 patients who received targeted therapy and/or immunotherapy are also summarized. The results show that the proportion of platelet recovery was similar among the different thrombopoietic agents. No new safety signals related to thrombopoietic agents were observed in this study. A higher proportion of physicians preferred to continue treatment with TPO-RA alone than with rhTPO and rhIL-11. CONCLUSIONS: This survey provides an overview of CTIT and the application of various thrombopoietic agents throughout China. Comparison of monotherapy with rhIL-11, rhTPO, and TPO-RA requires further randomized clinical trials. The appropriate application for thrombopoietic agents should depend on the pretreatment of platelets, treatment variables, and risk of bleeding. PLAIN LANGUAGE SUMMARY: To provide an overview of the outcome of cancer treatment-induced thrombocytopenia in China, our cross-sectional study analyzed 1437 cases treated with different thrombopoietic agents. Most of the patients were treated with recombinant interleukin 11 (rhIL-11) and recombinant thrombopoietin (rhTPO). rhTPO was associated with a significantly lower proportion of platelet recovery and platelet transfusion compared with rhIL-11.

Plasma proteomic characterization of colorectal cancer patients with FOLFOX chemotherapy by integrated proteomics technology.

BACKGROUND: Colorectal Cancer (CRC) is a prevalent form of cancer, and the effectiveness of the main postoperative chemotherapy treatment, FOLFOX, varies among patients. In this study, we aimed to identify potential biomarkers for predicting the prognosis of CRC patients treated with FOLFOX through plasma proteomic characterization. METHODS: Using a fully integrated sample preparation technology SISPROT-based proteomics workflow, we achieved deep proteome coverage and trained a machine learning model from a discovery cohort of 90 CRC patients to differentiate FOLFOX-sensitive and FOLFOX-resistant patients. The model was then validated by targeted proteomics on an independent test cohort of 26 patients. RESULTS: We achieved deep proteome coverage of 831 protein groups in total and 536 protein groups in average for non-depleted plasma from CRC patients by using a Orbitrap Exploris 240 with moderate sensitivity. Our results revealed distinct molecular changes in FOLFOX-sensitive and FOLFOX-resistant patients. We confidently identified known prognostic biomarkers for colorectal cancer, such as S100A4, LGALS1, and FABP5. The classifier based on the biomarker panel demonstrated a promised AUC value of 0.908 with 93% accuracy. Additionally, we established a protein panel to predict FOLFOX effectiveness, and several proteins within the panel were validated using targeted proteomic methods. CONCLUSIONS: Our study sheds light on the pathways affected in CRC patients treated with FOLFOX chemotherapy and identifies potential biomarkers that could be valuable for prognosis prediction. Our findings showed the potential of mass spectrometry-based proteomics and machine learning as an unbiased and systematic approach for discovering biomarkers in CRC.

Combination of automated sample preparation and micro-flow LC-MS for high-throughput plasma proteomics.

BACKGROUND: Non-invasive detection of blood-based markers is a critical clinical need. Plasma has become the main sample type for clinical proteomics research because it is easy to obtain and contains measurable protein biomarkers that can reveal disease-related physiological and pathological changes. Many efforts have been made to improve the depth of its identification, while there is an increasing need to improve the throughput and reproducibility of plasma proteomics analysis in order to adapt to the clinical large-scale sample analysis. METHODS: We have developed and optimized a robust plasma analysis workflow that combines an automated sample preparation platform with a micro-flow LC-MS-based detection method. The stability and reproducibility of the workflow were systematically evaluated and the workflow was applied to a proof-of-concept plasma proteome study of 30 colon cancer patients from three age groups. RESULTS: This workflow can analyze dozens of samples simultaneously with high reproducibility. Without protein depletion and prefractionation, more than 300 protein groups can be identified in a single analysis with micro-flow LC-MS system on a Orbitrap Exploris 240 mass spectrometer, including quantification of 35 FDA approved disease markers. The quantitative precision of the entire workflow was acceptable with median CV of 9%. The preliminary proteomic analysis of colon cancer plasma from different age groups could be well separated with identification of potential colon cancer-related biomarkers. CONCLUSIONS: This workflow is suitable for the analysis of large-scale clinical plasma samples with its simple and time-saving operation, and the results demonstrate the feasibility of discovering significantly changed plasma proteins and distinguishing different patient groups.

Toripalimab Plus Chemotherapy for Recurrent or Metastatic Nasopharyngeal Carcinoma: The JUPITER-02 Randomized Clinical Trial.

Importance: There are currently no therapies approved by the US Food and Drug Administration for nasopharyngeal carcinoma (NPC). Gemcitabine-cisplatin is the current standard of care for the first-line treatment of recurrent or metastatic NPC (RM-NPC). Objective: To determine whether toripalimab in combination with gemcitabine-cisplatin will significantly improve progression-free survival and overall survival as first-line treatment for RM-NPC, compared with gemcitabine-cisplatin alone. Design, Setting, and Participants: JUPITER-02 is an international, multicenter, randomized, double-blind phase 3 study conducted in NPC-endemic regions, including mainland China, Taiwan, and Singapore. From November 10, 2018, to October 20, 2019, 289 patients with RM-NPC with no prior systemic chemotherapy in the RM setting were enrolled from 35 participating centers. Interventions: Patients were randomized (1:1) to receive toripalimab (240 mg [n = 146]) or placebo (n = 143) in combination with gemcitabine-cisplatin for up to 6 cycles, followed by maintenance with toripalimab or placebo until disease progression, intolerable toxicity, or completion of 2 years of treatment. Main Outcome: Progression-free survival as assessed by a blinded independent central review. Secondary end points included objective response rate, overall survival, progression-free survival assessed by investigator, duration of response, and safety. Results: Among the 289 patients enrolled (median age, 46 [IQR, 38-53 years; 17% female), at the final progression-free survival analysis, toripalimab treatment had a significantly longer progression-free survival than placebo (median, 21.4 vs 8.2 months; HR, 0.52 [95% CI, 0.37-0.73]). With a median survival follow-up of 36.0 months, a significant improvement in overall survival was identified with toripalimab over placebo (hazard ratio [HR], 0.63 [95% CI, 0.45-0.89]; 2-sided P = .008). The median overall survival was not reached in the toripalimab group, while it was 33.7 months in the placebo group. A consistent effect on overall survival, favoring toripalimab, was found in subgroups with high and low PD-L1 (programmed death-ligand 1) expression. The incidence of all adverse events, grade 3 or greater adverse events, and fatal adverse events were similar between the 2 groups. However, adverse events leading to discontinuation of toripalimab or placebo (11.6% vs 4.9%), immune-related adverse events (54.1% vs 21.7%), and grade 3 or greater immune-related adverse events (9.6% vs 1.4%) were more frequent in the toripalimab group. Conclusions and Relevance: The addition of toripalimab to chemotherapy as first-line treatment for RM-NPC provided statistically significant and clinically meaningful progression-free survival and overall survival benefits compared with chemotherapy alone, with a manageable safety profile. These findings support the use of toripalimab plus gemcitabine-cisplatin as the new standard of care for this patient population. Trial Registration: ClinicalTrials.gov Identifier: NCT03581786.

Robust Capillary- and Micro-Flow Liquid Chromatography-Tandem Mass Spectrometry Methods for High-Throughput Proteome Profiling.

Capillary- and micro-flow liquid chromatography-tandem mass spectrometry (capLC-MS/MS and µLC-MS/MS) is becoming a valuable alternative to nano-flow LC-MS/MS due to its high robustness and throughput. The systematic comparison of capLC-MS/MS and µLC-MS/MS systems for global proteome profiling has not been reported yet. Here, the capLC-MS/MS (150 µm i.d. column, 1 µL/min) and µLC-MS/MS (1 mm i.d. column, 50 µL/min) systems were both established based on UltiMate 3000 RSLCnano coupled to an Orbitrap Exploris 240 by integrating with different flowmeters. We evaluated both systems in terms of sensitivity, analysis throughput, separation efficiency, and robustness. capLC-MS/MS was about 10 times more sensitive than µLC-MS/MS at different gradient lengths. Compared with capLC-MS/MS, µLC-MS/MS was able to achieve higher analysis throughput and separation efficiency. During the 7 days' long-term performance test, both systems showed good reproducibility of chromatographic full width (RSD < 3%), retention time (RSD < 0.4%), and protein identification (RSD < 3%). These results demonstrate that capLC-MS/MS is more suitable for high-throughput analysis of clinical samples with a limited starting material. When enough samples are available, µLC-MS/MS is preferred. Together, capLC and µLC coupled to Orbitrap Exploris 240 with moderate sensitivity should well meet the needs of large-cohort clinical proteomic analysis.

tert-Butylhydroquinone-induced formation of high-molecular-weight p62: A novel mechanism in the activation of Nrf2-Keap1.

The respiratory system is always exposed to air and is most vulnerable to attack by environmental free radicals. The nuclear factor E2-related factor 2-Kelch-like ECH-associated protein 1-antioxidant response element (Nrf2-Keap1-ARE) pathway and p62 are both involved in the oxidative stress response. However, the interplay between these two systems remains largely unknown. This study shows that treatment of L2 cells with tert-Butylhydroquinone (tBHQ) generates a high-molecular-weight (HMW) form of p62, leading to activation of the Nrf2-Keap1-ARE pathway. The levels of HMW-p62 increased as the tBHQ concentration increased, with concomitant decreases seen in the classical form of p62. Moreover, small interfering RNA targeting p62 increases Keap1 protein levels and inactivates the Nrf2-Keap1-ARE pathway. These results demonstrate that the Nrf2-Keap1 pathway is partially regulated by p62. tBHQ-induced HMW-p62 production may be a novel mechanism in the activation of the Nrf2-Keap1-ARE pathway.

A fully integrated sample preparation strategy for highly sensitive intact glycoproteomics.

A fully integrated sample preparation technology, termed Intact GlycoSISPROT, was developed for the highly sensitive analysis of site-specific glycopeptides. Through integrating all glycoproteomic sample preparation steps into a single spintip, Intact GlycoSISPROT provided a tool for site-specific glycosylation analysis with low micrograms to even nanograms of protein sample.

Chemosensitivity Evaluation using Circulating Tumor Cells Predicts Chemotherapy Outcomes in Patients with Advanced Cancer.

BACKGROUND: Chemotherapy is a clinically recognized effective technique for systemic treatment of malignant tumors. However, the tumor heterogeneity and multiple drug resistance (MDR) to the chemotherapeutic agents often lead to a failure of response to chemotherapy. We utilized a novel in vitro chemosensitivity test to identify sensitive and effective chemotherapeutic drugs and further elucidated the correlation between the in vitro chemosensitivity and clinical outcomes. MATERIALS AND METHODS: We developed a circulating tumor cell-based in vitro drug sensitivity test to evaluate the sensitivity of different chemotherapeutic agents. High glucose uptake combined with negative CD45 marker were exploited to distinguish the CTCs from leukocytes. The altered glucose metabolism of single cell was measured by custom-designed computational algorithm, and the toxicity of different drug combinations was assessed by different fluorescent intensity on CTCs in the treated and control group. RESULTS: We analyzed the potential of CTCs in predicting chemotherapy response in 92 patients with different cancer types. Our data showed that the isolated CTCs accurately predicted chemotherapy outcomes, especially in patients with late-stage cancer. CTC-based chemosensitivity evaluation can help guide clinical decision making and identify patients who are likely to benefit from chemotherapy. CONCLUSIONS: CTC-based chemosensitivity evaluation is an effective methodology to study the chemosensitivity of tumor cells in vitro. Our results using CTC-based chemosensitivity evaluation method were well correlated with the clinical outcomes of chemotherapy. The clinical implementation of our CTC-based chemosensitivity evaluation method can help spare patients with primary chemoresistance from the unnecessary toxicities of chemotherapy and improve chemotherapy outcomes.

Stable and EGF-Induced Temporal Interactome Profiling of CBL and CBLB Highlights Their Signaling Complex Diversity.

The epidermal growth factor receptor (EGFR) signal modulates cell proliferation, migration, and survival. Aberrant activation of EGFR constitutes the major cause of various cancers. Receptor ubiquitination and degradation mediated by CBL proteins play negative regulatory roles and control the intensity and duration of the signaling. With the construction of stable cell lines inducibly expressing FLAG-tagged CBL or CBLB, we identified 102 and 82 stable interacting proteins of CBL and CBLB, respectively, through the affinity purification followed by mass spectrometry (AP-MS) approach. Time-resolved profiling at six different time points combined with functional annotations of the temporal interactomes provides insights into the dynamic assembly of signal proteins upon EGFR signaling activation. Comparison between the interactomes of CBL and CBLB indicates their redundant but also complementary functions. Importantly, we validated the stable association of EPS15L1 and ITSN2 and temporal association of TNK2 to both CBL and CBLB through biochemical assays. Collectively, these results offer a useful resource for CBL and CBLB interactomes and highlight their prominent and diverse roles in the EGFR signaling network.

Photoaffinity-engineered protein scaffold for systematically exploring native phosphotyrosine signaling complexes in tumor samples.

Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. We developed a hybrid chemical proteomics approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. Dynamic SH2 domain-scaffolding protein complexes were efficiently cross-linked under mild UV light, captured by biotin tag, and identified by mass spectrometry. This approach was successfully used to profile native pTyr protein complexes from breast cancer tissue samples on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Among more than 1,000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer-associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. The Photo-pTyr-scaffold approach may become a generic tool for readily profiling dynamic pTyr signaling complexes in clinically relevant samples.

Combinatory Data-Independent Acquisition and Parallel Reaction Monitoring Method for Deep Profiling of Gangliosides.

Ganglioside is an important class of lipid species involved in intercellular signaling and various diseases, especially for neurodegenerative diseases. Systematic ganglioside profiling is challenging because of their naturally low abundance and highly diverse species. Herein, a new data-independent acquisition and parallel reaction monitoring (DIA/PRM) method with superior sensitivity was developed. The untargeted DIA acquisition consecutively records all the precursor ion and fragment ions at the same time, while the targeted PRM analysis with versatile higher collisional dissociation generates full MS/MS spectra for structure elucidation and verification. As compared with traditional data-dependent acquisition (DDA), the DIA/PRM method unbiasedly detected the majority of abundant ganglioside species and as low as 50 pg of ganglioside in an untargeted manner. Gangliosides in four kinds of biological samples including the mouse brain, mouse plasma, HeLa cell, and human colon cancer tissue were systematically identified, and low-abundance ganglioside species were further extended on the basis of linear chromatography retention rules of the most frequently detected ganglioside species. A total of 383 ganglioside features were defined with 329 of them derived from 32 ganglioside species. Taking advantage of the high-resolution MS analysis, rare ganglioside species were further elucidated according to their characteristic fragment ions and neutral losses. In total, 18 gangliosides with a ceramide carbon number from 20 to 25 and modified gangliosides, including 18 acetylated, 8 diacetylated, 1 phosphorylated, 36 N-glycolyneuraminic acid (NeuGc)-containing, and 7 di-NeuGc-containing gangliosides, were newly identified. The developed DIA/PRM method therefore generated a rich ganglioside resource for further functional exploration and is a unique alternative for DDA analysis for global ganglioside profiling in various biological systems.

Effect of aspirin on PET parameters in primary non-small cell lung cancer and its relationship with prognosis.

BACKGROUND: 18 F-FDG is a glucose analogue whose metabolic index SUV can effectively reflect the metabolic level of tumor microenvironment. Aspirin can affect the uptake of 18F-FDG by cancer cells, reducing the SUVmax value of primary tumors, exerting antitumor effect. This study aimed to evaluate the prognostic value of long-term aspirin and the relationship between aspirin intake and PET parameters value of primary tumor in non-small cell lung cancer (NSCLC). METHODS: Eighty-one NSCLC patients were recruited and divided into two groups: aspirin medication group and control group, who underwent surgery and had pathological diagnosis data between January 2012 and December 2016. Clinical characteristics were retrospective analyzed to evaluate the possibility of clinical prognosis, respectively. Kaplan-Meier curves and a Cox proportional hazard model were applied to evaluate the predictors of prognosis. RESULTS: The PET/CT SUVmax of the primary tumor in the aspirin group was lower than that in the control group (P < 0.05). Compared with the control group, the SUVmax, SUVmean and TLG of the primary tumor in aspirin group were lower, but the MTV value had no significant difference. Cox regression analysis showed that N stage and TNM stage were predictors of the prognosis. There was a significant difference in the use of aspirin in NSCLC patients. CONCLUSION: Aspirin can reduce SUVmax, SUVmean and TLG in primary tumor and aspirin can improve the prognosis of patients with NSCLC.

Multiomic analysis of a dried single-drop plasma sample using an integrated mass spectrometry approach.

An easy-to-use and fast approach was developed for integrated proteomic and metabolic profiling in a dried single-drop plasma sample. Plasma collection, room temperature storage, and sample preparation for both proteins and metabolites were seamlessly integrated in one spintip device. MS-based multiomic profiling using the same nano LC-MS system identified more than 150 proteins and 160 metabolites from the 1 µL plasma sample in 6 hours. Further combination with micro-flow LC and targeted MS made it a promising approach for the fast profiling of molecular biomarkers with high sensitivity and accuracy.

Long non-coding RNA LINC-PINT attenuates paclitaxel resistance in triple-negative breast cancer cells via targeting the RNA-binding protein NONO.

The treatment of triple-negative breast cancer (TNBC) relies largely on chemotherapies. However, it is frequent that TNBC patients develop resistance to the chemotherapy drugs. Generation of drug-resistant cell lines facilitates the identification of drug resistance. Here, we established two paclitaxel (PTX)-resistant TNBC cancer cell lines using an intermittent and stepwise method and found that long non-coding RNA long intergenic non-protein-coding RNA p53-induced transcript (LINC-PINT) was significantly decreased in PTX-resistant cancer cells. Ectopic expression of LINC-PINT sensitized both PTX-resistant TNBC and wild-type TNBC to PTX. Moreover, RNA immunoprecipitation showed that LINC-PINT bound to RNA-binding protein NONO. Overexpression of LINC-PINT resulted in the degradation of NONO in a proteasome-dependent manner and vice versa. Knockdown of NONO with siRNA sensitized TNBC to PTX. We further analyzed the expression level of LINC-PINT and NONO in patient samples via online database and found that LINC-PINT and NONO may function antagonistically in all types of breast cancers. Taken together, our data illustrated a tumor suppressor role of LINC-PINT in sensitizing TNBC to chemotherapies via destabilizing NONO.

Mutual regulation of JAG2 and PRAF2 promotes migration and invasion of colorectal cancer cells uncoupled from epithelial-mesenchymal transition.

BACKGROUND: Our previous studies revealed that Jagged 2 (JAG2) is involved in the regulation of migration and invasion of colon cancer cells without affecting cell proliferation. This study further explored the specific mechanism by which JAG2 promotes migration and invasion of colorectal cancer cells. METHODS: JAG2 mRNA expression in different clinical stages of colorectal cancer and normal intestinal tissues was detected by quantitative PCR (QPCR). QPCR and Western Blot were used to analyze the differential expression of JAG2 mRNA and protein between normal human colon tissue cells and various colorectal cancer cells. Co-expression status of JAG2 and epithelial-mesenchymal transition (EMT) markers in colon cancer tissues and cells was analyzed. The difference between TGF-ß-induced EMT model and the JAG2 overexpression model were compared in promoting migration and invasion of HT29 cells. HT29 cells were treated with EMT pathway inhibitors (LY2157299 and Slug siRNA) to identify a cross-talk between the JAG2 effect and the Notch pathway. Co-expressed genes of JAG2 in colorectal cancer cells were identified using siRNA and transcriptome microarray technology. The mutual regulation of JAG2 and the co-expressed gene PRAF2 and the regulation of the paracrine effect of exosomes were analyzed. RESULTS: JAG2 was abnormally expressed in colorectal cancer tissues and directly related to clinical stages. Similar to the findings in tissues, the expression of both JAG2 mRNA and protein was significantly increased in the colorectal cancer cell lines compared with that of normal colorectal cell line CCD18-Co. It was shown in our cell model that JAG2 was involved in the regulation of migration and invasion independent of the canonical Notch signaling pathway. More interestingly, JAG2 also promoted the migration and invasion of colon cancer cells in a non-EMT pathway. Further analysis revealed the co-expression of JAG2 with PRAF2 in colorectal cancer cells. JAG2-rich exosomes were released from colorectal cancer cells in a PRAF2-dependent way, while these exosomes regulated the metastasis of colorectal cancer cells in a paracrine manner. CONCLUSIONS: This is the evidence supporting the biological function of JAG2 through non-canonical Notch and non-EMT-dependent pathways and also the first demonstration of the functions of PRAF2 in colorectal cancer cells. These findings also provide theoretical basis for the development of small molecules or biological agents for therapeutic intervention targeting JAG2/PRAF2.

Primate-specific miRNA-637 inhibited tumorigenesis in human pancreatic ductal adenocarcinoma cells by suppressing Akt1 expression.

As a primate-specific microRNA, miR-637 has been discovered for nearly 10 years. Our previous study demonstrated that miR-637 acted as a suppressor in hepatocellular carcinoma. However, its biomedical significance in pancreatic cancer remains obscure. In the present study, miR-637 was found to be significantly downregulated in pancreatic ductal adenocarcinoma (PDAC) cell lines and most of the PDAC specimens. Furthermore, the enforced overexpression of miR-637 dramatically inhibited cell proliferation and induced apoptosis of PDAC cells. Akt1, as a serine/threonine-protein kinase, has been identified as an oncogene in multiple cancers including pancreatic cancer. Our data confirmed that Akt1 was a novel target for miR-637, and its knockdown also induced cell growth inhibition and apoptosis in PDAC cells. In conclusion, our data indicated that miR-637 acted as a tumor-suppressor in PDAC, and the suppressive effect was mediated, at least partially, by suppressing Akt1 expression.

Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes.

Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in incomplete digestion. Here, by seamlessly combining AP with the simple and integrated spintip-based proteomics technology (SISPROT), we developed an integrated AP-MS workflow for simultaneously processing more than 10 AP samples from cells cultured in six-well plates in 2 h. Moreover, we developed a quantitation-based data analysis workflow for differentiating potential interacting proteins from nonspecific interferences. The AP-SISPROT ensures high digestion efficiency especially for large transmembrane proteins such as EGFR and high quantification precision for profiling temporal interaction network of key EGFR signaling protein GRB2 across four time points of EGF treatment. More importantly, the integration feature allows minimum sample lose and helps the development of an ideal AP-MS workflow for studying endogenous protein complexes by the CRISPR Cas9 technology for the first time. By generating endogenously expressed bait protein fused with affinity tag, protein complexes associated with endogenous Integrin-linked kinase (ILK) was identified with much higher selectivity as compared with overexpressed and tagged ILK. The AP-SISPROT technology and its combination with CRISPR Cas9 technology should be generally applicable for studying protein complexes in a more efficient and physiologically relevant manner.