The RNA-binding protein MEX3A is a prognostic factor and regulator of resistance to gemcitabine in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer. Most patients present with advanced disease at diagnosis, which only permits palliative chemotherapeutic treatments. RNA dysregulation is a hallmark of most human cancers, including PDAC. To test the impact of RNA processing dysregulation on PDAC pathology, we performed a bioinformatics analysis to identify RNA-binding proteins (RBPs) associated with prognosis. Among the 12 RBPs associated with progression-free survival, we focused on MEX3A because it was recently shown to mark an intestinal stem cell population that is refractory to chemotherapeutic treatments, a typical feature of PDAC. Increased expression of MEX3A was correlated with higher disease stage in PDAC patients and with tumor development in a mouse model of PDAC. Depletion of MEX3A in PDAC cells enhanced sensitivity to chemotherapeutic treatment with gemcitabine, whereas its expression was increased in PDAC cells selected upon chronic exposure to the drug. RNA-sequencing analyses highlighted hundreds of genes whose expression is sensitive to MEX3A expression, with significant enrichment in cell cycle genes. MEX3A binds to its target mRNAs, like cyclin-dependent kinase 6 (CDK6), and promotes their stability. Accordingly, knockdown of MEX3A caused a significant reduction in PDAC cell proliferation and in progression to the S phase of the cell cycle. These findings uncover a novel role for MEX3A in the acquisition and maintenance of chemoresistance by PDAC cells, suggesting that it may represent a novel therapeutic target for PDAC

A bioluminescent mouse model of proliferation to highlight early stages of pancreatic cancer: A suitable tool for preclinical studies

Transgenic mouse models designed to recapitulate genetic and pathologic aspects of cancer are useful to study early stages of disease as well as its progression. Among several, two of the most sophisticated models for pancreatic ductal adenocarcinoma (PDAC) are the LSL-KrasG12D/+;Pdx-1-Cre (KC) and LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice, in which the Cre-recombinase regulated by a pancreas-specific promoter activates the expression of oncogenic Kras alone or in combination with a mutant p53, respectively. Non-invasive in vivo imaging offers a novel approach to preclinical studies introducing the possibility to investigate biological events in the spatio/temporal dimension. We recently developed a mouse model, MITO-Luc, engineered to express the luciferase reporter gene in cells undergoing active proliferation. In this model, proliferation events can be visualized non-invasively by bioluminescence imaging (BLI) in every body district in vivo. Here, we describe the development and characterization of MITO-Luc-KC- and -KPC mice. In these mice we have now the opportunity to follow PDAC evolution in the living animal in a time frame process. Moreover, by relating in vivo and ex vivo BLI and histopathological data we provide evidence that these mice could represents a suitable tool for pancreatic cancer preclinical studies. Our data also suggest that aberrant proliferation events take place early in pancreatic carcinogenesis, before tumour appearance

A bioluminescent mouse model of proliferation to highlight early stages of pancreatic cancer. A suitable tool for preclinical studies

Transgenic mouse models designed to recapitulate genetic and pathologic aspects of cancer are usefulto study early stages of disease as well as its progression. Among several, two of the most sophis-ticated models for pancreatic ductal adenocarcinoma (PDAC) are the LSL-KrasG12D/+;Pdx-1-Cre (KC)and LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice, in which the Cre-recombinase regulated by apancreas-specific promoter activates the expression of oncogenic Kras alone or in combination witha mutant p53, respectively. Non-invasive in vivo imaging offers a novel approach to preclinical stud-ies introducing the possibility to investigate biological events in the spatio/temporal dimension. Werecently developed a mouse model, MITO-Luc, engineered to express the luciferase reporter gene in cellsundergoing active proliferation. In this model, proliferation events can be visualized non-invasively bybioluminescence imaging (BLI) in every body district in vivo. Here, we describe the development andcharacterization of MITO-Luc-KC- and -KPC mice. In these mice we have now the opportunity to followPDAC evolution in the living animal in a time frame process. Moreover, by relating in vivo and ex vivoBLI and histopathological data we provide evidence that these mice could represents a suitable tool forpancreatic cancer preclinical studies. Our data also suggest that aberrant proliferation events take placeearly in pancreatic carcinogenesis, before tumour appearance

The advanced glycation end-product N\u3f5 -carboxymethyllysine promotes progression of pancreatic cancer: implications for diabetes-associated risk and its prevention

Diabetes is an established risk factor for pancreatic cancer (PaC), together with obesity, a Western diet, and tobacco smoking. The common mechanistic link might be the accumulation of advanced glycation end-products (AGEs), which characterizes all of the above disease conditions and unhealthy habits. Surprisingly, however, the role of AGEs in PaC has not been examined yet, despite the evidence of a tumour-promoting role of receptor for advanced glycation end-products (RAGE), the receptor for AGEs. Here, we tested the hypothesis that AGEs promote PaC through RAGE activation. To this end, we investigated the effects of the AGE N-epsilon-carboxymethyllysine (CML) in human pancreatic ductal adenocarcinoma (PDA) cell lines and in a mouse model of Kras-driven PaC interbred with a bioluminescent model of proliferation. Tumour growth was monitored in vivo by bioluminescence imaging and confirmed by histology. CML promoted PDA cell growth and RAGE expression, in a concentration-dependent and time-dependent manner, and activated downstream tumourigenic signalling pathways. These effects were counteracted by RAGE antagonist peptide (RAP). Exogenous AGE administration to PaC-prone mice induced RAGE upregulation in pancreatic intraepithelial neoplasias (PanINs) and markedly accelerated progression to invasive PaC. At 11 weeks of age (6 weeks of CML treatment), PaC was observed in eight of 11 (72.7%) CML-treated versus one of 11 (9.1%) vehicle-treated [control (Ctr)] mice. RAP delayed PanIN development in Ctr mice but failed to prevent PaC promotion in CML-treated mice, probably because of competition with soluble RAGE for binding to AGEs and/or compensatory upregulation of the RAGE homologue CD166/ activated leukocyte cell adhesion molecule, which also favoured tumour spread. These findings indicate that AGEs modulate the development and progression of PaC through receptor-mediated mechanisms, and might be responsible for the additional risk conferred by diabetes and other conditions characterized by increased AGE accumulation. Finally, our data suggest that an AGE reduction strategy, instead of RAGE inhibition, might be suitable for the risk management and prevention of PaC. Copyright (C) 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

MITO-Luc/GFP zebrafish model to assess spatial and temporal evolution of cell proliferation in vivo

Abstract We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence. In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. By in vitro and ex vivo experiments we demonstrate that luciferase activity in adult animals occurs in intestine, kidney and gonads, where detectable proliferating cells are located. Further, by time lapse experiments in live embryos, we observed a wave of GFP positive cells following fin clip. In adult zebrafish, in addition to a bright bioluminescence signal on the regenerating tail, an early unexpected signal coming from the kidney occurs indicating not only a fin cell proliferation, but also a systemic response to tissue damage. Finally, we observed that luciferase activity was inhibited by anti-proliferative interventions, i.e. 5FU, cell cycle inhibitors and X-Rays. In conclusion, MITO-Luc/GFP zebrafish is a novel animal model that may be crucial to assess the spatial and temporal evolution of cell proliferation in vivo

Multi-omic approach identifies a transcriptional network coupling innate immune response to proliferation in the blood of COVID-19 cancer patients

International audienceClinical outcomes of COVID-19 patients are worsened by the presence of co-morbidities, especially cancer leading to elevated mortality rates. SARS-CoV-2 infection is known to alter immune system homeostasis. Whether cancer patients developing COVID-19 present alterations of immune functions which might contribute to worse outcomes have so far been poorly investigated. We conducted a multi-omic analysis of immunological parameters in peripheral blood mononuclear cells (PBMCs) of COVID-19 patients with and without cancer. Healthy donors and SARS-CoV-2-negative cancer patients were also included as controls. At the infection peak, cytokine multiplex analysis of blood samples, cytometry by time of flight (CyTOF) cell population analyses, and Nanostring gene expression using Pancancer array on PBMCs were performed. We found that eight pro-inflammatory factors (IL-6, IL-8, IL-13, IL-1ra, MIP-1a, IP-10) out of 27 analyzed serum cytokines were modulated in COVID-19 patients irrespective of cancer status. Diverse subpopulations of T lymphocytes such as CD8 + T, CD4 + T central memory, Mucosal-associated invariant T (MAIT), natural killer (NK), and γδ T cells were reduced, while B plasmablasts were expanded in COVID-19 cancer patients. Our findings illustrate a repertoire of aberrant alterations of gene expression in circulating immune cells of COVID-19 cancer patients. A 19-gene expression signature of PBMCs is able to discriminate COVID-19 patients with and without solid cancers. Gene set enrichment analysis highlights an increased gene expression linked to Interferon α, γ, α/β response and signaling which paired with aberrant cell cycle regulation in cancer patients. Ten out of the 19 genes, validated in a real-world consecutive cohort, were specific of COVID-19 cancer patients independently from different cancer types and stages of the diseases, and useful to stratify patients in a COVID-19 disease severity-manner. We also unveil a transcriptional network involving gene regulators of both inflammation response and proliferation in PBMCs of COVID-19 cancer patients

Image_5_Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects.jpeg

BackgroundFew data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients.ObjectiveTo investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy.MethodsWe measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80–92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D.ResultsAn increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts.ConclusionFHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.</p

DataSheet_1_Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects.docx

BackgroundFew data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients.ObjectiveTo investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy.MethodsWe measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80–92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D.ResultsAn increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts.ConclusionFHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.</p

Image_3_Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects.jpeg

BackgroundFew data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients.ObjectiveTo investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy.MethodsWe measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80–92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D.ResultsAn increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts.ConclusionFHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.</p