The E3 ubiquitin ligase TRIP12 is required for pancreatic acinar cell plasticity and pancreatic carcinogenesis.

The E3 ubiquitin ligase thyroid hormone receptor interacting protein 12 (TRIP12) has been implicated in pancreatic adenocarcinoma (PDAC) through its role in mediating the degradation of pancreas transcription factor 1a (PTF1a). PTF1a is a transcription factor essential for the acinar differentiation state that is notably diminished during the early steps of pancreatic carcinogenesis. Despite these findings, the direct involvement of TRIP12 in the onset of pancreatic cancer has yet to be established. In this study, we demonstrated that TRIP12 protein was significantly upregulated in human pancreatic preneoplastic lesions. Furthermore, we observed that TRIP12 overexpression varied within PDAC samples and PDAC-derived cell lines. We further demonstrated that TRIP12 was required for PDAC-derived cell growth and for the expression of E2F-targeted genes. Acinar-to-ductal cell metaplasia (ADM) is a reversible process that reflects the high plasticity of acinar cells. ADM becomes irreversible in the presence of oncogenic Kras mutations and leads to the formation of preneoplastic lesions. Using two genetically modified mouse models, we showed that a loss of TRIP12 prevented acini from developing ADM in response to pancreatic injury. With two additional mouse models, we further discovered that a depletion of TRIP12 prevented the formation of KrasG12D-induced preneoplastic lesions and impaired metastasis formation in the presence of mutated KrasG12D and Trp53R172H genes. In summary our study identified an overexpression of TRIP12 from the early stages of pancreatic carcinogenesis and proposed this E3 ubiquitin ligase as a novel regulator of acinar plasticity with an important dual role in initiation and metastatic steps of PDAC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The antitumoral activity of TLR7 ligands is corrupted by the microenvironment of pancreatic tumors.

Toll-like receptors (TLRs) are key players in the innate immune system. Recent studies have suggested that they may affect the growth of pancreatic cancer, a disease with no cure. Among them, TLR7 shows promise for therapy but may also promotes tumor growth. Thus, we aimed to clarify the therapeutic potential of TLR7 ligands in experimental pancreatic cancer models, to open the door for clinical applications. In vitro, we found that TLR7 ligands strongly inhibit the proliferation of both human and murine pancreatic cancer cells, compared with TLR2 agonists. Hence, TLR7 treatment alters cancer cells' cell cycle and induces cell death by apoptosis. In vivo, TLR7 agonist therapy significantly delays the growth of murine pancreatic tumors engrafted in immunodeficient mice. Remarkably, TLR7 ligands administration instead increases tumor growth and accelerates animal death when tumors are engrafted in immunocompetent models. Further investigations revealed that TLR7 agonists modulate the intratumoral content and phenotype of macrophages and that depleting such tumor-associated macrophages strongly hampers TLR7 agonist-induced tumor growth. Collectively, our findings shine a light on the duality of action of TLR7 agonists in experimental cancer models and call into question their use for pancreatic cancer therapy.

Slow-Fast Model and Therapy Optimization for Oncolytic Treatment of Tumors.

The present work studies models of oncolytic virotherapy without space variable in which virus replication occurs at a faster time scale than tumor growth. We address the questions of the modeling of virus injection in this slow-fast system and of the optimal timing for different treatment strategies. To this aim, we first derive the asymptotic of a three-species slow-fast model and obtain a two-species dynamical system, where the variables are tumor cells and infected tumor cells. We fully characterize the behavior of this system depending on the various biological parameters. In the second part, we address the modeling of virus injection and its expression in the two-species system, where the amount of virus does not appear explicitly. We prove that the injection can be described by an instantaneous jump in the phase plane, where a certain amount of tumors cells are transformed instantly into infected tumor cells. This description allows discussing qualitatively the timing of different injections in the frame of successive treatment strategies. This work is illustrated by numerical simulations. The timing and amount of injected virus may have counterintuitive optimal values; nevertheless, the understanding is clear from the phase space analysis.

Upregulated Apelin Signaling in Pancreatic Cancer Activates Oncogenic Signaling Pathways to Promote Tumor Development.

Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated ß-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.

The Emerging Role of Cytidine Deaminase in Human Diseases: A New Opportunity for Therapy?

The recycling activity of cytidine deaminase (CDA) within the pyrimidine salvage pathway is essential to DNA and RNA synthesis. As such, CDA deficiency can lead to replicative stress, notably in Bloom syndrome. Alternatively, CDA also can deaminate cytidine and deoxycytidine analog-based therapies, such as gemcitabine. Thus, CDA overexpression is often associated with lower systemic, chemotherapy-related, adverse effects but also with resistance to treatment. Considering the increasing interest of CDA in cancer chemoresistance, the aims of this review are to describe CDA structure, regulation of expression, and activity, and to report the therapeutic strategies based on CDA expression that recently emerged for tumor treatment.

Gene Therapy for Pancreatic Cancer: Specificity, Issues and Hopes.

A recent death projection has placed pancreatic ductal adenocarcinoma as the second cause of death by cancer in 2030. The prognosis for pancreatic cancer is very poor and there is a great need for new treatments that can change this poor outcome. Developments of therapeutic innovations in combination with conventional chemotherapy are needed urgently. Among innovative treatments the gene therapy offers a promising avenue. The present review gives an overview of the general strategy of gene therapy as well as the limitations and stakes of the different experimental in vivo models, expression vectors (synthetic and viral), molecular tools (interference RNA, genome editing) and therapeutic genes (tumor suppressor genes, antiangiogenic and pro-apoptotic genes, suicide genes). The latest developments in pancreatic carcinoma gene therapy are described including gene-based tumor cell sensitization to chemotherapy, vaccination and adoptive immunotherapy (chimeric antigen receptor T-cells strategy). Nowadays, there is a specific development of oncolytic virus therapies including oncolytic adenoviruses, herpes virus, parvovirus or reovirus. A summary of all published and on-going phase-1 trials is given. Most of them associate gene therapy and chemotherapy or radiochemotherapy. The first results are encouraging for most of the trials but remain to be confirmed in phase 2 trials.

First-in-man phase 1 clinical trial of gene therapy for advanced pancreatic cancer: safety, biodistribution, and preliminary clinical findings.

This phase 1 trial was aimed to determine the safety, pharmacokinetics, and preliminary clinical activity of CYL-02, a nonviral gene therapy product that sensitizes pancreatic cancer cells to chemotherapy. CYL-02 was administrated using endoscopic ultrasound in 22 patients with pancreatic cancer that concomitantly received chemotherapy (gemcitabine). The maximum-tolerated dose (MTD) exceeded the maximal feasible dose of CYL-02 and was not identified. Treatment-related toxicities were mild, without serious adverse events. Pharmacokinetic analysis revealed a dose-dependent increase in CYL-02 DNA exposure in blood and tumors, while therapeutic RNAs were detected in tumors. No objective response was observed, but nine patients showed stable disease up to 6 months following treatment and two of these patients experienced long-term survival. Panels of plasmatic microRNAs and proteins were identified as predictive of gene therapy efficacy. We demonstrate that CYL-02 nonviral gene therapy has a favorable safety profile and is well tolerated in patients. We characterize CYL-02 biodistribution and demonstrate therapeutic gene expression in tumors. Treated patients experienced stability of disease and predictive biomarkers of response to treatment were identified. These promising results warrant further evaluation in phase 2 clinical trial.

The E3 ubiquitin ligase thyroid hormone receptor-interacting protein 12 targets pancreas transcription factor 1a for proteasomal degradation.

Pancreas transcription factor 1a (PTF1a) plays a crucial role in the early development of the pancreas and in the maintenance of the acinar cell phenotype. Several transcriptional mechanisms regulating expression of PTF1a have been identified. However, regulation of PTF1a protein stability and degradation is still unexplored. Here, we report that inhibition of proteasome leads to elevated levels of PTF1a and to the existence of polyubiquitinated forms of PTF1a. We used the Sos recruitment system, an alternative two-hybrid system method to detect protein-protein interactions in the cytoplasm and to map the interactome of PTF1a. We identified TRIP12 (thyroid hormone receptor-interacting protein 12), an E3 ubiquitin-protein ligase as a new partner of PTF1a. We confirmed PTF1a/TRIP12 interaction in acinar cell lines and in co-transfected HEK-293T cells. The protein stability of PTF1a is significantly increased upon decreased expression of TRIP12. It is reduced upon overexpression of TRIP12 but not a catalytically inactive TRIP12-C1959A mutant. We identified a region of TRIP12 required for interaction and identified lysine 312 of PTF1a as essential for proteasomal degradation. We also demonstrate that TRIP12 down-regulates PTF1a transcriptional and antiproliferative activities. Our data suggest that an increase in TRIP12 expression can play a part in PTF1a down-regulation and indicate that PTF1a/TRIP12 functional interaction may regulate pancreatic epithelial cell homeostasis.

Pancreatic preneoplastic lesions plasma signatures and biomarkers based on proteome profiling of mouse models.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a mortality that is almost identical to incidence. Because early detected PDAC is potentially curable, blood-based biomarkers that could detect currently developing neoplasia would improve patient survival and management. PDAC develops from pancreatic intraepithelial neoplasia (PanIN) lesions, graded from low grade (PanIN1) to high grade (PanIN3). We made the hypothesis that specific proteomic signatures from each precancerous stage exist and are detectable in plasma. METHODS: We explored the peptide profiles of microdissected PanIN cells and of plasma samples corresponding to the different PanIN grade from genetically engineered mouse models of PDAC using capillary electrophoresis coupled to mass spectrometry (CE-MS) and Chip-MS/MS. RESULTS: We successfully characterised differential peptides profiles from PanIN microdissected cells. We found that plasma from tumor-bearing mice and age-matched controls exhibit discriminative peptide signatures. We also determined plasma peptide signatures corresponding to low- and high-grade precancerous step present in the mice pancreas using the two mass spectrometry technologies. Importantly, we identified biomarkers specific of PanIN3. CONCLUSIONS: We demonstrate that benign and advanced PanIN lesions display distinct plasma peptide patterns. This strongly supports the perspectives of developing a non-invasive screening test for prediction and early detection of PDAC.

Endoscopic ultrasound-guided fine-needle aspiration biopsy coupled with a KRAS mutation assay using allelic discrimination improves the diagnosis of pancreatic cancer.

GOALS AND BACKGROUND: Mutation of the KRAS oncogene is present in 75% to 95% of pancreatic cancer tissues. This study aimed to evaluate whether endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), combined with analysis of the KRAS mutation, improves the diagnosis of pancreatic cancer in cases of inconclusive or doubtful cytopathologic analysis. PATIENTS AND METHODS: We prospectively included 186 patients with a pancreatic mass (103 men; mean age: 62 y). Cytopathology and KRAS mutations, using TaqMan MGB allelic discrimination, were performed on EUS-FNA material. A final diagnosis was obtained from EUS-FNA analysis and/or a subsequent biopsy if necessary, and/or surgery, and follow-up: these were pancreatic adenocarcinoma (n=104), other malignant pancreatic tumors (n=22), and benign lesions (n=60, including 35 cases of chronic pancreatitis). RESULTS: Inconclusive or doubtful (low-grade dysplasia or atypia) cytopathology was found in 68 cases. Of these, 29 patients who had adenocarcinoma were subsequently diagnosed, including 19 cases with a former KRAS mutation. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of cytopathology alone to diagnose adenocarcinoma were 73%, 100%, 100%, 75%, and 85%, respectively. When KRAS mutation analysis was combined with pathology, these values reached 88%, 99%, 99%, 89%, and 93%, respectively. The performance of EUS-FNA to diagnose malignancy was similarly improved after the KRAS-mutation assay (negative predictive value increased from 67% to 88%; accuracy increased from 85% to 94%). CONCLUSIONS: EUS-FNA plus KRAS-mutation analysis, using allelic discrimination, is accurate and improves the diagnosis of pancreatic adenocarcinoma when pathology is inconclusive or doubtful.

Tie1 deficiency induces endothelial-mesenchymal transition.

Endothelial-mesenchymal transition (EndMT) has a significant role in embryonic heart formation and in various pathologies. However, the molecular mechanisms that regulate EndMT induction remain to be elucidated. We show that suppression of receptor tyrosine kinase Tie1 but not Tie2 induces human endothelial cells to undergo EndMT and that Slug deficiency reverts this process. We find that Erk1/2, Erk5 and Akt cascades control Slug promoter activity induced by Tie1 deficiency. Interestingly, EndMT is present in human pancreatic tumour. We propose that EndMT associated with Tie1 downregulation participates in the pathological development of stroma observed in tumours.

Targeting miR-21 for the therapy of pancreatic cancer.

Despite tremendous efforts worldwide from clinicians and cancer scientists, pancreatic ductal adenocarcinoma (PDA) remains a deadly disease for which no cure is available. Recently, microRNAs (miRNAs) have emerged as key actors in carcinogenesis and we demonstrated that microRNA-21 (miR-21), oncomiR is expressed early during PDA. In the present study, we asked whether targeting miR-21 in human PDA-derived cell lines using lentiviral vectors (LVs) may impede tumor growth. We demonstrated that LVs-transduced human PDA efficiently downregulated miR-21 expression, both in vitro and in vivo. Consequently, cell proliferation was strongly inhibited and PDA-derived cell lines died by apoptosis through the mitochondrial pathway. In vivo, miR-21 depletion stopped the progression of a very aggressive model of PDA, to induce cell death by apoptosis; furthermore, combining miR-21 targeting and chemotherapeutic treatment provoked tumor regression. We demonstrate herein for the first time that targeting oncogenic miRNA strongly inhibit pancreatic cancer tumor growth both in vitro and in vivo. Because miR-21 is overexpressed in most human tumors; therapeutic delivery of miR-21 antagonists may still be beneficial for a large number of cancers for which no cure is available.

Discovery of SOCS7 as a versatile E3 ligase for protein-based degraders.

Targeted protein degradation (TPD) strategy harnesses the ubiquitin-proteasome system (UPS) to degrade a protein of interest (POI) by bringing it into proximity with an E3 ubiquitin ligase. However, the limited availability of functional E3 ligases and the emergence of resistance through mutations in UPS components restrict this approach. Therefore, identifying alternative E3 ligases suitable for TPD is important to develop new degraders and overcome potential resistance mechanisms. Here, we use a protein-based degrader method, by fusing an anti-tag intracellular antibody to an E3 ligase, to screen E3 ligases enabling the degradation of a tagged POI. We identify SOCS7 E3 ligase as effective biodegrader, able to deplete its target in various cell lines regardless of the POI's subcellular localization. We show its utility by generating a SOCS7-based KRAS degrader that inhibits mutant KRAS pancreatic cancer cells' proliferation. These findings highlight SOCS7 versatility as valuable E3 ligase for generating potent degraders.

Minute virus of mice shows oncolytic activity against pancreatic cancer cells exhibiting a mesenchymal phenotype.

Pancreatic cancer will soon become the second cause of death by cancer in Western countries. The main barrier to increase the survival of patients with this disease requires the development of novel and efficient therapeutic strategies that better consider tumor biology. In this context, oncolytic viruses emerge as promising therapeutics. Among them, the fibrotropic minute virus of mice prototype (MVMp) preferentially infects migrating and undifferentiated cells that highly resemble poorly differentiated, basal-like pancreatic tumors showing the worst clinical outcome. We report here that MVMp specifically infects, replicates in, and kills pancreatic cancer cells from murine and human origin with a mesenchymal, basal-like profile, while sparing cancer cells with an epithelial phenotype. Remarkably, MVMp infection, at a dose that does not provoke tumor growth inhibition in athymic mice, shows significant antitumoral effect in immune-competent models; extended mouse survival; and promoted the massive infiltration of tumors by innate, myeloid, and cytotoxic T cells that exhibit a less terminally exhausted phenotype. Collectively, we demonstrate herein for the first time that MVMp is specific and oncolytic for pancreatic tumors with mesenchymal, basal-like profile, paving the way for precision-medicine opportunities for the management of the most aggressive and lethal form of this disease.

TDP1 mutation causing SCAN1 neurodegenerative syndrome hampers the repair of transcriptional DNA double-strand breaks.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

Cytidine deaminase-dependent mitochondrial biogenesis as a potential vulnerability in pancreatic cancer cells.

Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine as part of the pyrimidine salvage pathway. Elevated levels of CDA are found in pancreatic tumors and associated with chemoresistance. Recent evidence suggests that CDA has additional functions in cancer cell biology. In this work, we uncover a novel role of CDA in pancreatic cancer cell metabolism. CDA silencing impairs mitochondrial metabolite production, respiration, and ATP production in pancreatic cancer cells, leading to a so-called Pasteur effect metabolic shift towards glycolysis. Conversely, we find that CDA expression promotes mitochondrial biogenesis and oxidative phosphorylation, independently of CDA deaminase activity. Furthermore, we observe that patient primary cells overexpressing CDA are more sensitive to mitochondria-targeting drugs. Collectively, this work shows that CDA plays a non-canonical role in pancreatic cancer biology by promoting mitochondrial function, which could be translated into novel therapeutic vulnerabilities.

Cytidine Deaminase Resolves Replicative Stress and Protects Pancreatic Cancer from DNA-Targeting Drugs.

Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA syntheses and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies. SIGNIFICANCE: Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.

[Kras oncogene and pancreatic cancer: thirty years after]. / Oncogène Kras et cancer du pancréas - trente ans plus tard.

Point mutations of the Kras oncogene induce in cancerous cells an uncontrolled increase of cell proliferation and invasiveness. Mutation of Kras appears early during the process of the pancreatic carcinogenesis and is the most frequent genetic alteration in pancreatic adenocarcinoma (75 to 95 % of cases) as well as in precancerous lesions such as PanIN and IMPN. These latter lesions and tumour microenvironment are reproduced in transgenic models developed in mice. These models are induced on the basis of Kras mutation (Pdx1-Cre ; Kras(G12D) mice) associated or not to the inactivation of tumour suppressor genes (TP53, DPC4, INK4A). Kras mutation assay is easily performed in human biological samples, especially in the cellular material sampled in pancreatic masses under endoscopic ultrasound by fine needle aspiration biopsy. In the near future, searching for Kras mutation could be useful in clinical practice either for positive diagnosis of pancreatic adenocarcinoma in case of unconclusive/doubtful cytopathological analysis or for the differential diagnosis with chronic pancreatitis especially in its pseudotumoural form.