Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110a PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110b, did not prevent preneoplastic lesion initiation. p110a signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110a was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110a cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110a is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment.

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Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micro-metastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas. We searched for a gene signature that discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell-free DNA (cfDNA) as an early biomarker of micro-metastatic disease to validate the identification of key signalling events. An unbiased approach identified, amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Ka activation signature as predictive of PDAC aggressiveness and prognosis. Pharmacological or tumour-restricted genetic PI3Kaselective inhibition prevented macro-metastatic evolution by hindering tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Ka inhibition altered the quantity and the species composition of the produced lipid second messenger PIP 3 , with a selective decrease of C36:2 PI-3,4,5-P 3 . Tumoural PI3Ka inactivation prevented the accumulation of protumoural CD206-positive macrophages in the tumour-adjacent tissue. Tumour cell-intrinsic PI3Ka promotes pro-metastatic features that could be pharmacologically targeted to delay macrometastatic evolution.

ABSTRACTp27 Kip1 (p27) is a negative regulator of proliferation and a tumor suppressor via the inhibition of cyclin-CDK activity in the nucleus. p27 is also involved in the regulation of other cellular processes, including transcription by acting as a transcriptional co-repressor. Loss of p27 expression is frequently observed in pancreatic adenocarcinomas in human and is associated with decreased patient survival. Similarly, in a mouse model of K-Ras-driven pancreatic cancer, loss of p27 accelerates tumor development and shortens survival, suggesting an important role for p27 in pancreatic tumorigenesis. Here, we sought to determine how p27 might contribute to early events leading to tumor development in the pancreas. We found that K-Ras activation in the pancreas causes p27 mislocalization at pre-neoplastic stages. Moreover, loss of p27 or expression of a mutant p27 that does not bind cyclinCDKs causes the mislocalization of several acinar polarity markers associated with metaplasia and induces the nuclear expression of Sox9 and Pdx1 two transcription factors involved in acinar-to-ductal metaplasia. Finally, we found that p27 directly represses transcription of Sox9, but not that of Pdx1. Thus, our results suggest that K-Ras activation, the earliest known event in pancreatic carcinogenesis, may cause loss of nuclear p27 expression which results in derepression of Sox9, triggering reprogrammation of acinar cells and metaplasia.

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.

Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micrometastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas.We researched a gene signature that could discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell-free DNA (cfDNA) as an early biomarker of micro-metastatic disease to validate the identification of key signalling events.Amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Kα activation signature predict PDAC aggressiveness and prognosis. Pharmacological or tumour-restricted genetic PI3Kα-selective inhibition prevented macro-metastatic evolution by inhibiting tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Kα inhibition altered the quantity and the species composition of the lipid second messenger PIP3 produced, with selective reduction of C36:2 PI-3,4,5-P3. PI3Kα inactivation prevented the accumulation of protumoural CD206-positive macrophages in the tumour-adjacent tissue.Tumour-cell intrinsic PI3Kα therefore promotes pro-metastatic features that could be pharmacologically targeted to delay macro-metastatic evolution.The paper explainedPROBLEM Pancreatic cancer is one of the most lethal solid cancers characterised by rapid progression after primary tumour detection by imaging. Key signalling events that specifically drives this rapid evolution into macro-metastatic disease are so far poorly understood.RESULT With two unbiased approaches to patient data analysis, higher PI3K pathway and more specifically higher PI3Kα activation signature can now be identified in the most aggressive pancreatic cancer primary tumours, that lead to earlier patient death. Our in vitro data showed that PI3Kα is a major positive regulator of tumour cell escape from the primary tumour: tumour-intrinsic PI3Kα activity enables actin cytoskeleton remodelling to escape the pancreatic tumour. We chose to use two preclinical models of pancreatic cancer to validate that PI3Kα is a target for delaying evolution of PDAC. The first one mimicked pancreatic patient micrometastatic disease that is undetected by echography and consisted in treating mice presenting echography detected primary tumours combined with increased circulating DNA as a blood biomarker of the most aggressive tumours. The second model consisted in studying the tumour cell implantation and their early proliferation in metastatic organ after injection in blood. We treated both preclinical models with a clinically relevant PI3K α-selective inhibitor (BYL-719/Alpelisib), that is currently being tested in pancreatic cancer patients (without any patient selection). We found that PI3Kα activity drives evolution of micrometastatic disease towards macro-metastatic stage in both models: inhibition of PI3Kα delayed primary tumour and micro-metastasis evolution. Finally, PI3Kα activity increases protumoural characteristics in peritumoural immune cells via tumour cell-intrinsic cytokine production that could facilitate metastatic evolution.IMPACT Circulating tumour DNA represents a strong independent biomarker linked to relapse and poor survival in solid cancer patients. A clinical study in resected PDAC patients with micrometastatic disease characterised by high circulating tumoural DNA levels is needed to assess if PI3Kα-selective inhibitors significantly delay metastatic progression and death.Graphical AbstractPancreatic ductal adenocarcinoma requires tumour-intrinsic PI3Kα activity to accelerate inflammatory metastatic disease.Biorender illustration.

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