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Supplementary Table S2

Pancreatic neuroendocrine tumors (PanNET) comprise two molecular subtypes, relatively benign islet tumors (IT) and invasive, metastasis-like primary (MLP) tumors. Until now, the origin of aggressive MLP tumors has been obscure. Herein, using multi-omics approaches, we revealed that MLP tumors arise from IT via dedifferentiation following a reverse trajectory along the developmental pathway of islet β cells, which results in the acquisition of a progenitor-like molecular phenotype. Functionally, the miR-181cd cluster induces the IT-to-MLP transition by suppressing expression of the Meis2 transcription factor, leading to upregulation of a developmental transcription factor, Hmgb3. Notably, the IT-to-MLP transition constitutes a distinct step of tumorigenesis and is separable from the classic proliferation-associated hallmark, temporally preceding accelerated proliferation of cancer cells. Furthermore, patients with PanNET with elevated HMGB3 expression and an MLP transcriptional signature are associated with higher-grade tumors and worse survival. Overall, our results unveil a new mechanism that modulates cancer cell plasticity to enable malignant progression.Significance:Dedifferentiation has long been observed as a histopathologic characteristic of many cancers, albeit inseparable from concurrent increases in cell proliferation. Herein, we demonstrate that dedifferentiation is a mechanistically and temporally separable step in the multistage tumorigenesis of pancreatic islet cells, retracing the developmental lineage of islet β cells.This article is highlighted in the In This Issue feature, p. 2355

Supplementary Figure S6

Many human cancers manifest the capability to circumvent attack by the adaptive immune system. In this work, we identified a component of immune evasion that involves frequent up-regulation of fragile X mental retardation protein (FMRP) in solid tumors. FMRP represses immune attack, as revealed by cancer cells engineered to lack its expression. FMRP-deficient tumors were infiltrated by activated T cells that impaired tumor growth and enhanced survival in mice. Mechanistically, FMRP’s immunosuppression was multifactorial, involving repression of the chemoattractant C-C motif chemokine ligand 7 (CCL7) concomitant with up-regulation of three immunomodulators—interleukin-33 (IL-33), tumor-secreted protein S (PROS1), and extracellular vesicles. Gene signatures associate FMRP’s cancer network with poor prognosis and response to therapy in cancer patients. Collectively, FMRP is implicated as a regulator that orchestrates a multifaceted barrier to antitumor immune responses.

Significance The capabilities for invasion and metastasis underlie the mortality and morbidity of most forms of human cancer. Currently, there are no effective therapies specifically targeting these cancer phenotypes, in part due to the paucity of dominant mutations that induce them, and indeed losses of suppressors of invasion and metastasis are increasingly recognized as determinants, posing challenges for drug development. Our results implicate epigenetic gene regulation mediated by elevated expression of distinct microRNAs in orchestrating invasion and metastasis, evidently by abrogating distinctive suppressor mechanisms. Therefore, targeting such microRNAs holds promise as a strategy to combat malignant cancers with epigenetically disrupted tumor suppressor mechanisms., MicroRNA-mediated gene regulation has been implicated in various diseases, including cancer. This study examined the role of microRNAs (miRNAs) during tumorigenesis and malignant progression of pancreatic neuroendocrine tumors (PanNETs) in a genetically engineered mouse model. Previously, a set of miRNAs was observed to be specifically up-regulated in a highly invasive and metastatic subtype of mouse and human PanNET. Using functional assays, we now implicate different miRNAs in distinct phenotypes: miR-137 stimulates tumor growth and local invasion, whereas the miR-23b cluster enables metastasis. An algorithm, Bio-miRTa, has been developed to facilitate the identification of biologically relevant miRNA target genes and applied to these miRNAs. We show that a top-ranked miR-137 candidate gene, Sorl1, has a tumor suppressor function in primary PanNETs. Among the top targets for the miR-23b cluster, Acvr1c/ALK7 has recently been described to be a metastasis suppressor, and we establish herein that it is down-regulated by the miR-23b cluster, which is crucial for its prometastatic activity. Two other miR-23b targets, Robo2 and P2ry1, also have demonstrable antimetastatic effects. Finally, we have used the Bio-miRTa algorithm in reverse to identify candidate miRNAs that might regulate activin B, the principal ligand for ALK7, identifying thereby a third family of miRNAs—miRNA-130/301—that is congruently up-regulated concomitant with down-regulation of activin B during tumorigenesis, suggestive of functional involvement in evasion of the proapoptotic barrier. Thus, dynamic up-regulation of miRNAs during multistep tumorigenesis and malignant progression serves to down-regulate distinctive suppressor mechanisms of tumor growth, invasion, and metastasis.

Summary Metastasis - the disseminated growth of tumours in distant organs – underlies cancer mortality. Breast-to-brain metastasis (B2BM) is disconcertingly common and disruptive, being prevalent in the aggressive basal-like subtype, albeit evident at varying frequencies in all subtypes. Previous studies revealed parameters of breast cancer metastasis to brain, but its preference for this site remains an enigma. Herein we show that B2BM cells co-opt a neuronal signaling pathway recently implicated in invasive tumour growth, involving activation by glutamate ligand of an N-methyl-D-aspartate receptor (NMDAR), whose signaling is demonstrably instrumental in model systems for metastatic colonization of the brain, and associated with poor prognosis. While NMDAR receptor activation is autocrine in some primary tumour types, human and mouse B2BM cells express receptors but secrete insufficient glutamate to activate signaling, which is instead supplied via the formation of pseudo-tripartite synapses between cancer cells and glutamatergic neurons, presenting an insidious rationale for brain metastasis.

Background Pancreatic Neuroendocrine Tumours (PanNETs) are the second most common form of pancreatic cancer. We previously identified two principal subtypes of PanNET: insulinomas (islet tumors; IT) and metastasis-like primaries (MLP), corresponding to the low- and high-grade classification of human PanNETs. This study describes the mechanisms by which PanNETs progress from IT to more aggressive MLP tumors and eventually metastasize. Methods We profiled single cell transcriptomes, bulk mRNA and miRNA transcriptomes and the proteomes of primary and metastasis specimens from the genetically engineered mouse model of PanNETs (Rip1 Tag2). Results We found that the IT tumours maintained the expression of mature s cell markers. In contrast, the MLP tumours expressed pancreatic progenitor markers. By integrating the data on s-cell differentiation from pancreatic progenitors to mature s cells, we demonstrated that the tumour progression from IT to MLP follows the reverse embryonic and postnatal developmental path. Furthermore, we identified Hmgb3 and miR-181cd cluster as the MLP master regulators. Over-expression of the miR-181cd cluster in IT-like cancer cell-lines resulted in the acquisition of the MLP gene signature and MLP morphologic phenotypes, in addition to activation Hmgb3 expression. This suggested a central role for Hmgb3 in initiating the MLP program. Furthermore, inhibiting the expression of Hmgb3 in MLP-like cancer cell-lines resulted in a significant growth decrease, demonstrating the importance of Hmgb3 in the maintenance of MLP-like cell state in vitro. Using transcriptomic data from human patients, we evaluated the relevance of the MLP program in human tumours. We established that aggressive human PanNET tumours also follow the same reverse developmental trajectory of dedifferentiation. Notably, patients with high MLP genes expression had a worse prognosis. Conclusions These data demonstrate dedifferentiation as a mechanism by which malignant neuroendocrine cancer cells acquire progenitor-like features, enabling them to become more aggressive and metastatic. In addition, miR-181cd cluster and Hmgb3 act as the core regulators in the initiation of dedifferentiation and maintenance of progenitor-like features in tumour cells. Legal entity responsible for the study CMSO Group at ISREC, EPFL. Funding Swiss National Science Foundation. Disclosure All authors have declared no conflicts of interest.

Cancer evolves through the emergence and selection of molecular alterations. Cancer genome profiling has revealed that specific events are more or less likely to be co-selected, suggesting that the selection of one event depends on the others. However, the nature of these evolutionary dependencies and their impact remain unclear. Here, we designed SELECT, an algorithmic approach to systematically identify evolutionary dependencies from alteration patterns. By analyzing 6,456 genomes from multiple tumor types, we constructed a map of oncogenic dependencies associated with cellular pathways, transcriptional readouts, and therapeutic response. Finally, modeling of cancer evolution shows that alteration dependencies emerge only under conditional selection. These results provide a framework for the design of strategies to predict cancer progression and therapeutic response.

Development of orbital shaking technology for enhanced mixing with lower mechanical demand has been receiving significant attention since the advent of advanced mixing schemes. Amongst shaken bioreactors,microtiter plates play significant role both for research and industrial purposes due to their capability of handling tiny amount of liquid in parallel experimentations. Detailed understanding of complicated Flow hydrodynamics thus seems to be considered a continual effort, as it is responsible for efficient gas-liquid mass transfer. Computational fluid dynamics (CFD) technique is shown to be a suitable numerical method in particular for discovering concealed flow facts, which can reliably predict the unsteady state flow within individual plate cells. In this study, CFD is employed to account for gas-liquid interface shape determination to reflect details of hydrodynamics for a novel geometrically modified microtiterplate, which would address diverse unknown flow characteristics vital for accurate mass transfer calculations.

Penicillin G is an active pharmaceutical ingredient of great importance in health sectors. Meanwhile, because of its huge quantity production and resistance to biodegradability, this antibiotic is ubiquitously presented in aquatic environment. In this study, the degradation of effluent wastewater from Penicillin G production plant by Photo- Fenton process and Ultrasound process (Sonolysis) was investigated, the effects of different process variables in both methods were evaluated and at last the degradation of Pen-G by these two processes in the optimum conditions was compared. Pen-G concentration and Chemical Oxygen Demand (COD) were selected as the environmental parameters to follow the performance of Photo-Fenton process and Sonolysis. Optimum condition of Photo-Fenton process is in H2O2/Fe2+ molar ratio 20 (20 mM H2O2 and 1 mM Fe2+) and pH 3.5 and for Sonolysis (35 KHz) is in pH 3. Complete degradation of Pen-G occurred in 30 and 70 min for Photo-Fenton process and Sonolysis, respectively. Based on the results, Photo-Fenton process is more efficient in terms of both COD degradation and Pen-G removal than Sonolysis (35 KHz).

Summary Therapeutic targeting of tumor angiogenesis with VEGF inhibitors results in demonstrable, but transitory efficacy in certain human tumors and mouse models of cancer, limited by unconventional forms of adaptive/evasive resistance. In one such mouse model, potent angiogenesis inhibitors elicit compartmental reorganization of cancer cells around remaining blood vessels. The glucose and lactate transporters GLUT1 and MCT4 are induced in distal hypoxic cells in a HIF1α-dependent fashion, indicative of glycolysis. Tumor cells proximal to blood vessels instead express the lactate transporter MCT1, and p-S6, the latter reflecting mTOR signaling. Normoxic cancer cells import and metabolize lactate, resulting in upregulation of mTOR signaling via glutamine metabolism enhanced by lactate catabolism. Thus, metabolic symbiosis is established in the face of angiogenesis inhibition, whereby hypoxic cancer cells import glucose and export lactate, while normoxic cells import and catabolize lactate. mTOR signaling inhibition disrupts this metabolic symbiosis, associated with upregulation of the glucose transporter GLUT2., Graphical Abstract, Highlights • Angiogenesis inhibitors causing acute hypoxia elicit metabolic compartmentalization • Hypoxic cancer cells import and metabolize glucose, secreting lactate • Normoxic vessel-proximal cancer cells import and metabolize lactate, involving mTOR • Co-inhibiting mTOR disrupts the metabolic symbiosis, Allen et al. find that inhibitors of tumor angiogenesis that cause acute hypoxia elicit compartmentalization of cancer cells into symbiotic clusters. Hypoxic cancer cells metabolize glucose and secrete lactate; normoxic, vessel-proximal cancer cells import and metabolize the lactate in a process involving mTOR signaling. Co-inhibition of mTOR disrupts the symbiosis, in part by upregulating glucose transport in normoxic cells.

Reference EPFL-CONF-232279View record in Web of Science Record created on 2017-11-08, modified on 2017-11-08

Reference EPFL-CONF-232278View record in Web of Science Record created on 2017-11-08, modified on 2017-11-08

Pancreatic neuroendocrine tumors (PanNET) comprise two molecular subtypes, relatively benign islet tumors (IT) and invasive, metastasis-like primary (MLP) tumors. Until now, the origin of aggressive MLP tumors has been obscure. Herein, using multi-omics approaches, we revealed that MLP tumors arise from IT via dedifferentiation following a reverse trajectory along the developmental pathway of islet beta cells, which results in the acquisition of a progenitor-like molecular phenotype. Functionally, the miR-181cd cluster induces the IT-to-MLP transition by suppressing expression of the Meis2 transcription factor, leading to upregulation of a developmental transcription factor, Hmgb3. Notably, the IT-to-MLP transition constitutes a distinct step of tumorigenesis and is separable from the classic proliferation-associated hallmark, temporally preceding accelerated proliferation of cancer cells. Furthermore, patients with PanNET with elevated HMGB3 expression and an MLP transcriptional signature are associated with higher-grade tumors and worse survival. Overall, our results unveil a new mechanism that modulates cancer cell plasticity to enable malignant progression., SIGNIFICANCE: Dedifferentiation has long been observed as a histopathologic characteristic of many cancers, albeit inseparable from concurrent increases in cell proliferation. Herein, we demonstrate that dedifferentiation is a mechanistically and temporally separable step in the multistage tumorigenesis of pancreatic islet cells, retracing the developmental lineage of islet beta cells.

Summary Herein, we report that the TGFs superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and luminal breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not “authorized” to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand.

Lung carcinoids are variably aggressive and mechanistically understudied neuroendocrine neoplasms (NENs). Here, we identified and elucidated the function of a miR-375/yes-associated protein (YAP) axis in lung carcinoid (H727) cells. miR-375 and YAP are respectively high and low expressed in wild-type H727 cells. Following lentiviral CRISPR/Cas9-mediated miR-375 depletion, we identified distinct transcriptomic changes including dramatic YAP upregulation. We also observed a significant decrease in neuroendocrine differentiation and substantial reductions in cell proliferation, transformation, and tumor growth in cell culture and xenograft mouse disease models. Similarly, YAP overexpression resulted in distinct and partially overlapping transcriptomic changes, phenocopying the effects of miR-375 depletion in the same models as above. Transient YAP knockdown in miR-375-depleted cells reversed the effects of miR-375 on neuroendocrine differentiation and cell proliferation. Pathways analysis and confirmatory real-time PCR studies of shared dysregulated target genes indicate that this axis controls neuroendocrine related functions such as neural differentiation, exocytosis, and secretion. Taken together, we provide compelling evidence that a miR-375/YAP axis is a critical mediator of neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.