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Figure S5 shows the effects of the combination of streptozotocin with mTOR inhibitors on the size of intrahepatic tumor nodules.

Streptozotocin-based chemotherapy is the first-line chemotherapy recommended for advanced pancreatic neuroendocrine tumors (pNETs), whereas targeted therapies, including mTOR inhibitors, are available in second-line treatment. Unfortunately, objective response rates to both treatments are limited. Because mTOR pathway activation, commonly observed in pNETs, has been reported as one of the major mechanisms accounting for chemoresistance, we investigated the potential benefit of mTOR inhibition combined with streptozotocin treatment in a subset of pNETs, namely insulinomas. To evaluate the potential of mTOR inhibition in combination with streptozotocin, we selected four different inhibitors acting at various levels of the pathway (everolimus: inhibition of mTORC1; MK-2206: inhibition of AKT; BKM120: inhibition of PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of mTORC1 and mTORC2). Effects on cell viability and apoptosis were assessed in insulinoma cell lines INS-1E (rat) and MIN6 (mouse) in vitro and were confirmed in vivo by using a mouse model of hepatic tumor dissemination after intrasplenic xenograft. In vitro, all four combinations display synergistic effects. These combinations lead to heterogeneous mTOR pathway inhibition, in agreement with their respective target, and increased apoptosis. In vivo, tumor growth in the liver was significantly inhibited by combining streptozotocin with everolimus (P = 0.0014), BKM120 (P = 0.0092), or BEZ235 (P = 0.008) as compared to each agent alone. These results suggest that targeting the mTOR pathway in combination with streptozotocin could be of potential benefit for insulinomas and pNET patients and thus support further clinical investigations. Mol Cancer Ther; 17(1); 60–72. ©2017 AACR.

This file gathers all supplementary data (material and methods, supplementary figures and table)

Figure S3 shows the bliss analyses for combinations of streptozotocin with everolimus, MK-2206, BKM120 or BEZ235 in MIN6 cells.

Table S1 provides results from the whole-genome analysis of the INS-1E cell line.

Figure S2 shows the bliss analyses for combinations of streptozotocin with everolimus, MK-2206, BKM120 or BEZ235 in INS-1E cells.

Figure S4 shows how streptozotocin and mTOR inhibitors affect the mTOR pathway and apoptosis in MIN6 cells.

Figure S7 shows that the β-cell mass of xenografted mice is not affected by treatment with streptozotocin or mTOR inhibitors.

Figure S6 shows the effects of the combination of streptozotocin with mTOR inhibitors on the number of intrahepatic tumor nodules.

Figure S1 shows sensitivities of INS-1E and MIN6 cells to streptozotocin, everolimus, MK-22006, BKM120 and BEZ235.

Supplementary Materials and Methods

CRMP5 depletion does not promote apoptosis.(A): Apoptosis is monitored in GL15 cells transfected with CRMP5-siRNA1 and control-siRNA using caspase 3 antibody (1/400, BD Pharmingen, 559565). 5-bromo-2'-deoxyuridine (BrdU) indicates proliferating cells in both conditions. (B): TUNEL staining (marker of apoptosis) (Promega, G3250) in CRMP5-siRNA1 transfected GL15 cells Scale bar=20µm. (C): TUNEL staining quantification in CRMP5-siRNA transfected GL15 cells after 4 days. The total number of cells counted is indicated by DAPI quantification; TUNEL-stained cells were counted for each condition. The results were obtained from 3 independent experiments.

CRMP5 does not modulate Notch ligand expression. Western blot analysis of three Notch ligands-Jagged 1, DLL1 and DNER-using specific antibodies after CRMP5 silencing; representative western blots of three independent experiments are shown.

Clinicopathological characteristics of patients according to CRMP5 status

CRMP5 expression, Notch signaling and Akt activations in GBM-stem cells of GBM_SC1 neurospheres. A) Flow cytometry analyses of double-fluorescence staining for CRMP5-Nestin, CRMP5-IQGAP1, and Nestin-Notch1 positive cells. Cells were gated on forward light scatter (FSC) and side scatter (SSC) to exclude dead cells and debris. The percentage of double labeling cells was represented on dot plot. Axes were determined accordind to the control gate. Analysis was performed with FlowJo software. B) Immunofluorescence analysis of CRMP5 expression of GBM_SC1 cells cultured in neurosphere (left panel) and differentiation medium (right panel). C) Western blot analysis of stem-cell markers expression (CD133, Musashi1, IQGAP1, Nestin, Notch1,2) and CRMP5 in GBM_SC1 cells cultured in neurospheres or differentiation medium. Notch signaling activation was evidenced by Hes and Akt activation by p-Akt expression.

Sub-cellular fractionation of (A): GL15 , (B): A172 cell lines and (C): GBM xenograft GBM_SC1. CRMP5 sub-cellular expression was analyzed by western blot, and the fraction purity was assessed using markers of each compartment: Calpain, cytosol; EGFR, membrane; histone H1, nucleus; vimentin, cytoskeleton.

CRMP5 depletion with CRMP5 siRNA2, inhibits proliferation. (A): CRMP5 expression using western blot after CRMP5 siRNA2 transfection. (B): Proliferation analysis in A172 cells transfected with CRMP5-siRNA2 or control-siRNA (p

Working model of CRMP5 mechanism to control Notch signaling pathway and cellular proliferation in GBM. (1) Notch receptors are expressed in GBM and activated by interaction with their ligands (2) After activation, notch is cleaved by the γ-secretase complex and (3) the Notch intracellular domain (NICD) is translocated in the nucleus. (4) activates its target genes (hes1, hey1) promoting tumor cells proliferation. Notch activates, by an unknown mechanism, Akt which is another event increasing GBM proliferation and survival and leading to poor patient survival. (6) Notch signaling pathway inhibition is mediated by the E3 ubiquitin ligase (Itch) recruitment which (7) targets the Notch receptors into lysosomal degradation, resulting in Notch signaling pathway inhibition. These events are correlated with increased survival of patients. (8) CRMP5 is expressed in GBM and promotes the Notch signaling pathway over Itch-induced degradation of the Notch receptors thereby leading to sustained activation of the Notch signaling pathway, increased proliferation resulting in decreased overall survival of patients

CRMP1-5 mRNA expressions do not predict patients survival in glioblastoma using a subset of TCGA GBM cohort (N=173). (A): Comparison of CRMP1-5 mRNA levels among GBMs of patient with different survival. No significant difference in mRNA levels was observed in GBMs from long survivors (Survival > 33 months, N=35) to patients with normal survival (

Streptozotocin-based chemotherapy is the first-line chemotherapy recommended for advanced pancreatic neuroendocrine tumors (pNETs), whereas targeted therapies, including mTOR inhibitors, are available in second-line treatment. Unfortunately, objective response rates to both treatments are limited. Because mTOR pathway activation, commonly observed in pNETs, has been reported as one of the major mechanisms accounting for chemoresistance, we investigated the potential benefit of mTOR inhibition combined with streptozotocin treatment in a subset of pNETs, namely insulinomas. To evaluate the potential of mTOR inhibition in combination with streptozotocin, we selected four different inhibitors acting at various levels of the pathway (everolimus: inhibition of mTORC1; MK-2206: inhibition of AKT; BKM120: inhibition of PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of mTORC1 and mTORC2). Effects on cell viability and apoptosis were assessed in insulinoma cell lines INS-1E (rat) and MIN6 (mouse) in vitro and were confirmed in vivo by using a mouse model of hepatic tumor dissemination after intrasplenic xenograft. In vitro, all four combinations display synergistic effects. These combinations lead to heterogeneous mTOR pathway inhibition, in agreement with their respective target, and increased apoptosis. In vivo, tumor growth in the liver was significantly inhibited by combining streptozotocin with everolimus (P = 0.0014), BKM120 (P = 0.0092), or BEZ235 (P = 0.008) as compared to each agent alone. These results suggest that targeting the mTOR pathway in combination with streptozotocin could be of potential benefit for insulinomas and pNET patients and thus support further clinical investigations. Mol Cancer Ther; 17(1); 60–72. ©2017 AACR.

The identification of novel regulators of tumor progression is a key challenge to gain knowledge on the biology of small intestinal neuroendocrine tumors (SI-NETs). We recently identified the loss of the axon guidance protein semaphorin 3F as a protumoral event in SI-NETs. Interestingly the expression of its receptor neuropilin-2 (NRP-2) was still maintained. This study aimed at deciphering the potential role of NRP-2 as a contributor to SI-NET progression. The role of NRP-2 in SI-NET progression was addressed using an approach integrating human tissue and serum samples, cell lines and in vivo models. Data obtained from human SI-NET tissues showed that membranous NRP-2 expression is present in a majority of tumors, and is correlated with invasion, metastatic abilities, and neovascularization. In addition, NRP-2 soluble isoform was found elevated in serum samples from metastatic patients. In preclinical mouse models of NET progression, NRP-2 silencing led to a sustained antitumor effect, partly driven by the downregulation of VEGFR2. In contrast, its ectopic expression conferred a gain of aggressiveness, driven by the activation of various oncogenic signaling pathways. Lastly, NRP-2 inhibition led to a decrease of tumor cell viability, and sensitized to therapeutic agents. Overall, our results point out NRP-2 as a potential therapeutic target for SI-NETs, and will foster the development of innovative strategies targeting this receptor. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objectives Several targeted therapies are available for metastatic neuroendocrine tumours (NETs) but no predictive factor of response to these treatments has been identified yet. Our aim was to identify and evaluate clinical, biological, histological and functional markers of response to everolimus. Methods We retrospectively reviewed 53 patients with NETs treated with everolimus (68 % in clinical trials). Clinical, biological and histological data were analyzed. The functional marker p-p70S6K, a main effector of the mTOR pathway, was studied by immunohistochemistry in 43 cases. Prognostic factors of progression-free survival (PFS) were studied by Kaplan Meier analysis. Results All patients had metastatic and progressive disease before everolimus treatment. Objective response was 9 % and median PFS was 8.1 (4.7-11.5) months. Hypercholesterolemia (HR = 0.13, p

// Julien Bollard 1, * , Patrick Massoma 1, * , Cecile Vercherat 1 , Martine Blanc 1 , Florian Lepinasse 2 , Nicolas Gadot 3 , Christophe Couderc 1 , Gilles Poncet 4 , Thomas Walter 4 , Marie-Odile Joly 1, 2, 5 , Valerie Hervieu 1, 2, 5 , Jean-Yves Scoazec 1, 2, 3, 5 , Colette Roche 1 1 Centre de Recherche en Cancerologie de Lyon, INSERM U1052, CNRS UMR5286, Equipe «Differenciation endocrine et tumorigenese», Faculte Laennec, F-69372 Lyon, France 2 Hospices Civils de Lyon, Hopital Edouard Herriot, Service Central d’Anatomie et de Cytologie Pathologiques, F-69437 Lyon, France 3 Universite Lyon 1, Federation de Recherche Sante Lyon-Est, ANIPATH, Faculte Laennec, F-69372 Lyon, France 4 Hospices Civils de Lyon, Hopital Edouard Herriot, Federation des Specialites Digestives, F-69437 Lyon, France 5 Universite de Lyon, Universite Lyon 1, F-69622 Villeurbanne, France * These authors have contributed equally to this work Correspondence to: Colette Roche, e-mail: colette.roche@inserm.fr Keywords: small intestine neuroendocrine tumor, semaphorin, proliferation, tumor progression Received: May 20, 2015 Accepted: September 21, 2015 Published: October 02, 2015 ABSTRACT Gastro-intestinal neuroendocrine tumors (GI-NETs) are rare neoplasms, frequently metastatic, raising difficult clinical and therapeutic challenges due to a poor knowledge of their biology. As neuroendocrine cells express both epithelial and neural cell markers, we studied the possible involvement in GI-NETs of axon guidance molecules, which have been shown to decrease tumor cell proliferation and metastatic dissemination in several tumor types. We focused on the role of Semaphorin 3F (SEMA3F) in ileal NETs, one of the most frequent subtypes of GI-NETs. SEMA3F expression was detected in normal neuroendocrine cells but was lost in most of human primary tumors and all their metastases. SEMA3F loss of expression was associated with promoter gene methylation. After increasing endogenous SEMA3F levels through stable transfection, enteroendocrine cell lines STC-1 and GluTag showed a reduced proliferation rate in vitro . In two different xenograft mouse models, SEMA3F-overexpressing cells exhibited a reduced ability to form tumors and a hampered liver dissemination potential in vivo . This resulted, at least in part, from the inhibition of mTOR and MAPK signaling pathways. This study demonstrates an anti-tumoral role of SEMA3F in ileal NETs. We thus suggest that SEMA3F and/or its cellular signaling pathway could represent a target for ileal NET therapy.

Collapsin response mediator protein 5 (CRMP5) belongs to a family of five cytosolic proteins that play a major role in nervous system development. This protein was first described in cancer-induced autoimmune processes, causing neurodegenerative disorders (paraneoplastic neurologic syndromes). CRMP5 expression has been reported to serve as a biomarker for high-grade lung neuroendocrine carcinomas; however, its functional roles have not been examined in any setting of cancer pathophysiology. In this study, we report two different CRMP5 expression patterns observed in human glioblastoma (GBM) biopsies that establish connections between CRMP5 expression, Notch receptor signaling, and GBM cell proliferation. We demonstrated that elevated CRMP5 promotes Notch receptor expression and Akt activation in human tumor cell lines, GBM stem cells, and primary tumor biopsies. We have shown that the high CRMP5 and Notch expression in GBM xenograft is related to stem cells. This suggests that high CRMP5 expression pattern in GBM biopsies encompasses a subset of stem cells. Mechanistically, CRMP5 functioned by hijacking Notch receptors from Itch-dependent lysosomal degradation. Our findings suggest that CRMP5 serves as a major mediator of Notch signaling and Akt activation by controlling the degradation of the Notch receptor, with implications for defining a biomarker signature in GBM that correlates with and may predict patient survival. Cancer Res; 75(17); 3519–28. ©2015 AACR.

Streptozotocin-based chemotherapy is the first-line chemotherapy recommended for advanced pancreatic neuroendocrine tumors (pNETs), whereas targeted therapies, including mTOR inhibitors, are available in second-line treatment. Unfortunately, objective response rates to both treatments are limited. Because mTOR pathway activation, commonly observed in pNETs, has been reported as one of the major mechanisms accounting for chemoresistance, we investigated the potential benefit of mTOR inhibition combined with streptozotocin treatment in a subset of pNETs, namely insulinomas. To evaluate the potential of mTOR inhibition in combination with streptozotocin, we selected four different inhibitors acting at various levels of the pathway (everolimus: inhibition of mTORC1; MK-2206: inhibition of AKT; BKM120: inhibition of PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of mTORC1 and mTORC2). Effects on cell viability and apoptosis were assessed in insulinoma cell lines INS-1E (rat) and MIN6 (mouse)

International audience; The diagnosis of high-grade neuroendocrine tumors has strong clinical relevance because it identifies patients at higher risk of an unfavorable Outcome Who Should receive multimodal treatment. However, these tumors call be mistaken for poorly differentiated nonsmall cell carcinoma or carcinoid lung tumors. In fact, no immunohistochemical marker can currently distinguish between histologic lung subtypes. Because the collapsin response mediator protein (CRMP) family is involved in an autoimmune disease associated with small cell lung carcinoma, we explored the relationship between CRMP5 expression and lung tumor behavior. Using World Health Organization morphologic criteria, 123 lung neuroendocrine tumors and 41 randomly selected non-neuroendocrine tumors were classified. CRMP5 protein expression in tumors, metastases, and healthy lung tissue was assessed using immunostaining method. Strong and extensive CRMP5 expression was seen in 98.6% of high-grade neuroendocrine lung tumors, including small cell lung carcinoma and large Cell lung neuroendocrine carcinoma, but not in any of the squamous cell carcinomas or lung adenocarcinomas in our series. In contrast, the majority of low-grade neuroendocrine lung tumors were negative for CRMP5 staining, although weak CRMP5 expression was seen in some, with 2 different staining patterns of either scattered positive cells or small foci of positive cells. Our findings point at CRMP5 as a novel marker for routine pathologic evaluation Of lung tumors surgical samples in distinguishing between highly aggressive neuroendocrine carcinoma and the other lung cancers.

Small-intestinal neuroendocrine tumors (SI-NETs) are defined as locally invasive only after extension to the muscularis propria. To gain further insight into the molecular mechanisms, we applied a proteomic approach to an orthotopic xenograft model to identify candidate proteins evaluable in human SI-NETs. After grafting STC-1 neuroendocrine tumor cells on the caecum of nude mice, comparative proteomic studies were performed between the pre-invasive and the invasive stages, respectively 2 and 8 weeks after grafting. We identified 24 proteins displaying at least a 1.5-fold differential expression between 2 and 8 week-stages. Most were cytoskeleton-associated proteins, among which five showed decreasing expression levels (CRMP2, TCP1e, TPM2, vimentin, desmin) and two increasing expression levels (14-3-3γ, CK8). Changes for CRMP2, TCP1e, TPM2 and 14-3-3γ were confirmed in experimental tumors and in a series of 28 human SI-NETs. In conclusion, our results underline the relevance of proteomics to identify novel biomarkers of tissue invasion.

Brain banks manage and store fully clinically and pathologically characterised brains. The diversity of techniques used in research projects increases. These biological resource centres are made to adapt brain tissue processing. Furthermore, the development of more sensitive techniques to analyse nucleic acids and proteins offers new fields of exploration when combined with laser capture microdissection in order to decipher the physiopathology of diseases at the cell level. In this study, our goal was to evaluate procedures and set a workflow compatible with the constraints of brain banks, from brain sampling to laser capture microdissection and pre-analytical quality assessment. We compared various methods of freezing brain tissue, focused on morphological quality preservation of brain microscopical structures and on the quality of nucleic acid or protein yields. Staining protocols combined with strategies to lower neurones autofluorescence were adapted for the same purpose. Finally, we found that laser capture microdissection is possible in the setting of brain banks. However, the entire process has to be envisioned from the autopsy to the analysis. The impact on protein or nucleic acid quality is a limitation that restricts the amount of samples available for this purpose.