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Purpose: Given that nerve growth factor has previously been shown to be involved in breast cancer progression, we have tested here the hypothesis that the other neurotrophins (NT) are expressed and have an influence in breast tumor growth.Experimental Design: The expression of brain-derived neurotrophic factor (BDNF), NT-3 and NT-4/5, as well as the neurotrophin receptor p75NTR, TrkB, and TrkC, was studied by RT-PCR, Western blotting, and immunohistochemistry in cell lines and tumor biopsies. The biological impacts of neurotrophins, and associated mechanisms, were analyzed in cell cultures and xenografted mice.Results: BDNF and NT-4/5 were expressed and secreted by breast cancer cells, and the use of blocking antibodies suggested an autocrine loop mediating cell resistance to apoptosis. The corresponding tyrosine kinase receptor TrkB was only rarely observed at full length, whereas the expression of TrkB-T1, lacking the kinase domain, as well as p75NTR, were detected in all tested breast cancer cell lines and tumor biopsies. In contrast, NT-3 and TrkC were not detected. SiRNA against p75NTR and TrkB-T1 abolished the antiapoptotic effect of BDNF and NT-4/5, whereas the pharmacological inhibitors K252a and PD98059 had no effect, suggesting the involvement of p75NTR and TrkB-T1, but not kinase activities from Trks and MAPK. In xenografted mice, anti-BDNF, anti-NT-4/5, anti-p75NTR, or anti-TrkB-T1 treatments resulted in tumor growth inhibition, characterized by an increase in cell apoptosis, but with no change in proliferation.Conclusion: BDNF and NT-4/5 contribute to breast cancer cell survival and can serve as prospective targets in attempts to inhibit tumor growth. Clin Cancer Res; 17(7); 1741–52. ©2011 AACR.

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Supplementary Figure 2 from Nerve Growth Factor Is a Potential Therapeutic Target in Breast Cancer

Supplementary Figure 3 from Nerve Growth Factor Is a Potential Therapeutic Target in Breast Cancer

We show here that nerve growth factor (NGF), the prototypic neurotrophin, can be targeted in breast cancer to inhibit tumor cell proliferation, survival, and metastasis. Analysis of a series of biopsies revealed widespread expression of NGF in the majority of human breast tumors, with anti-NGF immunoreactivity concentrated in the epithelial cancer cells. Moreover, immunodeficient mice xenografted with human breast cancer cells and treated with either anti-NGF antibodies or small interfering RNA against NGF displayed inhibited tumor growth and metastasis. Such treatments directed against NGF induced a decrease in cell proliferation with a concomitant increase in apoptosis of breast cancer cells and an inhibition of tumor angiogenesis. Together, these data indicate that targeting NGF in breast cancer may have therapeutic ramifications. [Cancer Res 2008;68(2):346–51]

Supplementary Figure 1 from Nerve Growth Factor Is a Potential Therapeutic Target in Breast Cancer

Motivation Nowadays, epigenetic gene regulations are studied in each part of the biology, from embryonic development to diseases such as cancers and neurodegenerative disorders. Currently, to quantify and compare CpG methylation levels of a specific region of interest, the most accessible technique is the bisulfite sequencing PCR (BSP). However, no existing user-friendly tool is able to analyze data from all approaches of BSP. Therefore, the most convenient way to process results from the direct sequencing of PCR products (direct-BSP) is to manually analyze the chromatogram traces, which is a repetitive and prone to error task. Results Here, we implement a new R-based tool, called ABSP for analysis of bisulfite sequencing PCR, providing a complete analytic process of both direct-BSP and cloning-BSP data. It uses the raw sequencing trace files (.ab1) as input to compute and compare CpG methylation percentages. It is fully automated and includes a user-friendly interface as a built-in R shiny app, quality control steps and generates publication-ready graphics. Availability and implementation The ABSP tool and associated data are available on GitHub at https://github.com/ABSP-methylation-tool/ABSP. Supplementary information Supplementary data are available at Bioinformatics online.

Polycomb repressive complex 2 (PRC2) mediates histone H3K27me3 methylation and the stable transcriptional repression of a number of gene expression programs involved in the control of cellular identity during development and differentiation. Here, we report on the generation and on the characterization of a zebrafish line harboring a null allele of eed, a gene coding for an essential component of the PRC2. Homozygous eed-deficient mutants present a normal body plan development but display strong defects at the level of the digestive organs, such as reduced size of the pancreas, hepatic steatosis, and a loss of the intestinal structures, to die finally at around 10–12 days post fertilization. In addition, we found that PRC2 loss of function impairs neuronal differentiation in very specific and discrete areas of the brain and increases larval activity in locomotor assays. Our work highlights that zebrafish is a suited model to study human pathologies associated with PRC2 loss of function and H3K27me3 decrease.

Cancer cells exhibit hallmarks in terms of proliferation, resistance to cell death, angiogenesis, invasion, metastasis, and genomic instability. Despite the progress in cancer research and the comprehension of tumorigenesis mechanisms, cancer remains a major issue in public health. A better understanding of the molecular factors associated with the appearance or progression of cancer may allow the development of therapeutic alternatives. Increasing data highlight the role of long non-coding RNAs in many diseases, including cancer. The long non-coding RNA H19 was the first discovered riboregulator, and it has been shown to be involved at multiple steps of tumorigenesis. Indeed, this lncRNA exert its action at various molecular scales. Understanding the role of H19 in cancer progression may allow to set up therapeutic strategies to prevent tumor expansion and metastatic dissemination. In this review, we will summarize the overexpression of the long non-coding RNA H19 in several types of cancer and the multiple implications of the long non-coding RNA H19 in the different hallmarks that define human cancer.

Among pediatric brain tumors, Diffuse Intrinsic Pontine Gliomas (DIPGs) display a particularly dismal prognosis, highlighted by their median survival lower than one year. Indeed, DIPGs’ location and infiltrative properties preclude their surgical resection. Moreover, DIPGs poorly respond to chemotherapeutic agents. In this context, the only treatment for these tumors remains palliative radiotherapy, systematically followed by tumor progression. In addition to their resistance to therapies, DIPGs are characterized by recurrent histone H3 mutations. The H3.3K27M mutation is the most frequent and results from a heterozygous single nucleotide variant in the H3F3A gene, inducing the lysine 27 substitution by a methionine. Although H3.3K27M’s driver role in DIPGs tumorigenesis is now established, its role in their chemo- and radioresistance remains unclear. Aiming to decipher the potential role of this mutation in pediatric gliomas’ resistance to therapies, we established isogenic cellular models of H3.3K27M induction and reversion.We first induced H3.3K27M mutation in three initially non-mutated supratentorial pediatric glioma cell lines. Thus, we generated models that stably expressed the dominant-negative H3.3K27M or the wild type H3.3 as controls. Complementarily, to study H3.3K27M roles in a DIPG cell context, we also developed H3.3K27M reversion models in two initially mutated DIPG cell lines by applying a gene-editing strategy based on the combinatorial use of the CRISPR/Cas9 technology and an insert.We showed that H3.3K27M induction in Res259 and KNS42 cells conferred a radioresistant phenotype to a fractionated radiotherapy schedule. Besides, we performed a screening of 80 anti-cancer drugs, which revealed a differential impact of the mutation on the drug sensitivity profiles of our three H3.3K27M-induced cell lines. These results indicate that H3.3K27M can control pediatric glioma cells’ resistance to therapies, but in a heterogeneous way depending on the cellular context. Along this line, we are currently characterizing the chemo- and radiotherapy response of our new DIPG H3.3K27M-reversed models. Altogether, our first results support a role for H3.3K27M in pediatric gliomas resistance to treatments, and our complementary models pave the way for identifying new H3.3K27M-dependent mechanisms and promising targets to sensitize DIPGs to therapies. Citation Format: Andria Rakotomalala, Paul Lewandowski, Quentin Bailleul, Clara Savary, Mélanie Arcicasa, Christine Bal, Maud Hamadou, Paul Huchedé, Audrey Restouin, Remy Castellano, Yves Collette, Audrey Vincent, Pierre-Olivier Angrand, Eric Adriaenssens, Xuefen Le Bourhis, Pierre Leblond, Marie Castets, Eddy Pasquier, Alessandro Furlan, Samuel Meignan. Engineering new cellular models to decipher H3.3K27M mutation role in DIPGs' resistance to therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1671.

Tremendous data have been accumulated in the effort to understand chemoresistance of triple negative breast cancer (TNBC). However, modifications in cancer cells surviving combined and sequential treatment still remain poorly described. In order to mimic clinical neoadjuvant treatment, we first treated MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide for 2 days, and then with paclitaxel for another 2 days. After 4 days of recovery, persistent cells surviving the treatment were characterized at both cellular and molecular level. Persistent cells exhibited increased growth and were more invasive in vitro and in zebrafish model. Persistent cells were enriched for vimentinhigh sub-population, vimentin knockdown using siRNA approach decreased the invasive and sphere forming capacities as well as Akt phosphorylation in persistent cells, indicating that vimentin is involved in chemotherapeutic treatment-induced enhancement of TNBC aggressiveness. Interestingly, ectopic vimentin overexpression in native cells increased cell invasion and sphere formation as well as Akt phosphorylation. Furthermore, vimentin overexpression alone rendered the native cells resistant to the drugs, while vimentin knockdown rendered them more sensitive to the drugs. Together, our data suggest that vimentin could be considered as a new targetable player in the ever-elusive status of drug resistance and recurrence of TNBC.

Simple Summary Despite many therapeutic approaches attempted over the last years, the prognosis of children with high-grade glioma or diffuse intrinsic pontine glioma remains dismal. Hypoxia-activated prodrugs (HAPs) were developed to target hypoxic areas within solid tumors as gliomas. Evofosfamide (Evo) is a 2nd generation HAP exhibiting significant preclinical and clinical activities against adult glioblastoma. We thus investigated the potential of Evo in six pediatric glioma cell lines. Interestingly, we showed that the growth of all cell lines was inhibited by Evo, mainly under hypoxia as expected. We also evidenced a significant synergism between Evo and three drugs widely used in pediatric oncology. Finally, Evo appeared able to potentiate the effect of ionizing radiations. Since these tumors are highly hypoxic and Evo appears effective in hypoxic glioma cells as a single drug and in combination with radio- and chemotherapy, hypoxia-activated prodrugs could represent a promising therapeutic option for children with brain tumors. Abstract Hypoxia is a hallmark of many solid tumors and is associated with resistance to anticancer treatments. Hypoxia-activated prodrugs (HAPs) were developed to target the hypoxic regions of these tumors. Among 2nd generation HAPs, Evofosfamide (Evo, also known as TH-302) exhibits preclinical and clinical activities against adult glioblastoma. In this study, we evaluated its potential in the field of pediatric neuro-oncology. We assessed the efficacy of Evo in vitro as a single drug, or in combination with SN38, doxorubicin, and etoposide, against three pediatric high-grade glioma (pHGG) and three diffuse intrinsic pontine glioma (DIPG) cell lines under hypoxic conditions. We also investigated radio-sensitizing effects using clonogenic assays. Evo inhibited the growth of all cell lines, mainly under hypoxia. We also highlighted a significant synergism between Evo and doxorubicin, SN38, or etoposide. Finally, Evo radio-sensitized the pHGG cell line tested, both with fractionated and single-dose irradiation schedules. Altogether, we report here the first preclinical proof of evidence about Evofosfamide efficiency against hypoxic pHGG and DIPG cells. Since such tumors are highly hypoxic, and Evo potentiates the effect of ionizing radiation and chemotherapy, it appears as a promising therapeutic strategy for children with brain tumors.

Accumulating data highlight the role of neurotrophins and their receptors in human breast cancer. This family includes nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), both synthetized as proneurotrophins (proNGF and proBDNF). (pro)NGF and (pro)BDNF initiate their biological effects by binding to both their specific receptors TrkA and TrkB, respectively, and the common receptor p75NTR. Currently, no data are available about their expression and potential role in canine mammary tumors. The aim of this study was to investigate expression of proNGF and BDNF as well as their receptors TrkA, TrkB, and p75NTR in canine mammary carcinomas, and to correlate them with clinicopathological parameters (grade, histological type, lymph node status, recurrence, and distant metastasis) and survival. Immunohistochemistry was performed on serial sections of 96 canine mammary carcinomas with antibodies against proNGF, BDNF, TrkA, TrkB, and p75NTR. Of the 96 carcinomas, proNGF expression was detected in 71 (74%), BDNF in 79 (82%), TrkA in 94 (98%), TrkB in 35 (37%), and p75NTR in 44 (46%). No association was observed between proNGF, BDNF, or TrkA expression and either clinicopathological parameters or survival. TrkB and p75NTR expression were associated with favorable clinicopathological parameters as well as better overall survival.

The Polycomb Repressive Complex 1 (PRC1) is a chromatin-associated protein complex involved in transcriptional repression of hundreds of genes controlling development and differentiation processes, but also involved in cancer and stem cell biology. Within the canonical PRC1, members of Pc/CBX protein family are responsible for the targeting of the complex to specific gene loci. In mammals, the Pc/CBX protein family is composed of five members generating, through mutual exclusion, different PRC1 complexes with potentially distinct cellular functions. Here, we performed a global analysis of the cbx gene family in 68 teleost species and traced the distribution of the cbx genes through teleost evolution in six fish super-orders. We showed that after the teleost-specific whole genome duplication, cbx4, cbx7 and cbx8 are retained as pairs of ohnologues. In contrast, cbx2 and cbx6 are present as pairs of ohnologues in the genome of several teleost clades but as singletons in others. Furthermore, since zebrafish is a widely used vertebrate model for studying development, we report on the expression of the cbx family members during zebrafish development and in adult tissues. We showed that all cbx genes are ubiquitously expressed with some variations during early development.

Primarily used in genetic studies of development, the zebrafish (Danio rerio) has rapidly emerged as a promising animal model of human cancer. Cancer cell transplantation in zebrafish constitutes a key platform for clinical research since it allows to study cellular and molecular events involved in various aspects of tumorigenesis and to evaluate the efficacy of therapeutic molecules in vivo. Applied to patient-derived cells, the xenotransplantation approach in zebrafish allows to define the most appropriate therapeutic strategies for specific alterations found in patients in the context of personalized medicine. This review discusses the zebrafish transplantation model for the study of cancer development and drug discovery.

Supplemental Material, Combined_supplemental_materials-Rogez_et_al for Expression and Prognostic Significance of Neurotrophins and Their Receptors in Canine Mammary Tumors by Bernadette Rogez, Quentin Pascal, Audrey Bobillier, François Machuron, Robert-Alain Toillon, Dominique Tierny, Valérie Chopin and Xuefen Le Bourhis in Veterinary Pathology

En raison de sa simplicité d’utilisation, la culture cellulaire reste le matériel de choix pour l’étude des mécanismes impliqués dans le cancer. Toutefois, la mise en culture des cellules est souvent responsable de la perte de certaines de leurs caractéristiques et de l’acquisition de nouvelles propriétés. Aussi, les modèles animaux sont essentiels à l’étude des phénomènes physiologiques et pathologiques comme la tumorigenèse. Parmi les modèles animaux utilisés en cancérologie, le poisson zèbre apparaît comme un modèle particulièrement pertinent. En effet, la facilité et les faibles coûts d’élevage du poisson zèbre, tout comme le large éventail de possibilités en termes de manipulation génétique et d’imageriein vivoclassent ce modèle vertébré parmi les outils les plus remarquables pour l’étude du cancer. Cette revue a pour objectif de faire le point sur les apports et le potentiel du poisson zèbre dans la lutte contre le cancer.

The histone lysine methyltransferase EZH2, as part of the Polycomb Repressive Complex 2 (PRC2), mediates H3K27me3 methylation which is involved in gene expression program repression. Through its action, EZH2 controls cell-fate decisions during the development and the differentiation processes. Here, we report the generation and the characterization of an ezh2 -deficient zebrafish line. In contrast to its essential role in mouse early development, loss of ezh2 function does not affect zebrafish gastrulation. Ezh2 zebrafish mutants present a normal body plan but die at around 12 dpf with defects in the intestine wall, due to enhanced cell death. Thus, ezh2 -deficient zebrafish can initiate differentiation toward the different developmental lineages but fail to maintain the intestinal homeostasis. Expression studies revealed that ezh2 mRNAs are maternally deposited. Then, ezh2 is ubiquitously expressed in the anterior part of the embryos at 24 hpf, but its expression becomes restricted to specific regions at later developmental stages. Pharmacological inhibition of Ezh2 showed that maternal Ezh2 products contribute to early development but are dispensable to body plan formation. In addition, ezh2 -deficient mutants fail to properly regenerate their spinal cord after caudal fin transection suggesting that Ezh2 and H3K27me3 methylation might also be involved in the process of regeneration in zebrafish.

Resume L’efficacite des therapies ciblees est souvent compromise par l’emergence de resistance et, dans ces situations, l’inhibition du recepteur a activite tyrosine kinase MET apparait etre une option therapeutique pertinente. En effet, differentes etudes ont identifie un rationnel fort a l’association d’un inhibiteur de MET a une autre therapie ciblee. En clinique, cette association pourrait etre une perspective therapeutique interessante en premiere ligne, pour dejouer une resistance primaire, comme en nieme ligne pour pallier une resistance acquise. L’utilisation d’inhibiteurs diriges contre MET se revele particulierement prometteuse lorsqu’ils sont associes notamment aux therapies ciblant l’ epidermal growth factor receptor (EGFR), le vascular endothelial growth factor (VEGF), HER2 et BRAF. En effet, l’amplification genique de met est la deuxieme alteration la plus frequente conduisant a une resistance aux TKI EGFR dans les cancers bronchiques non a petites cellules (CBNPC) EGFR mutes. D’autre part, l’hypoxie peut entrainer une augmentation de l’expression de MET et cette activation aberrante semble impliquee dans l’echappement aux anti-VEGF. Dans le cancer du sein HER2 positif, l’activation de MET conduit a une persistance de la croissance tumorale. Enfin, 14 % des patients porteurs d’un melanome BRAF V600E mute, presentent une resistance primaire aux inhibiteurs de BRAF liee a une secretion d’HGF (ligand de MET) par le stroma, induisant l’activation du recepteur. Dans ce contexte, de nombreux essais cliniques, ici repertories, sont en cours dans differents cancers associant un anti-MET a une autre therapie ciblant EGFR, BRAF, VEGF ou HER2. Cette revue met en lumiere le benefice que l’inhibition de MET devrait apporter aux patients atteints d’un cancer refractaire aux therapies ciblees.

International audience; Many solid cancers are hierarchically organized with a small number of cancer stem cells (CSCs) able to regrow a tumor, while their progeny lacks this feature. Breast CSC is known to contribute to therapy resistance. The study of those cells is usually based on their cell-surface markers like CD44high /CD24low/neg or their aldehyde dehydrogenase (ALDH) activity. However, these markers cannot be used to track the dynamics of CSC. Here, a transcriptomic analysis is performed to identify segregating gene expression in CSCs and non-CSCs, sorted by Aldefluor assay. It is observed that among ALDH-associated genes, only ALDH1A1 isoform is increased in CSCs. A CSC reporter system is then developed by using a far red-fluorescent protein (mNeptune) under the control of ALDH1A1 promoter. mNeptune-positive cells exhibit higher sphere-forming capacity, tumor formation, and increased resistance to anticancer therapies. These results indicate that the reporter identifies cells with stemness characteristics. Moreover, live tracking of cells in a microfluidic system reveals a higher extravasation potential of CSCs. Live tracking of non-CSCs under irradiation treatment show, for the first time, live reprogramming of non-CSCs into CSCs. Therefore, the reporter will allow for cell tracking to better understand the implication of CSCs in breast cancer development and recurrence.

International audience; ProNGF expression has been linked to several types of cancers including breast cancer, and we have previously shown that proNGF stimulates breast cancer invasion in an autocrine manner through membrane receptors sortilin and TrkA. However, little is known regarding TrkA-associated protein partners upon proNGF stimulation. By proteomic analysis and proximity ligation assays, we found that proNGF binding to sortilin induced sequential formation of the functional sortilin/TrkA/EphA2 complex, leading to TrkA-phosphorylation dependent Akt activation and EphA2-dependent Src activation. EphA2 inhibition using siRNA approach abolished proNGF-stimulated clonogenic growth of breast cancer cell lines. Combinatorial targeting of TrkA and EphA2 dramatically reduced colony formation in vitro, primary tumor growth and metastatic dissemination towards the brain in vivo. Finally, proximity ligation assay in breast tumor samples revealed that increased TrkA/EphA2 proximity ligation assay signals were correlated with a decrease of overall survival in patients. All together, these data point out the importance of TrkA/EphA2 functional association in proNGF-induced tumor promoting effects, and provide a rationale to target proNGF/TrkA/EphA2 axis by alternative methods other than the simple use of tyrosine kinase inhibitors in breast cancer.

Trimethylation on H3K27 mediated by Polycomb Repressive Complex 2 (PRC2) is required to control gene repression programs involved in development, regulation of tissue homeostasis or maintenance and lineage specification of stem cells. In Drosophila, the PRC2 catalytic subunit is the single protein E(z), while in mammals this function is fulfilled by two proteins, Ezh1 and Ezh2. Based on database searches, we propose that Ezh1 arose from an Ezh2 gene duplication that has occurred in the common ancestor to elasmobranchs and bony vertebrates. Expression studies in zebrafish using in situ hybridization and RT-PCR followed by the sequencing of the amplicon revealed that ezh1 mRNAs are maternally deposited. Then, ezh1 transcripts are ubiquitously distributed in the entire embryo at 24 hpf and become more restricted to anterior part of the embryo at later developmental stages. To unveil the function of ezh1 in zebrafish, a mutant line was generated using the TALEN technology. Ezh1-deficient mutant fish are viable and fertile, but the loss of ezh1 function is responsible for the earlier death of ezh2 mutant larvae indicating that ezh1 contributes to zebrafish development in absence of zygotic ezh2 gene function. Furthermore, we show that presence of ezh1 transcripts from the maternal origin accounts for the delayed lethality of ezh2-deficient larvae.

CD44+/CD24–phenotype has been used to identify human and canine mammary cancer stem-like cells. In canine mammary tumors, CD44+/CD24–phenotype has been associated with high grade and lymph node infiltration. However, several studies have reported opposing results regarding the clinical significance of phenotypic groups formed by the combination of CD44 and CD24 in both human and canine mammary tumors. So far, no study has investigated the correlation between these phenotypes and survival in dogs. The aim of this study was to investigate the expression and distribution of CD44 and CD24 in canine mammary carcinomas and to correlate them with histological diagnosis and survival in a well-characterized cohort. Immunohistochemistry was performed in 96 mammary carcinomas with antibodies against CD44 and CD24. Expression of CD44+and CD44+/CD24–phenotype was detected in 75 of 96 (78%) and 63 of 96 (65.6%) carcinomas, respectively. Their expression was associated with tumor type, occurring more often in tubular complex carcinomas than in solid carcinomas. CD44+/CD24–phenotype was associated with a better overall survival ( P = .001). CD24+expression was detected in 52 of 96 tumors (54%) and CD44–/CD24+phenotype in 39 of 96 tumors (40.6%). Both were associated with poor clinicopathological parameters (high grade, and emboli). No correlation with overall survival was observed. CD44+/CD24–expression was associated with a better prognosis and occurred at high frequency and high level, indicating that this phenotype is not suitable to detect cancer stem cells in canine mammary carcinomas. Although further studies are needed, our results suggest that CD24 may constitute a valuable marker of poor prognosis for canine mammary carcinomas.

Cancer stem cells (CSCs), are thought to be at the origin of tumor development and resistance to therapies. Thus, a better understanding of the molecular mechanisms involved in the control of CSC stemness is essential to the design of more effective therapies for cancer patients. Cancer cell stemness and the subsequent expansion of CSCs are regulated by micro-environmental signals including neurotrophins. Over the years, the roles of neurotrophins in tumor development have been well established and regularly reviewed. Especially, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are reported to stimulate tumor cell proliferation, survival, migration and/or invasion, and favors tumor angiogenesis. More recently, neurotrophins have been reported to regulate CSCs. This review briefly presents neurotrophins and their receptors, summarizes their roles in different cancers, and discusses the emerging evidence of neurotrophins-induced enrichment of CSCs as well as the involved signaling pathways.

Diffuse Intrinsic Pontine Glioma is one of the worst pediatric brain tumors regarding prognosis due notably to intrinsic cell resistance to radio and chemotherapy. One of the main characteristics of DIPG cells is the presence of a mono-allelic mutation on the lysine 27 of histone H3 (H3K27M). This mutation inhibits the trimethylation of this lysine that leads to strong modifications of gene expression. Until now, even though this mutation seems to be a driver event in tumorigenesis, its role in cell resistance to treatment has not been deciphered, due to a lack of relevant cellular models. This way, in order to evaluate the role of the mutation on resistance to treatment, we first induced the mutation in three H3K27-unmutated pediatric glioma cell lines. In parallel, using the CRISPR/Cas9 technology, we are establishing DIPG cellular models in which the mutation will be reversed. By gene trapping approach, we aim to restore an H3F3Awt/wt genotype. After validation, these models would result in original tools to study the impact of H3K27M mutation in DIPG cells resistance to treatment. For the model of induction, the transfected cell lines exhibit the mutation accompanied by a loss of H3K27me3 mark and H3.3 overexpression. For now, we showed an increased cell growth due to the mutation in two cell lines, under normoxia as well as under hypoxia. On contrary there was no impact on resistance to chemotherapy or ionizing radiation. In the third cell line, we didn't observe any impact on cell growth, but an increase of cell radioresistance. Concerning the mutation reversion, our preliminary results show homologous recombination at the right locus in the genome, and some clones present a loss of the mutation confirmed by sequencing. After the removal of resistance cassette by action of a recombinase protein, we will be able to evaluate the biological effects of mutation reversion. To sum up, these different models would allow us to decipher cellular and molecular mechanism induced by the H3.3K27M mutation in DIPG cells including resistance to treatment, and thus, to possibly identify putative therapeutic targets. Citation Format: Quentin Bailleul, Mélanie Arcicasa, Audrey Hochart, Andria Rakotomalala, Marie Castets, Eddy Pasquier, Pierre-Olivier Angrand, Eric Adriaenssens, Xuefen Le Bourhis, Pierre Leblond, Samuel Meignan. Impact of H3.3K27M mutation on diffuse intrinsic pontine glioma's resistance to treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5005.

Zebrafish (Danio rerio) is an excellent model to study a wide diversity of human cancers. In this review, we provide an overview of the genetic and reverse genetic toolbox allowing the generation of zebrafish lines that develop tumors. The large spectrum of genetic tools enables the engineering of zebrafish lines harboring precise genetic alterations found in human patients, the generation of zebrafish carrying somatic or germline inheritable mutations or zebrafish showing conditional expression of the oncogenic mutations. Comparative transcriptomics demonstrate that many of the zebrafish tumors share molecular signatures similar to those found in human cancers. Thus, zebrafish cancer models provide a unique in vivo platform to investigate cancer initiation and progression at the molecular and cellular levels, to identify novel genes involved in tumorigenesis as well as to contemplate new therapeutic strategies.

Glioblastoma is the most common malignant brain tumor with extremely poor patient survival of approximately 15 months (1). This poor prognosis reflects therapeutic resistance and is attributed, at least in part, to the existence of a subset of cells named glioma stem-like cells (GSCs) (2,3). Numerous studies indicate that glioblastoma is heterogeneous, composing of a rare population of GSCs and more differentiated glioma cells (DGCs).

Although cell culture and mouse models will remain a cornerstone of cancer research, the unique capabilities of the zebrafish outline the potential of this model for shedding light on cancer biology in vivo. Zebrafish develops cancers spontaneously, after chemical mutagenesis or through genetic manipulations. Furthermore, zebrafish cancers are similar to human tumors at the histological and molecular levels allowing the study of tumor initiation, progression and heterogeneity. Xenotransplantation of human cancer cells in embryos or adult zebrafish presents the advantage of following cancer cell behavior in vivo. Finally, zebrafish embryos are used in molecule screens and contribute to the identification of novel anti-cancer therapeutic strategies. Here, we review different involvements of the zebrafish model in cancer research.

Supplemental Material, DS1_VET_10.1177_0300985818813653 for CD44 and CD24 Expression and Prognostic Significance in Canine Mammary Tumors by Bernadette Rogez, Quentin Pascal, Audrey Bobillier, François Machuron, Chann Lagadec, Dominique Tierny, Xuefen Le Bourhis, and Valérie Chopin in Veterinary Pathology

The discovery of cancer stem cells (CSCs) fundamentally advanced our understanding of the mechanisms governing breast cancer development. However, the stimuli that control breast CSC self-renewal and differentiation have still not been fully detailed. We previously showed that nerve growth factor (NGF) and its precursor proNGF can stimulate breast cancer cell growth and invasion in an autocrine manner. In this study, we investigated the effects of NGF and proNGF on the breast CSC compartment and found that NGF or proNGF enrich for CSCs in several breast cancer cell lines. This enrichment appeared to be achieved by increasing the number of symmetric divisions of quiescent/slow-proliferating CSCs. Interestingly, in vitro NGF pretreatment of MCF-7 luminal breast cancer cells promoted epithelial to mesenchymal transition in tumors of severe combined immunodeficient mice. Furthermore, p75NTR, the common receptor for both neurotrophins and proneurotrophins, mediated breast CSC self-renewal by regulating the expression of pluripotency transcription factors. Our data indicate, for the first time, that the NGF/proNGF/p75NTR axis plays a critical role in regulating breast CSC self-renewal and plasticity. Stem Cells 2015;33:342–353

p75(NTR), the common receptor for both neurotrophins and proneurotrophins, has been widely studied because of its role in many tissues, including the nervous system. More recently, a close relationship between p75(NTR) expression and pluripotency has been described. p75(NTR) was shown to be expressed in various types of stem cells and has been used to prospectively isolate stem cells with different degrees of potency. Here, we give an overview of the current knowledge on p75(NTR) in stem cells, ranging from embryonic to adult stem cells, and cancer stem cells. In an attempt to address its potential role in the control of stem cell biology, the molecular mechanisms underlying p75(NTR) signaling in different models are also highlighted. p75(NTR)-mediated functions include survival, apoptosis, migration, and differentiation, and depend on cell type, (pro)neurotrophin binding, interacting transmembrane co-receptors expression, intracellular adaptor molecule availability, and post-translational modifications, such as regulated proteolytic processing. It is therefore conceivable that p75(NTR) can modulate cell-fate decisions through its highly ramified signaling pathways. Thus, elucidating the potential implications of p75(NTR) activity as well as the underlying molecular mechanisms of p75(NTR) will shed new light on the biology of both normal and cancer stem cells.

Distant metastases account for the majority of cancer-related deaths in breast cancer. The rate and site of metastasis differ between estrogen receptor (ER)-negative and ER-positive tumours, and metastatic fate can be very diverse even within the ER-negative group. Characterisation of new pro-metastatic markers may help to identify patients with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metastasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist in carbohydrate molecules, such as sialyl-Lewis x antigen (sLex), borne by glycoproteins or glycolipids on the cancer cell surface. We have previously demonstrated that the molecular scaffold presenting sLex to selectins (e.g. glycolipid vs. glycoproteins) was crucial for these interactions to occur. Moreover, we reported that detection of sLex alone in breast carcinomas was only of limited prognostic value. However, since sLex was found to be carried by several glycoproteins in cancer cells, we hypothesized that the combination of the carbohydrate with its carriers could be more relevant than each marker independently. In this study, we addressed this question by analysing sLex expression together with two glycoproteins (BST-2 and LGALS3BP), shown to interact with E-selectin in a carbohydrate-dependent manner, in a cohort of 249 invasive breast cancers. We found both glycoproteins to be associated with distant metastasis risk and poorer survival. Importantly, concomitant high expression of BST-2 with sLex defined a sub-group of patients with ER-negative tumours displaying higher risks of liver and brain metastasis and a 3-fold decreased survival rate.

Overcoming the drug resistance remains a crucial issue in cancer treatment. For refractory patients, the use of MET receptor tyrosine kinase inhibitors seems to be hopeful. Indeed, important mechanisms underlying drug resistance argue for association of MET inhibitors with targeted therapies, both on first-line to prevent a primary resistance and on the second line to overcoming acquired resistance. Indeed, met gene amplification is the second most common alteration involved in acquired resistance to anti-epidermal growth factor receptor (EGFR) therapies in non-small cells lung cancer (NSCLC). Hypoxia, for its part, can activate MET transcription and amplifies HGF signaling resulting in MET activation, which could be involved in vascular endothelial growth factor (VEGF) inhibitors escape. In HER2 positive breast cancers, MET amplification may also induce tumor cells a hatch escape, resulting in secondary resistance. Finally, some patients with BRAF mutated melanoma exhibit primary resistance to BRAF inhibition by stromal HGF (ligand of MET) secretion resulting in MET receptor activation. Experimental data highlight the role of MET in primary and secondary resistance and encourage combined treatments including MET inhibitors. In this context, several promising clinical trials are in progress in numerous cancers (NSCLC, melanoma, breast cancer, glioblastoma…) using combination of anti-MET and other specific therapies targeting EGFR, BRAF, VEGF or HER2. This review summarizes the potential benefits that MET inhibition should provide to patients with cancer refractory to targeted therapies.

The post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is regulated by a unique couple of enzymes. O-GlcNAc transferase (OGT) transfers the GlcNAc residue from UDP-GlcNAc, the final product of the hexosamine biosynthetic pathway (HBP), whereas O-GlcNAcase (OGA) removes it. This study and others show that OGT and O-GlcNAcylation levels are increased in cancer cell lines. In that context, we studied the effect of OGT silencing in the colon cancer cell lines HT29 and HCT116 and the primary colon cell line CCD841CoN. Herein, we report that OGT silencing diminished proliferation, in vitro cell survival and adhesion of primary and cancer cell lines. SiOGT dramatically decreased HT29 and CCD841CoN migration, CCD841CoN harboring high capabilities of migration in Boyden chamber system when compared to HT29 and HCT116. The expression levels of actin and tubulin were unaffected by OGT knockdown but siOGT seemed to disorganize microfilament, microtubule, and vinculin networks in CCD841CoN. While cancer cell lines harbor higher levels of OGT and O-GlcNAcylation to fulfill their proliferative and migratory properties, in agreement with their higher consumption of HBP main substrates glucose and glutamine, our data demonstrate that OGT expression is not only necessary for the biological properties of cancer cell lines but also for normal cells.