ProNGF promotes brain metastasis through TrkA/EphA2 induced Src activation in triple negative breast cancer cells.

BACKGROUND: Triple-Negative Breast Cancer is particularly aggressive, and its metastasis to the brain has a significant psychological impact on patients' quality of life, in addition to reducing survival. The development of brain metastases is particularly harmful in triple-negative breast cancer (TNBC). To date, the mechanisms that induce brain metastasis in TNBC are poorly understood. METHODS: Using a human blood-brain barrier (BBB) in vitro model, an in vitro 3D organotypic extracellular matrix, an ex vivo mouse brain slices co-culture and in an in vivo xenograft experiment, key step of brain metastasis were recapitulated to study TNBC behaviors. RESULTS: In this study, we demonstrated for the first time the involvement of the precursor of Nerve Growth Factor (proNGF) in the development of brain metastasis. More importantly, our results showed that proNGF acts through TrkA independent of its phosphorylation to induce brain metastasis in TNBC. In addition, we found that proNGF induces BBB transmigration through the TrkA/EphA2 signaling complex. More importantly, our results showed that combinatorial inhibition of TrkA and EphA2 decreased TBNC brain metastasis in a preclinical model. CONCLUSIONS: These disruptive findings provide new insights into the mechanisms underlying brain metastasis with proNGF as a driver of brain metastasis of TNBC and identify TrkA/EphA2 complex as a potential therapeutic target.

Influence of EGF and pro-NGF on EGFR/SORTILIN interaction and clinical impact in head and neck squamous cell carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) remains a cancer with a poor prognosis, with a 5-year survival rate of less than 50%. Although epidermal growth factor receptor (EGFR) is almost always overexpressed, targeted anti-EGFR therapies have modest efficacy and are mainly used in palliative care. Growth factors such as Nerve Growth Factor (NGF) and its precursor proNGF have been shown in our laboratory to play a role in tumor growth and aggressiveness. Interestingly, an interaction between Sortilin, a proNGF receptor, and EGFR has been observed. This interaction appears to interfere with the pro-oncogenic signaling of EGF and modulate the membrane expression of EGFR. The aim of this study was to characterize this interaction biologically, to assess its impact on clinical prognosis and to analyze its role in the cellular trafficking of EGFR. Using immunohistochemical staining on tumor sections from patients treated at our university center and PLA (Proximity Ligation Assay) labeling, we showed that Sortilin expression is significantly associated with reduced 5-year survival. However, when Sortilin was associated with EGFR, this association was not found. Using the Cal-27 and Cal-33 cancer cell lines, we observed that proNGF reduces the effects of EGF on cell growth by inducing the internalization of its receptor. These results therefore suggest a regulatory role for Sortilin in the degradation or renewal of EGFR on the membrane. It would be interesting in future work to show the intracellular fate of EGFR and the role of (pro)neurotrophins in these mechanisms.

TrkA Co-Receptors: The Janus Face of TrkA?

Larotrectinib and Entrectinib are specific pan-Trk tyrosine kinase inhibitors (TKIs) approved by the Food and Drug Administration (FDA) in 2018 for cancers with an NTRK fusion. Despite initial enthusiasm for these compounds, the French agency (HAS) recently reported their lack of efficacy. In addition, primary and secondary resistance to these TKIs has been observed in the absence of other mutations in cancers with an NTRK fusion. Furthermore, when TrkA is overexpressed, it promotes ligand-independent activation, bypassing the TKI. All of these clinical and experimental observations show that genetics does not explain all therapeutic failures. It is therefore necessary to explore new hypotheses to explain these failures. This review summarizes the current status of therapeutic strategies with TrkA inhibitors, focusing on the mechanisms potentially involved in these failures and more specifically on the role of TrkA.

ABSP: an automated R tool to efficiently analyze region-specific CpG methylation from bisulfite sequencing PCR.

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.

TRPM8-Rap1A Interaction Sites as Critical Determinants for Adhesion and Migration of Prostate and Other Epithelial Cancer Cells.

Emerging evidence indicates that the TRPM8 channel plays an important role in prostate cancer (PCa) progression, by impairing the motility of these cancer cells. Here, we reveal a novel facet of PCa motility control via direct protein-protein interaction (PPI) of the channel with the small GTPase Rap1A. The functional interaction of the two proteins was assessed by active Rap1 pull-down assays and live-cell imaging experiments. Molecular modeling analysis allowed the identification of four putative residues involved in TRPM8-Rap1A interaction. Point mutations of these sites impaired PPI as shown by GST-pull-down, co-immunoprecipitation, and PLA experiments and revealed their key functional role in the adhesion and migration of PC3 prostate cancer cells. More precisely, TRPM8 inhibits cell migration and adhesion by trapping Rap1A in its GDP-bound inactive form, thus preventing its activation at the plasma membrane. In particular, residues E207 and Y240 in the sequence of TRPM8 and Y32 in that of Rap1A are critical for the interaction between the two proteins not only in PC3 cells but also in cervical (HeLa) and breast (MCF-7) cancer cells. This study deepens our knowledge of the mechanism through which TRPM8 would exert a protective role in cancer progression and provides new insights into the possible use of TRPM8 as a new therapeutic target in cancer treatment.

Direct interaction of TrkA/CD44v3 is essential for NGF-promoted aggressiveness of breast cancer cells.

BACKGROUND: CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer. METHODS: After determining which CD44 isoforms (variants) are involved in forming the TrkA/CD44 complex using proximity ligation assays, we investigated the molecular determinants of this interaction. By molecular modeling, we isolated the amino acids involved and confirmed their involvement using mutations. A CD44v3 mimetic peptide was then synthesized to block the TrkA/CD44v3 interaction. The effects of this peptide on the growth, migration and invasion of xenografted triple-negative breast cancer cells were assessed. Finally, we investigated the correlations between the expression of the TrkA/CD44v3 complex in tumors and histo-pronostic parameters. RESULTS: We demonstrated that isoform v3 (CD44v3), but not v6, binds to TrkA in response to NGF stimulation. The final 10 amino acids of exon v3 and the TrkA H112 residue are necessary for the association of CD44v3 with TrkA. Functionally, the CD44v3 mimetic peptide impairs not only NGF-induced RhoA activation, clonogenicity, and migration/invasion of breast cancer cells in vitro but also tumor growth and metastasis in a xenograft mouse model. We also detected TrkA/CD44v3 only in cancerous cells, not in normal adjacent tissues. CONCLUSION: Collectively, our results suggest that blocking the CD44v3/TrkA interaction can be a new therapeutic option for triple-negative breast cancers.

Loss of Polycomb Repressive Complex 2 Function Alters Digestive Organ Homeostasis and Neuronal Differentiation in Zebrafish.

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.

Vimentin Promotes the Aggressiveness of Triple Negative Breast Cancer Cells Surviving Chemotherapeutic Treatment.

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.

ORAI3 silencing alters cell proliferation and promotes mitotic catastrophe and apoptosis in pancreatic adenocarcinoma.

Changes in cytosolic free Ca2+ concentration play a central role in many fundamental cellular processes including muscle contraction, neurotransmission, cell proliferation, differentiation, gene transcription and cell death. Many of these processes are known to be regulated by store-operated calcium channels (SOCs), among which ORAI1 is the most studied in cancer cells, leaving the role of other ORAI channels yet inadequately addressed. Here we demonstrate that ORAI3 channels are expressed in both normal (HPDE) and pancreatic ductal adenocarcinoma (PDAC) cell lines, where they form functional channels, their knockdown affecting store operated calcium entry (SOCE). More specifically, ORAI3 silencing increased SOCE in PDAC cell lines, while decreasing SOCE in normal pancreatic cell line. We also show the role of ORAI3 in proliferation, cell cycle, viability, mitotic catastrophe and cell death. Finally, we demonstrate that ORAI3 silencing impairs pancreatic tumor growth and induces cell death in vivo, suggesting that ORAI3 could represent a potential therapeutic target in PDAC treatment.

Expression and Prognostic Significance of Neurotrophins and Their Receptors in Canine Mammary 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.

Small Structural Differences between Two Ferrocenyl Diphenols Determine Large Discrepancies of Reactivity and Biological Effects.

The ferrocenyl diphenol complexes 1,1-bis(4'-hydroxyphenyl)-2-ferrocenyl-but-1-ene (1) and 1,2-bis(4'-hydroxyphenyl)-1-ferrocenyl-but-1-ene [(Z)-2], which differ by the relative position of the two phenolic substituents, display dramatically different antiproliferative activities on cancer cells (1 is far more cytotoxic than 2). In this study, our goal was to discover the origin of this difference by comparing their reactivity and biological behaviour. In terms of common behaviour, we found that 1 and 2 are both efficient inhibitors of thioredoxin reductase (TrxR) in vitro after oxidation by a horseradish peroxidase/H2 O2 system. However, as 1 is only a moderate inhibitor of TrxR in MDA-MB-231 cells, TrxR is probably not the major target responsible for the cytotoxicity of 1. In terms of differences, we noted that 1 induced a significant redox imbalance characterised by lipid peroxidation and thiol oxidation, and a moderate decrease of the mitochondrial membrane potential in breast cancer cells, whereas 2 has almost no effect. These results underline the importance of the trans configuration in the ferrocenyl-double bond-phenol motif, which is present in 1 but is cis in (Z)-2.

ProNGF increases breast tumor aggressiveness through functional association of TrkA with EphA2.

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.

Differential recruitment of CD44 isoforms by ErbB ligands reveals an involvement of CD44 in breast cancer.

Members of the CD44 family of transmembrane glycoproteins control cell signaling pathways from numerous cell surface receptors, including receptor tyrosine kinases (RTKs). The decisive factor (ligand, RTKs or both) that controls the recruitment of specific CD44 isoforms is still unknown. We investigated this question by using the EGFR signaling pathway, in which one receptor can be activated by a broad range of ligands. By means of siRNA-mediated downregulation of CD44 expression and blocking experiments, we identified CD44v6 as a co-receptor for EGF- and ER-induced ErbB1 activation and for NRG1-induced ErbB3 and ErbB4 activation. In contrast, TGFα is independent of all CD44 isoforms, even though it addresses the same receptor pairs as EGF. Moreover, the heparin-sulfated CD44v3 isoform is required for HB-EGF-induced EGFR signaling. These data suggest that specific CD44 isoforms are recruited in a ligand-dependent manner as co-receptors in the EGFR signaling pathways and that the specificity is determined by the ligand and not by the receptors themselves. The in vivo relevance of this interplay between CD44 isoforms and EGFR ligands is underlined by the decreased metastatic spreading of mammary carcinomas in mice treated with a CD44v6-specific peptide. Most importantly, we found a clear correlation between the presence of CD44v6/ErbB1 complexes in breast cancer patients and lymph node metastases.

The histone lysine methyltransferase Ezh2 is required for maintenance of the intestine integrity and for caudal fin regeneration in zebrafish.

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.

Silencing the Nucleocytoplasmic O-GlcNAc Transferase Reduces Proliferation, Adhesion, and Migration of Cancer and Fetal Human Colon Cell Lines.

The post-translational modification of proteins by O-linked ß-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.

Neurotrophin signaling in cancer stem cells.

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.

NGF-induced TrkA/CD44 association is involved in tumor aggressiveness and resistance to lestaurtinib.

There is accumulating evidence that TrkA and its ligand Nerve Growth Factor (NGF) are involved in cancer development. Staurosporine derivatives such as K252a and lestaurtinib have been developed to block TrkA kinase signaling, but no clinical trial has fully demonstrated their therapeutic efficacy. Therapeutic failures are likely due to the existence of intrinsic signaling pathways in cancer cells that impede or bypass the effects of TrkA tyrosine kinase inhibitors. To verify this hypothesis, we combined different approaches including mass spectrometry proteomics, co-immunoprecipitation and proximity ligation assays. We found that NGF treatment induced CD44 binding to TrkA at the plasma membrane and subsequent activation of the p115RhoGEF/RhoA/ROCK1 pathway to stimulate breast cancer cell invasion. The NGF-induced CD44 signaling was independent of TrkA kinase activity. Moreover, both TrkA tyrosine kinase inhibition with lestaurtinib and CD44 silencing with siRNA inhibited cell growth in vitro as well as tumor development in mouse xenograft model; combined treatment significantly enhanced the antineoplastic effects of either treatment alone. Altogether, our results demonstrate that NGF-induced tyrosine kinase independent TrkA signaling through CD44 was sufficient to maintain tumor aggressiveness. Our findings provide an alternative mechanism of cancer resistance to lestaurtinib and indicate that dual inhibition of CD44 and TrkA tyrosine kinase activity may represent a novel therapeutic strategy.

[Radiation-induces increased tumor cell aggressiveness of tumors of the glioblastomas?]. / La radiothérapie induit-elle une agressivité accrue des cellules tumorales du glioblastome ?

Glioblastoma multiform is the most common and aggressive brain tumor with a worse prognostic. Ionizing radiation is a cornerstone in the treatment of glioblastome with chemo-radiation association being the actual standard. As a paradoxal effect, it has been suggested that radiotherapy could have a deleterious effect on local recurrence of cancer. In vivo studies have studied the effect of radiotherapy on biological modification and pathogenous effect of cancer cells. It seems that ionizing radiations with photon could activate oncogenic pathways in glioblastoma cell lines. We realized a review of the literature of photon-enhanced effect on invasion and migration of glioblastoma cells by radiotherapy.

Radiation-enhanced cell migration/invasion process: a review.

Radiation therapy is a keystone treatment in cancer. Photon radiation has proved its benefits in overall survival in many clinical studies. However, some patients present local recurrences or metastases when cancer cells survive to treatment. Metastasis is a process which includes adhesion of the cell to the extracellular matrix, degradation of the matrix by proteases, cell motility, intravasation in blood or lymphatic vessels, extravasation in distant parenchyma and development of cell colonies. Several studies demonstrated that ionizing radiation might promote migration and invasion of tumor cells by intricate implications in the micro-environment, cell-cell junctions, extracellular matrix junctions, proteases secretion, and induction of epithelial-mesenchymal transition. This review reports various cellular pathways involved in the photon-enhanced cell invasion process for which potential therapeutic target may be employed for enhancing antitumor effectiveness. Understanding these mechanisms could lead to therapeutic strategies to counter the highly invasive cell lines via specific inhibitors or carbon-ion therapy.