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.

Macrophage-Colony-Stimulating Factor Receptor Enhances Prostate Cancer Cell Growth and Aggressiveness In Vitro and In Vivo and Increases Osteopontin Expression.

Prostate cancer is a major public health concern and one of the most prevalent forms of cancer worldwide. The definition of altered signaling pathways implicated in this complex disease is thus essential. In this context, abnormal expression of the receptor of Macrophage Colony-Stimulating Factor-1 (M-CSF or CSF-1) has been described in prostate cancer cells. Yet, outcomes of this expression remain unknown. Using mouse and human prostate cancer cell lines, this study has investigated the functionality of the wild-type CSF-1 receptor in prostate tumor cells and identified molecular mechanisms underlying its ligand-induced activation. Here, we showed that upon CSF-1 binding, the receptor autophosphorylates and activates multiple signaling pathways in prostate tumor cells. Biological experiments demonstrated that the CSF-1R/CSF-1 axis conferred significant advantages in cell growth and cell invasion in vitro. Mouse xenograft experiments showed that CSF-1R expression promoted the aggressiveness of prostate tumor cells. In particular, we demonstrated that the ligand-activated CSF-1R increased the expression of spp1 transcript encoding for osteopontin, a key player in cancer development and metastasis. Therefore, this study highlights that the CSF-1 receptor is fully functional in a prostate cancer cell and may be a potential therapeutic target for the treatment of prostate cancer.

The long non-coding RNA H19: an active player with multiple facets to sustain the hallmarks of cancer.

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.

Telomerase regulation by the long non-coding RNA H19 in human acute promyelocytic leukemia cells.

BACKGROUND: Since tumor growth requires reactivation of telomerase (hTERT), this enzyme is a challenging target for drug development. Therefore, it is of great interest to identify telomerase expression and activity regulators. Retinoids are well-known inducers of granulocytic maturation associated with hTERT repression in acute promyelocytic leukemia (APL) blasts. In a maturation-resistant APL cell line, we have previously identified a new pathway of retinoid-induced hTERT transcriptional repression independent of differentiation. Furthermore, we reported the isolation of a cell variant resistant to this repression. Those cell lines could serve as unique tools to identify new telomerase regulators. METHODS: Using a microarray approach we identified the long non-coding RNA, H19 as a potential candidate playing a role in telomerase regulation. Expression of H19, hTERT, and hTR were examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Telomerase activity was quantified by quantitative telomeric repeats amplification protocol (qTRAP). In vitro and in vivo assays were performed to investigate H19 function on telomerase expression and activity. RESULTS: We showed both in retinoid-treated cell lines and in APL patient cells an inverse relationship between the expression of H19 and the expression and activity of hTERT. Exploring the mechanistic link between H19 and hTERT regulation, we showed that H19 is able to impede telomerase function by disruption of the hTERT-hTR interaction. CONCLUSIONS: This study identifies a new way of telomerase regulation through H19's involvement and thereby reveals a new function for this long non-coding RNA that can be targeted for therapeutic purpose.

Regulation of Human Breast Cancer by the Long Non-Coding RNA H19.

Breast cancer is one of the most common causes of cancer related deaths in women. Despite the progress in early detection and use of new therapeutic targets associated with development of novel therapeutic options, breast cancer remains a major problem in public health. Indeed, even if the survival rate has improved for breast cancer patients, the number of recurrences within five years and the five-year relative survival rate in patients with metastasis remain dramatic. Thus, the discovery of new molecular actors involved in breast progression is essential to improve the management of this disease. Numerous data indicate that long non-coding RNA are implicated in breast cancer development. The oncofetal lncRNA H19 was the first RNA identified as a riboregulator. Studying of this lncRNA revealed its implication in both normal development and diseases. In this review, we summarize the different mechanisms of action of H19 in human breast cancer.

Nerve growth factor and proNGF simultaneously promote symmetric self-renewal, quiescence, and epithelial to mesenchymal transition to enlarge the breast cancer stem cell compartment.

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, p75(NTR), 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/p75(NTR) axis plays a critical role in regulating breast CSC self-renewal and plasticity.

Inhibition of latent membrane protein 1 impairs the growth and tumorigenesis of latency II Epstein-Barr virus-transformed T cells.

Epstein-Barr virus (EBV) is a common human herpesvirus. Infection with EBV is associated with several human malignancies in which the virus expresses a set of latent proteins, among which is latent membrane protein 1 (LMP1). LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry of activated members of the tumor necrosis factor (TNF) receptor superfamily, through the ability of LMP1 to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic cell line and a lymphocytic (NC5) cell line immortalized by EBV that expresses the type II latency program. Here we generated LMP1 dominant negative forms (DNs), based on fusion between green fluorescent protein (GFP) and transformation effector site 1 (TES1) or TES2 of LMP1. Then we generated cell lines conditionally expressing these DNs. These DNs inhibit NF-κB and Akt pathways, resulting in the impairment of survival processes and increased apoptosis in these cell lines. This proapoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs, which enable the generation of an apoptotic complex involving TRADD, FADD, and caspase 8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that synthetic peptides display similar inhibitory effects in EBV-infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches for malignant diseases associated with EBV.

Mitochondrial adaptation decreases drug sensitivity of persistent triple negative breast cancer cells surviving combinatory and sequential chemotherapy.

Triple negative breast cancer (TNBC) is an aggressive malignancy for which chemotherapy remains the standard treatment. However, between 3 and 5 years after chemotherapy, about half patients will relapse and it is essential to identify vulnerabilities of cancer cells surviving neoadujuvant therapy. In this study, we established persistent TNBC cell models after treating MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide, and then with paclitaxel, for a total of 18 weeks. The resulting chemo-persistent cell lines were more proliferative, both in vitro and in xenografted mice. Interestingly, MDA-MB-231 persistent cells became less sensitive to chemotherapeutic drugs, whereas SUM159-PT persistent cells kept similar sensitivity compared to control cells. The reduced sensitivity to chemotherapy in MDA-MB-231 persistent cells was found to be associated with an increased oxidative phosphorylation (OXPHOS) and modified levels of tricarboxylic acid cycle (TCA) intermediates. Integration of data from proteomics and metabolomics demonstrated TCA cycle among the most upregulated pathways in MDA-MB-231 persistent cells. The absence of glucose and pyruvate impeded OXPHOS in persistent cells, while the absence of glutamine did not. In contrast, OXPHOS was not modified in control cells independently of TCA substrates, indicating that MDA-MB-231 persistent cells evolved towards a more pyruvate dependent profile. Finally, the inhibition of pyruvate entry into mitochondria with UK-5099 reduced OXPHOS and re-sensitized persistent cells to therapeutic agents. Together, these findings suggest that targeting mitochondrial pyruvate metabolism may help to overcome mitochondrial adaptation of chemo-persistent TNBC.

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.

Cancer Cells Upregulate Tau to Gain Resistance to DNA Damaging Agents.

Recent reports suggested a role for microtubules in double-strand-DNA break repair. We herein investigated the role of the microtubule-associated protein Tau in radio- and chemotherapy. Noticeably, a lowered expression of Tau in breast cancer cell lines resulted in a significant decrease in mouse-xenograft breast tumor volume after doxorubicin or X-ray treatments. Furthermore, the knockdown of Tau impaired the classical nonhomologous end-joining pathway and led to an improved cellular response to both bleomycin and X-rays. Investigating the mechanism of Tau's protective effect, we found that one of the main mediators of response to double-stranded breaks in DNA, the tumor suppressor p53-binding protein 1 (53BP1), is sequestered in the cytoplasm as a consequence of Tau downregulation. We demonstrated that Tau allows 53BP1 to translocate to the nucleus in response to DNA damage by chaperoning microtubule protein trafficking. Moreover, Tau knockdown chemo-sensitized cancer cells to drugs forming DNA adducts, such as cisplatin and oxaliplatin, and further suggested a general role of Tau in regulating the nuclear trafficking of DNA repair proteins. Altogether, these results suggest that Tau expression in cancer cells may be of interest as a molecular marker for response to DNA-damaging anti-cancer agents. Clinically targeting Tau could sensitize tumors to DNA-damaging treatments.

Dimerization of kringle 1 domain from hepatocyte growth factor/scatter factor provides a potent MET receptor agonist.

Hepatocyte growth factor/scatter factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in postnatal life of epithelial organs such as the liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search of HGF/SF mimics for therapy. In this work, we describe the rational design, production, and characterization of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF in tandem orientation. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent of MET activation.

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.

Nerve growth factor promotes breast cancer angiogenesis by activating multiple pathways.

BACKGROUND: Although several anti-angiogenic therapies have been approved in the treatment of cancer, the survival benefits of such therapies are relatively modest. Discovering new molecules and/or better understating signaling pathways of angiogenesis is therefore essential for therapeutic improvements. The objective of the present study was to determine the involvement of nerve growth factor (NGF) in breast cancer angiogenesis and the underlying molecular mechanisms. RESULTS: We showed that both recombinant NGF and NGF produced by breast cancer cells stimulated angiogenesis in Matrigel plugs in immunodeficient mice. NGF strongly increased invasion, cord formation and the monolayer permeability of endothelial cells. Moreover, NGF-stimulated invasion was under the control of its tyrosine kinase receptor (TrkA) and downstream signaling pathways such as PI3K and ERK, leading to the activation of matrix metalloprotease 2 and nitric oxide synthase. Interestingly, NGF increased the secretion of VEGF in both endothelial and breast cancer cells. Inhibition of VEGF, with a neutralizing antibody, reduced about half of NGF-induced endothelial cell invasion and angiogenesis in vivo. CONCLUSIONS: Our findings provided direct evidence that NGF could be an important stimulator for breast cancer angiogenesis. Thus, NGF, as well as the activated signaling pathways, should be regarded as potential new targets for anti-angiogenic therapy against breast cancer.

Propagation and Maintenance of Cancer Stem Cells: A Major Influence of the Long Non-Coding RNA H19.

Cancer stem cells (CSCs) represent a rare population of tumor cells that exhibit stem cell properties with the abilities of self-renewal and differentiation. These cells are now widely accepted to be responsible for tumor initiation, development, resistance to conventional therapies, and recurrence. Thus, a better understanding of the molecular mechanisms involved in the control of CSCs is essential to improve patient management in terms of diagnostics and therapies. CSCs are regulated by signals of the tumor microenvironment as well as intrinsic genetic and epigenetic modulators. H19, the first identified lncRNA is involved in the development and progression of many different cancer types. Recently, H19 has been demonstrated to be implicated in the regulation of CSCs in different types of cancers. The aim of this review is to provide an overview of the role and mechanisms of action of H19 in the regulation of CSCs. We summarize how H19 may regulate CSC division and cancer cell reprogramming, thus affecting metastasis and drug resistance. We also discuss the potential clinical implications of H19.

Enhancement of Breast Cancer Cell Aggressiveness by lncRNA H19 and its Mir-675 Derivative: Insight into Shared and Different Actions.

Breast cancer is a major public health problem and the leading world cause of women death by cancer. Both the recurrence and mortality of breast cancer are mainly caused by the formation of metastasis. The long non-coding RNA H19, the precursor of miR-675, is involved in breast cancer development. The aim of this work was to determine the implication but, also, the relative contribution of H19 and miR-675 to the enhancement of breast cancer metastatic potential. We showed that both H19 and miR-675 increase the invasive capacities of breast cancer cells in xenografted transgenic zebrafish models. In vitro, H19 and miR-675 enhance the cell migration and invasion, as well as colony formation. H19 seems to induce the epithelial-to-mesenchymal transition (EMT), with a decreased expression of epithelial markers and an increased expression of mesenchymal markers. Interestingly, miR-675 simultaneously increases the expression of both epithelial and mesenchymal markers, suggesting the induction of a hybrid phenotype or mesenchymal-to-epithelial transition (MET). Finally, we demonstrated for the first time that miR-675, like its precursor H19, increases the stemness properties of breast cancer cells. Altogether, our data suggest that H19 and miR-675 could enhance the aggressiveness of breast cancer cells through both common and different mechanisms.

2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations.

Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2'-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations.

Molecular basis of cytotoxicity of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) in EBV latency III B cells: LMP1 induces type II ligand-independent autoactivation of CD95/Fas with caspase 8-mediated apoptosis.

The Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) is thought to act as the major transforming protein in various cell types, by rerouting the tumor necrosis factor receptor family signaling pathway. Despite this implication in EBV-associated transformation of cells, LMP1 toxicity is a well-known but poorly studied feature, perhaps because it contradicts its role in transformation. We show that LMP1 physiological levels are very heterogeneous and that the highest levels of LMP1 correlate with Fas overexpression and spontaneous apoptosis in lymphoblastoid cell lines (LCLs). To understand the cytotoxic effect of LMP1 in LCLs, we cloned wild-type LMP1 into a doxycycline double-inducible episomal vector pRT-1, with a truncated version of NGFR as a surrogate marker of inducibility. We found that LMP1 overexpression induced apoptosis in LCL B cells, as shown by annexin V labeling, sub-G(1) peak, and poly(ADP ribose) polymerase cleavage. Knocking down Fas expression by small interfering RNA abolished LMP1-induced apoptosis. The absence of detectable levels of Fas ligand mRNA suggested a ligand-independent activation of Fas. LMP1 induced Fas overexpression with its relocalization in lipid raft microdomains of the membrane. Fas immunoprecipitation detected FADD (Fas-associated death domain protein) and caspase 8, suggesting a Fas-dependent formation of the death-inducing signaling complex. Caspases 8, 9, 3, and 7 were activated by LMP1. Caspase 8 activation was associated with BID cleavage and truncated-BID mitochondrial relocalization, consistent with type II apoptosis. Therefore, our results are in agreement with a model where LMP1-dependent NF-kappaB activation induces Fas overexpression and autoactivation that could overwhelm the antiapoptotic effect of NF-kappaB, revealing an ambivalent function of LMP1 in cell survival and programmed cell death.

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.