Diffuse midline glioma (DMG) is a rare and aggressive pediatric brain tumor, with a median survival of less than 12 months. Due to its location in the pons, surgical resection is impossible, leaving radiation therapy as the only palliative treatment option. Unfortunately, radiation therapy yields minimal improvement in survival. Thus, characterization of the vascular component of the DMG microenvironment at the cellular and molecular levels following radiotherapy to improve therapeutic strategies. A human syngeneic blood-brain tumor barrier (BBTB) in vitro model, comprising endothelial cells, pericytes and DMG cells was submitted to a single dose of radiation (2 Gγ to 6 Gγ) and was characterized for its physical and metabolic properties over a period of 7 days post-exposure. The results were then compared to the effects of the same irradiation protocol on a physiologic blood-brain barrier (BBB) model. Following irradiation, the endothelial permeability of the BBB ECs and the BBTB ECs was preserved for up to 7 days but associated with Claudin-5 heterogeneous distribution at the ECs borders and decrease of expression after irradiation. Nevertheless, irradiation was found to potentiate the effect of TNFα on the physical integrity of the BBB, which was less important for the BBTB. The metabolic properties of the BBB and BBTB were modulated by radiation at the transcriptional level. Interestingly, different regulations were observed in endothelial cells and pericytes. Notably, pericytes have demonstrated compensatory effects. Immunoblots confirmed the decrease of BCRP, MRP4 and MFSD2A in BBTB endothelial cells after irradiation. Despite significant reduced efficiency, P-gp/BCRP efflux pump activity remains functional in endothelial cells and pericytes following irradiation. Irradiation sensitizes the BBB, but to a lesser extent the BBTB, to the effects of pro-inflammatory cytokines. The observed decrease in P-gp/BCRP activity, as well as the involvement of MFSD2A, MRP4 and Claudin-5 regulation, warrant further investigations.

The majority of breast cancer-related deaths result from metastatic progression, especially brain metastasis, associated with the poorest prognosis. Patients with triple-negative breast cancer (TNBC) are particularly prone to developing brain metastases. In this context, the blood-brain barrier (BBB) plays a pivotal role; however, the contribution of brain pericytes in mediating interactions between TNBC cells and the brain endothelial cells (ECs) remains unclear. To investigate the role of pericytes in modulating these interactions, we used a syngeneic human in vitro BBB model, composed of human brain-like endothelial cells (hBLECs) co-cultured with human brain pericytes (hBPs). Our findings demonstrate that hBPs secretions significantly reduce TNBC cell adhesion to hBLECs and protect the endothelium from TNBC cell-induced damage, both under normoxic and hypoxic conditions. Interestingly, we found that hBPs promote TNBC cell migration, invasion, and increase clonogenicity. Together, these findings uncover a dual role of brain pericytes in TNBC brain metastasis: they protect ECs and maintain BBB integrity against TNBC cells while simultaneously promoting tumor aggressiveness. Understanding this balance between protective and pro-metastatic functions may provide insights for developing novel therapeutic approaches to strengthen BBB defenses and limit brain colonization in TNBC patients.

Signal transducer and activator of transcription 3 (STAT3) is a promising yet challenging anticancer drug target due to its complex signaling and limited "druggability". To this end, we herein highlight a target engagement-focused screening and optimization pipeline pursuing the discovery of novel STAT3 inhibitors. From a STAT3 differential scanning fluorimetry high-throughput screen, we identified compounds that appeared to stabilize STAT3 toward thermal aggregation and moderately inhibited cellular STAT3 activity. Subsequent evaluation using complementary and orthogonal assays revealed their high affinity for tropomyosin receptor kinase A (TrkA). Applying a similar target engagement-inspired approach, we refined inhibitor binding and selectivity toward TrkA, showing efficacy in cellular TrkA cancer models. Top compound, PI-15, demonstrated successful target engagement in a cellular thermal shift assay and potently inhibited TrkA activity in cancer cells. These approaches highlight the importance of prioritizing rigorous target engagement validation early in the drug discovery pipeline, resulting in promising new inhibitors.

Phenotypic plasticity is a major factor in tumor heterogeneity and treatment resistance. In particular, cancer stem cells (CSCs) represent a small subpopulation within tumors that possesses self-renewal and tumor-forming capabilities. Understanding reprogramming, maintenance, and lineage properties of CSCs requires dedicated tools to disentangle the respective influences of phenotypic inheritance and cell-cell interactions. Here, we set up ultra-wide field microscopy to image breast cancer cell lines expressing a stemness fluorescent reporter over several days. The fluorescent reporter distinguishes three phenotypes: CSCs, cancer differentiated cells (CDCs), and intermediate/transiting cancer cells (iCCs). Spatial statistics indicate significant zonation in which CSCs cluster together and are spatially separated from CDCs, forming patterns resembling niches. Surprisingly, single-cell time series reveal spontaneous reprogramming events from CDC to CSC even in unperturbed populations. We identify that such transitions are prone to arise during the cell cycle. Moreover, lineage analysis shows that the phenotype is partially inherited from ancestor cells. However, such heredity is not sufficient to explain the spatial properties of the cell population, which also depend on cell-cell interactions. Indeed, we find that phenotypic transitions of cancer cells are influenced by the phenotypic state of neighboring cells. Reprogramming into CSCs is respectively promoted and inhibited by the presence of CSCs and CDCs in the neighborhood. Altogether, our results disentangle how phenotypic inheritance and intercellular interactions orchestrate the spatio-temporal self-organization of cancer cell heterogeneity, maintaining a subpopulation of CSCs within niches.

Kv10.1 potassium channel has been shown to be involved in breast cancer luminal cell proliferation and survival as well as migration in basal cell model in normoxia. Moreover, it is clearly established that solid tumors present a hypoxic center. Currently, few information is available about the involvement of the Kv10.1 channel in hypoxic context in breast cancer progression. We explored the role of Kv10.1 in the hypoxia-induced aggressiveness promotion of different breast cancer cell models: luminal A (MCF-7 cells), HER2-overexpressing (SKBr3 cells) and basal subtype (MDA-MB-231 cells) by using complementary approaches of cell biology assay, western blot and qPCR. Our results show that Kv10.1 expression and function are related to hypoxia and HIF-1α. Under hypoxia, we demonstrated that Kv10.1 is a major actor of the migration and it regulates the hypoxia-induced EMT in the three cells models by promoting the mesenchymal phenotype. Finally, after RNAseq comparison of cells expressing or not the channel in hypoxia condition, we highlighted the involvement of the β1-integrin / FAK, AKT and ERK pathways in the MDA-MB-231 breast cancer cell aggressiveness. We demonstrated, for the first time, the impact of Kv10.1 channel in hypoxia-induced aggressivity of breast cancer cells demonstrating its potential use as a complementary target to fight against metastasis development.

Cancer stem cell (CSC) has paved the way to many fundamental and translational studies. Recent studies have highlighted differentiated breast cancer cells (non-CSCs) switching phenotype to CSCs in response to various stimuli, depicting the existence of cancer stem cell plasticity. Although strategies to reduce the phenotypic plasticity of non-CSCs into CSCs are likely to prevent treatment-resilient cancer cells driving recurrence, most phenotypic plasticity mechanisms involve Notch, Wnt or MAPK signaling pathways. In this study, breast cancer cells were irradiated to identify soluble reprogramming factors. Using conditioned medias, protein arrays analyses, flow cytometry and in cellulo/in vivo functional assays, we demonstrated, for the first time, that radiation-induced chemokine expression, especially CXCL1 and CCL5 and their receptors CXCR2, CCR1 and CCR5, stimulates reprogramming of breast non-CSCs into CSCs. Treatment of non-CSCs with recombinant CXCL1 and CCL5 is sufficient to induce cell reprogramming, while their inhibition can be used to prevent reprogramming and sensitize tumor to radiation. Moreover, analysis of gene expression profiles from 38 public merged databases demonstrated that combined over-expression of CXCL1/CXCR2, CCL5/CCR1 or CCL5/CCR5 has a poorer prognosis in patients treated with radiotherapy, suggesting a promising way for patient stratification, where individuals with elevated cytokine levels could benefit from radiotherapy in conjunction with cytokine inhibitors. Taken together, our findings provide a rationale to consider these axes as potential targets and predictive biomarkers in breast cancer patients.

Extracellular matrix (ECM) is a three-dimensional (3D) structure found around cells in the tissues of many organisms. It is composed mainly of fibrous proteins, such as collagen and elastin, and adhesive glycoproteins, such as fibronectin and laminin—as well as proteoglycans, such as hyaluronic acid. The ECM performs several essential functions, including structural support of tissues, regulation of cell communication, adhesion, migration, and differentiation by providing biochemical and biomechanical cues to the cells. Pancreatic β-cells have been previously shown to be responsive to the surrounding mechanical stress, impacting their insulin-secreting function. We aimed to derive a physiologically relevant in vitro model of pancreatic tissue by using an innovative synthesised porous ECM that mimics the native tissue microenvironment and mechanical properties. Here we performed mechanical, physico-chemical and functional characterisation of a synthetic hydrogel ECM, composed of hyaluronic acid cross-linked with collagen types I and VI and modified with fibronectin. The hydrogel was used as a 3D cell culture scaffold for the MIN6 insulinoma cell line. Cell proliferation, viability, gene expression, and insulin secretion in response to glucose stimulus were assessed and contrasted with classic monolayer culture. The biomaterial exhibited a shear modulus of 815.37 kPa and a distinctive viscoelastic response. MIN6 cells showed a higher proliferation and viability rates and maintained insulin secretion in response to glucose stimulus and β-cell identity gene expression when cultured in the 3D hydrogel compared to monolayer culture. Our study demonstrated the potential of this biomimetic hydrogel scaffold as an innovative matrix enabling better in vitro models to study disease physiopathology.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mitochondria are important cell death checkpoints, and mitochondrial Ca2+ overload is considered as a potent apoptotic intrinsic pathway inducer. Here, we report that this Ca2+ apoptosis link is largely ineffective in inducing cell-death just by itself and required a concomitant inhibition of autophagy to counteract its pro-survival action. In such condition, an acute mitochondrial stress revealed by a DRP1-mediated mitochondrial dynamic remodeling is observed concomitantly with mitochondrial depolarization, release of cytochrome c, and efficient apoptosis induction. We also uncover that mitochondrial Ca2+ status modulates the function of autophagy as a sensitizer for chemotherapies. This priming mediated by mitochondrial Ca2+ overload and inhibition of autophagy sensitizes many cancer cells types to different chemotherapies with independent mechanisms of action. Collectively, our results redefine an important cell signaling pathway, uncovering new combined therapies for the treatment of diseases associated with mitochondrial Ca2+ homeostasis disorders such as cancer.

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 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 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.