The Identification of New c-FLIP Inhibitors for Restoring Apoptosis in TRAIL-Resistant Cancer Cells.

The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.

Simple elaboration of drug-SPION nanocapsules (hybridosomes®) by solvent shifting: Effect of the drug molecular structure and concentration.

Drug nanocapsules coated with iron oxide nanoparticles (SPION) were elaborated by the simultaneous nanoprecipitation of the drug and the nanoparticles, through solvent shifting. We examined four drugs: sorafenib, sorafenib tosylate, α-tocopherol and paclitaxel, to cover the cases of molecular solids, ionic solids, and molecular liquids. We first investigated the formation of the drug core in the final mixture of solvents at different concentrations. A Surfactant-Free Micro-Emulsion domain (SFME, thermodynamically stable) was observed at low drug concentration and an Ouzo domain (metastable) at high drug concentration, except for the case of paclitaxel which crystallizes at high concentration without forming an Ouzo domain. When co-nanoprecipitated with the molecular drugs in the Ouzo domain (sorafenib or α-tocopherol), the SPION limited the coalescence of the drug particles to less than 100 nm, forming capsules with a drug encapsulation efficiency of ca 80 %. In contrast, larger capsules were formed from the SFME or when using the ionic form (sorafenib tosylate). Finally, the sorafenib-SPION capsules exhibit a similar chemotherapeutic effect as the free drug on the hepatocellular carcinoma in vitro.

Genomic characteristics and clinical significance of CD56+ circulating tumor cells in small cell lung cancer.

Circulating tumor cells (CTC) have been studied in various solid tumors but clinical utility of CTC in small cell lung cancer (SCLC) remains unclear. The aim of the CTC-CPC study was to develop an EpCAM-independent CTC isolation method allowing isolation of a broader range of living CTC from SCLC and decipher their genomic and biological characteristics. CTC-CPC is a monocentric prospective non-interventional study including treatment-naïve newly diagnosed SCLC. CD56+ CTC were isolated from whole blood samples, at diagnosis and relapse after first-line treatment and submitted to whole-exome-sequencing (WES). Phenotypic study confirms tumor lineage and tumorigenic properties of isolated cells for the 4 patients analyzed with WES. WES of CD56+ CTC and matched tumor biopsy reveal genomic alteration frequently impaired in SCLC. At diagnosis CD56+ CTC were characterized by a high mutation load, a distinct mutational profile and a unique genomic signature, compared to match tumors biopsies. In addition to classical pathways altered in SCLC, we found new biological processes specifically affected in CD56+ CTC at diagnosis. High numeration of CD56+ CTC (> 7/ml) at diagnosis was associated with ES-SCLC. Comparing CD56+ CTC isolated at diagnosis and relapse, we identify differentially altered oncogenic pathways (e.g. DLL3 or MAPK pathway). We report a versatile method of CD56+ CTC detection in SCLC. Numeration of CD56+ CTC at diagnosis is correlated with disease extension. Isolated CD56+ CTC are tumorigenic and show a distinct mutational profile. We report a minimal gene set as a unique signature of CD56+ CTC and identify new affected biological pathways enriched in EpCAM-independent isolated CTC in SCLC.

GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1.

Upon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process.

Newly identified tumor suppressor functions of ING proteins.

The INhibitor of Growth (ING) proteins (ING1, ING2, ING3, ING4 and ING5) are a family of epigenetic regulators. Their decreased expression in numerous cancers led to identifying the ING proteins as gatekeeper tumor suppressors as they regulate cell cycle progression, apoptosis and senescence. Subsequently, they were also described as caretaker tumor suppressors through their involvement in DNA replication and the DNA damage response (DDR). Recent studies have identified new interactions of the ING proteins with proteins or pathways implicated in cell proliferation, the maintenance of stem cells pluripotency or the DDR. Furthermore, the ING proteins have been identified as regulators of ribosomal RNA synthesis and of mRNA stability and as regulators of mitochondrial DNA transcription resulting in the regulation of metabolism. These new findings highlight new antitumorigenic activities of the ING proteins that are potential targets for cancer treatment.

Characterization of the Peri-Membrane Fluorescence Phenomenon Allowing the Detection of Urothelial Tumor Cells in Urine.

Urine cytology is non-invasive, easy to collect, with medium sensitivity and a high specificity. It is an effective way to detect high-grade bladder cancer (BC), but it is less effective on low-grade BC because the rate of equivocal results is much higher. Recently, the fluorescent properties of plasma membranes of urothelial tumor cells (UTC) found in urine cytology have been shown to be useful in improving the early detection of BC. This phenomenon is called peri-membrane fluorescence (PMF). Based on previous studies that have identified the PMF on UTCs, the main objective was to characterize this phenomenon. For this study, a software was specially created to quantify the PMF of all tested cells and different treatments performed. PMF was not found to be a morphological and discriminating feature of UTCs, all cells in shape and not from urine show PMF. We were able to highlight the crucial role of plasma membrane integrity in the maintenance of PMF. Finally, it was found that the induction of a strong cellular stress induced a decrease in PMF, mimicking what was observed in non-tumor cells collected from urine. These results suggest that PMF is found in cells able to resist this stress, such as tumor cells.

Preclinical Models for Acquired Resistance to Third-Generation EGFR Inhibitors in NSCLC: Functional Studies and Drug Combinations Used to Overcome Resistance.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are currently recommended as first-line treatment for advanced non-small-cell lung cancer (NSCLC) with EGFR-activating mutations. Third-generation (3rd G) EGFR-TKIs, including osimertinib, offer an effective treatment option for patients with NSCLC resistant 1st and 2nd EGFR-TKIs. However, the efficacy of 3rd G EGFR-TKIs is limited by acquired resistance that has become a growing clinical challenge. Several clinical and preclinical studies are being carried out to better understand the mechanisms of resistance to 3rd G EGFR-TKIs and have revealed various genetic aberrations associated with molecular heterogeneity of cancer cells. Studies focusing on epigenetic events are limited despite several indications of their involvement in the development of resistance. Preclinical models, established in most cases in a similar manner, have shown different prevalence of resistance mechanisms from clinical samples. Clinically identified mechanisms include EGFR mutations that were not identified in preclinical models. Thus, NRAS genetic alterations were not observed in patients but have been described in cell lines resistant to 3rd G EGFR-TKI. Mainly, resistance to 3rd G EGFR-TKI in preclinical models is related to the activation of alternative signaling pathways through tyrosine kinase receptor (TKR) activation or to histological and phenotypic transformations. Yet, preclinical models have provided some insight into the complex network between dominant drivers and associated events that lead to the emergence of resistance and consequently have identified new therapeutic targets. This review provides an overview of preclinical studies developed to investigate the mechanisms of acquired resistance to 3rd G EGFR-TKIs, including osimertinib and rociletinib, across all lines of therapy. In fact, some of the models described were first generated to be resistant to first- and second-generation EGFR-TKIs and often carried the T790M mutation, while others had never been exposed to TKIs. The review further describes the therapeutic opportunities to overcome resistance, based on preclinical studies.

Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs.

ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

Radiosensitizing Fe-Au nanocapsules (hybridosomes®) increase survival of GL261 brain tumor-bearing mice treated by radiotherapy.

Glioblastoma remains a cancer for which the effectiveness of treatments has shown little improvement over the last decades. For this pathology, multiple therapies combining resection, chemotherapy and radiotherapy remain the norm. In this context, the use of high-Z nanoparticles such as gold or hafnium to potentiate radiotherapy is attracting more and more attention. Here, we evaluate the potentiating effect of hollow shells made of gold and iron oxide nanoparticles (hybridosomes®) on the radiotherapy of glioblastoma, using murine GL261-Luc+ brain tumor model. While iron oxide seems to have no beneficial effect for radiotherapy, we observe a real effect of gold nanoparticles-despite their low amount-with a median survival increase of almost 20% compared to radiotherapy only and even 33% compared to the control group. Cellular and in vivo studies show that a molecule of interest nano-precipitated in the core of the hybridosomes® is released and internalized by the surrounding brain cells. Finally, in vivo studies show that hybridosomes® injected intra-tumorally are still present in the vicinity of the brain tumor more than 5 days after injection (duration of the Stupp protocol's radiation treatment). Interestingly, one mouse treated with radiotherapy in the presence of gold-containing hybridosomes® survived 78 days. Monitoring of the tumoral growth of this long-term survivor using both MRI and bioluminescence revealed a decrease of the tumor size after treatment. These very encouraging results are a proof-of-concept that hybridosomes® are really effective tools for the development of combined therapies (chemo-radiotherapy).

Orthotopic model of lung cancer: isolation of bone micro-metastases after tumor escape from Osimertinib treatment.

BACKGROUND: Osimertinib is a third generation tyrosine kinase inhibitor (TKI) that targets the epidermal growth factor receptor (EGFR) in lung cancer. However, although this molecule is not subject to some of the resistance mechanisms observed in response to first generation TKIs, ultimately, patients relapse because of unknown resistance mechanisms. New relevant non-small cell lung cancer (NSCLC) mice models are therefore required to allow the analysis of these resistance mechanisms and to evaluate the efficacy of new therapeutic strategies. METHODS: Briefly, PC-9 cells, previously modified for luciferase expression, were injected into the tail vein of mice. Tumor implantation and longitudinal growth, almost exclusively localized in the lung, were evaluated by bioluminescence. Once established, the tumor was treated with osimertinib until tumor escape and development of bone metastases. RESULTS: Micro-metastases were detected by bioluminescence and collected for further analysis. CONCLUSION: We describe an orthotopic model of NSCLC protocol that led to lung primary tumor nesting and, after osimertinib treatment, by metastases dissemination, and that allow the isolation of these small osimertinib-resistant micro-metastases. This model provides new biological tools to study tumor progression from the establishment of a lung tumor to the generation of drug-resistant micro-metastases, mimicking the natural course of the disease in human NSCLC patients.

ING2 tumor suppressive protein translocates into mitochondria and is involved in cellular metabolism homeostasis.

ING2 (Inhibitor of Growth 2) is a tumor suppressor gene that has been implicated in critical biological functions (cell-cycle regulation, replicative senescence, DNA repair and DNA replication), most of which are recognized hallmarks of tumorigenesis occurring in the cell nucleus. As its close homolog ING1 has been recently observed in the mitochondrial compartment, we hypothesized that ING2 could also translocate into the mitochondria and be involved in new biological functions. In the present study, we demonstrate that ING2 is imported in the inner mitochondrial fraction in a redox-sensitive manner in human cells and that this mechanism is modulated by 14-3-3η protein expression. Remarkably, ING2 is necessary to maintain mitochondrial ultrastructure integrity without interfering with mitochondrial networks or polarization. We observed an interaction between ING2 and mtDNA under basal conditions. This interaction appears to be mediated by TFAM, a critical regulator of mtDNA integrity. The loss of mitochondrial ING2 does not impair mtDNA repair, replication or transcription but leads to a decrease in mitochondrial ROS production, suggesting a detrimental impact on OXPHOS activity. We finally show using multiple models that ING2 is involved in mitochondrial respiration and that its loss confers a protection against mitochondrial respiratory chain inhibition in vitro. Consequently, we propose a new tumor suppressor role for ING2 protein in the mitochondria as a metabolic shift gatekeeper during tumorigenesis.

Noninvasive Urine-Based Tests to Diagnose or Detect Recurrence of Bladder Cancer.

Liquid biopsies are increasingly used for the diagnosis and follow-up of cancer patients. Urine is a body fluid that can be used to detect cancers and others diseases. It is noninvasive and easy to collect. To detect Bladder Cancer (BC), cytology is the first assay used. It is an effective way to detect high grade BC but has a high rate of equivocal results, especially for low grade BC. Furthermore, cystoscopy is used to confirm cytology results and to determine cancer status. Cystoscopy is also effective but highly invasive, and not well accepted by patients, especially for BC follow-up. In this review we survey the numerous assays recently developed in order to diagnose BC at an early stage, and to facilitate the follow-up of patients. We discuss their effectiveness, ease of use, and applications. Finally, we discuss assays that, in the future, could improve the diagnosis and management of BC patients.

When Endoplasmic Reticulum Proteostasis Meets the DNA Damage Response.

Sustaining both proteome and genome integrity (GI) requires the integration of a wide range of mechanisms and signaling pathways. These comprise, in particular, the unfolded protein response (UPR) and the DNA damage response (DDR). These adaptive mechanisms take place respectively in the endoplasmic reticulum (ER) and in the nucleus. UPR and DDR alterations are associated with aging and with pathologies such as degenerative diseases, metabolic and inflammatory disorders, and cancer. We discuss the emerging signaling crosstalk between UPR stress sensors and the DDR, as well as their involvement in cancer biology.

Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response.

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.

Focus-ING on DNA Integrity: Implication of ING Proteins in Cell Cycle Regulation and DNA Repair Modulation.

The ING family of tumor suppressor genes is composed of five members (ING1-5) involved in cell cycle regulation, DNA damage response, apoptosis and senescence. All ING proteins belong to various HAT or HDAC complexes and participate in chromatin remodeling that is essential for genomic stability and signaling pathways. The gatekeeper functions of the INGs are well described by their role in the negative regulation of the cell cycle, notably by modulating the stability of p53 or the p300 HAT activity. However, the caretaker functions are described only for ING1, ING2 and ING3. This is due to their involvement in DNA repair such as ING1 that participates not only in NERs after UV-induced damage, but also in DSB repair in which ING2 and ING3 are required for accumulation of ATM, 53BP1 and BRCA1 near the lesion and for the subsequent repair. This review summarizes evidence of the critical roles of ING proteins in cell cycle regulation and DNA repair to maintain genomic stability.

Exploiting ING2 Epigenetic Modulation as a Therapeutic Opportunity for Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) has been the leading cause of cancer-related death worldwide, over the last few decades. Survival remains extremely poor in the metastatic setting and, consequently, innovative therapeutic strategies are urgently needed. Inhibitor of Growth Gene 2 (ING2) is a core component of the mSin3A/Histone deacetylases complex (HDAC), which controls the chromatin acetylation status and modulates gene transcription. This gene has been characterized as a tumor suppressor gene and its status in cancer has been scarcely explored. In this review, we focused on ING2 and other mSin3A/HDAC member statuses in NSCLC. Taking advantage of existing public databases and known pharmacological properties of HDAC inhibitors, finally, we proposed a therapeutic model based on an ING2 biomarker-guided strategy.

ING3 is required for ATM signaling and DNA repair in response to DNA double strand breaks.

Inhibitor of Growth 3 (ING3) is a candidate tumor suppressor gene whose expression is lost in tumors such as hepatocellular carcinoma, head and neck squamous cell carcinoma and melanoma. In the present study, we show that ING3-depleted human cells and yeast cells deleted for its ortholog YNG2 are sensitive to DNA damage suggesting a conserved role in response to such stress. In human cells, ING3 is recruited to DNA double strand breaks and is required for ATM activation. Remarkably, in response to doxorubicin, ATM activation is dependent on ING3 but not on TIP60, whose recruitment to DNA breaks also depends on ING3. These events lead to ATM-mediated phosphorylation of NBS1 and the subsequent recruitment of RNF8, RNF168, 53BP1, and BRCA1, which are major mediators of the DNA damage response. Accordingly, upon genotoxic stress, DNA repair by non-homologous end joining (NHEJ) or homologous recombination (HR) were impaired in absence of ING3. Finally, immunoglobulin class switch recombination (CSR), a physiological mechanism requiring NHEJ repair, was impaired in the absence of ING3. Since deregulation of DNA double strand break repair is associated with genomic instability, we propose a novel function of ING3 as a caretaker tumor suppressor involved in the DNA damage signaling and repair.

Regulat-INGs in tumors and diseases: Focus on ncRNAs.

ING family genes (Inhibitor of Growth) are tumor suppressor genes that play a vital role in cell homeostasis. It has been shown that their expression is lost or diminished in many cancers and other diseases. The main mechanisms by which they are regulated in oncogenesis have not yet been fully elucidated. The involvement of non-coding RNAs (ncRNAs) and in particular microRNAs (miRNAs) in post-transcriptional gene regulation is well established. miRNAs are short sequences (18-25 nucleotides) that can bind to the 3 'UTR sequence of the targeted messenger RNA (mRNA), leading to its degradation or translational repression. Interactions between the ING family and miRNAs have been described in some cancers but also in other diseases. The involvement of miRNAs in ING family regulation opens up new fields of investigation, particularly for targeted therapies. In this review, we will summarize the regulatory mechanisms at the RNA and protein level of the ING family and focus on the interactions with ncRNAs.

tert-Butylphenolic Derivatives from Paenibacillus odorifer-A Case of Bioconversion.

Two compounds (1) and (2) containing tert-butylphenol groups were, for the first time, produced during the culture of Paenibacillus odorifer, a bacterial strain associated with the crustose lichen, Rhizocarpon geographicum. Their entire structures were identified by one-dimensional (1D) and two-dimensional (2D) NMR and high-resolution electrospray ionisation mass spectrometry (HRESIMS) spectroscopic analyses. Among them, Compound 1 exhibited significant cytotoxicity against B16 murine melanoma and HaCaT human keratinocyte cell lines with micromolar half maximal inhibitory concentration (IC50) values. Furthermore, after supplementation studies, a putative biosynthesis pathway was proposed for Compound 1 throughout a bioconversion by this bacterial strain of butylated hydroxyanisole (BHA), an antioxidant polymer additive.