TFEB and TFE3 drive kidney cystogenesis and tumorigenesis.

Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors.

Targeting the USP7/RRM2 axis drives senescence and sensitizes melanoma cells to HDAC/LSD1 inhibitors.

Deubiquitinating enzymes are key regulators of the ubiquitin-proteasome system and cell cycle, and their dysfunction leads to tumorigenesis. Our in vivo drop-out screens in patient-derived xenograft models identify USP7 as a regulator of melanoma. We show that USP7 downregulation induces cellular senescence, arresting melanoma growth in vivo and proliferation in vitro in BRAF- and NRAS-mutant melanoma. We provide a comprehensive understanding of targets and networks affected by USP7 depletion by performing a global transcriptomic and proteomics analysis. We show that RRM2 is a USP7 target and is regulated by USP7 during S phase of the cell cycle. Ectopic expression of RRM2 in USP7-depleted cells rescues the senescent phenotype. Pharmacological inhibition of USP7 by P5091 phenocopies the shUSP7-induced senescent phenotype. We show that the bifunctional histone deacetylase (HDAC)/LSD1 inhibitor domatinostat has an additive antitumor effect, eliminating P5091-induced senescent cells, paving the way to a therapeutic combination for individuals with melanoma.

ShcD Binds DOCK4, Promotes Ameboid Motility and Metastasis Dissemination, Predicting Poor Prognosis in Melanoma.

Metastases are the primary cause of cancer-related deaths. The underlying molecular and biological mechanisms remain, however, elusive, thus preventing the design of specific therapies. In melanomas, the metastatic process is influenced by the acquisition of metastasis-associated mutational and epigenetic traits and the activation of metastatic-specific signaling pathways in the primary melanoma. In the current study, we investigated the role of an adaptor protein of the Shc family (ShcD) in the acquisition of metastatic properties by melanoma cells, exploiting our cohort of patient-derived xenografts (PDXs). We provide evidence that the depletion of ShcD expression increases a spread cell shape and the capability of melanoma cells to attach to the extracellular matrix while its overexpression switches their morphology from elongated to rounded on 3D matrices, enhances cells' invasive phenotype, as observed on collagen gel, and favors metastasis formation in vivo. ShcD overexpression sustains amoeboid movement in melanoma cells, by suppressing the Rac1 signaling pathway through the confinement of DOCK4 in the cytoplasm. Inactivation of the ShcD signaling pathway makes melanoma cells more sensitive to therapeutic treatments. Consistently, ShcD expression predicts poor outcome in a cohort of 183 primary melanoma patients.

A Na+ /Ca2+ exchanger of the olive pathogen Pseudomonas savastanoi pv. savastanoi is critical for its virulence.

In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na+ /Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2-AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+ /Ca2+ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na+ /Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a 'host signal' that allows and promotes Psav pathogenicity on olive plants.

CDC25 as a common therapeutic target for triple-negative breast cancer - the challenges ahead.

The dual phosphatase CDC25 has recently been identified as a target for diverse triple-negative breast cancers including RB1/PTEN/P53-deficient tumors. Moreover, CDC25 inhibitors effectively synergize with PI3K inhibitors to suppress tumor growth. We discuss these findings and the challenges that lie ahead in bringing CDC25 inhibitors to the clinic.

Identification of CDC25 as a Common Therapeutic Target for Triple-Negative Breast Cancer.

CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC), RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs.

Aquaporin-4 antibody titration in NMO patients treated with rituximab: A retrospective study.

OBJECTIVE: We undertook an observational retrospective study to investigate the usefulness of aquaporin-4 (AQP4) antibodies (Ab) titration in the management of patients with neuromyelitis optica (NMO) treated with rituximab (RTX) by studying (1) the correlation between AQP4-Ab titer and disease activity, (2) the influence of RTX on antibody levels, and (3) the association between AQP4-Ab levels and responsiveness to RTX. METHODS: A cell-based assay was used for AQP4-Ab titration in 322 serum samples from 7 patients with NMO treated with RTX (median follow-up 65 months), according to a treatment-to-target approach. Serum samples were collected every month following standardized procedures. RESULTS: (1) In group analysis, AQP4-Ab titers correlated with the disease activity, showing higher titers during and preceding relapses than during remission. However, in individual analysis, an increase in AQP4-Ab titers and CD19+ B cells did not always precede a relapse. (2) A reduction of AQP4-Ab titers in the short-term and long-term period was observed during RTX treatment. (3) Reduction of AQP4-Ab titers was observed in responder patients both 3 months after RTX infusion and in the long-term follow-up. In one nonresponder patient, AQP4-Ab levels never decreased during the treatment period. CONCLUSIONS: Titration of AQP4-Abs could be useful in the clinical management of patients with NMO treated with RTX: titration before each reinfusion and 3 months after each reinfusion may provide information about responsiveness to RTX. Although a relationship among AQP4-Ab levels, disease activity, and response to RTX was observed, the usefulness of AQP4-Ab titration to predict relapses is limited.

Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica.

OBJECTIVE: Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). RESULTS: Results of tests on 92 controls identified 12assays as highly specific (0-1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. CONCLUSIONS: The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology.

High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor.

We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer.

Antagonistic Effect of a Salivary Proline-Rich Peptide on the Cytosolic Ca2+ Mobilization Induced by Progesterone in Oral Squamous Cancer Cells.

A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

Synthesis of new indole-based bisphosphonates and evaluation of their chelating ability in PE/CA-PJ15 cells.

Bisphosphonates are the most important class of antiresorptive agents used against osteoclast-mediated bone loss, and, more recently, in oncology. These compounds have high affinity for calcium ions (Ca(2+)) and therefore target bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function. They are extensively used in healthcare, however they are affected by severe side effects; pharmacological properties of bisphosphonates depend on their molecular structure, which is frequently the cause of poor intestinal adsorption and low distribution. In this work we synthesized six novel bisphosphonate compounds having a variably substituted indole moiety to evaluate their extra- and intracellular calcium chelating ability in PE/CA-PJ15 cells. Preliminary in silico and in vitro ADME studies were also performed and the results suggested that the indole moiety plays an important role in cell permeability and metabolism properties.

Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis.

BACKGROUND: Gene expression analyses in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) from patients with multiple sclerosis (MS) are restrained by the low RNA amounts from CSF cells and low expression levels of certain genes. Here, we applied a Taqman-based pre-amplification real-time reverse-transcription polymerase chain reaction (RT-PCR) (PreAmp RT-PCR) to cDNA from CSF cells and PBMC of MS patients and analyzed multiple genes related to immune system function and genes expressed by Epstein-Barr virus (EBV), a herpesvirus showing strong association with MS. Using this enhanced RT-PCR method, we aimed at the following: (1) identifying gene signatures potentially useful for patient stratification, (2) understanding whether EBV infection is perturbed in CSF and/or blood, and (3) finding a link between immune and EBV infection status. METHODS: Thirty-one therapy-free patients with relapsing-remitting MS were included in the study. Paired CSF cells and PBMC were collected and expression of 41 immune-related cellular genes and 7 EBV genes associated with latent or lytic viral infection were determined by PreAmp RT-PCR. Clinical, radiological, CSF, and gene expression data were analyzed using univariate and multivariate (cluster analysis, factor analysis) statistical approaches. RESULTS: Several immune-related genes were differentially expressed between CSF cells and PBMC from the whole MS cohort. By univariate analysis, no or only minor differences in gene expression were found associated with sex, clinical, or radiological condition. Cluster analysis on CSF gene expression data grouped patients into three clusters; clusters 1 and 2 differed by expression of genes that are related mainly to innate immunity, irrespective of sex and disease characteristics. By factor analysis, two factors grouping genes involved in antiviral immunity and immune regulation, respectively, accurately discriminated cluster 1 and cluster 2 patients. Despite the use of an enhanced RT-PCR method, EBV transcripts were detected in a minority of patients (5 of 31), with evidence of viral latency activation in CSF cells or PBMC and of lytic infection in one patient with active disease only. CONCLUSIONS: Analysis of multiple cellular and EBV genes in paired CSF cell and PBMC samples using PreAmp RT-PCR may yield new information on the complex interplay between biological processes underlying MS and help in biomarker identification.

CD19 mRNA quantification improves rituximab treatment-to-target approach: a proof of concept study.

We compared pre-amplification (PA) RT-PCR blood CD19 mRNA quantification with flow cytometry (FC), to personalize rituximab re-treatment in neuromyelitis optica spectrum disorders (NMOSDs) patients. 47 blood samples from 3 NMOSDs patients were studied. PA-RT-PCR quantified CD19 in all samples, and a positivity threshold was defined, whereas CD19+ B cells were under threshold in 31/47 samples by FC. In all samples where CD19+ B cells were above FC threshold, they resulted above the PA-RT-PCR threshold. CD19 mRNA was above threshold in 8 other samples, resulted negative by FC, and preceded the FC positivity in 7/8 samples by 1-3 months, showing major sensitivity.

Evaluation of a multiparametric immunofluorescence assay for standardization of neuromyelitis optica serology.

BACKGROUND: Neuromyelitis optica (NMO) is a severely disabling autoimmune disorder of the central nervous system, which predominantly affects the optic nerves and spinal cord. In a majority of cases, NMO is associated with antibodies to aquaporin-4 (AQP4) (termed NMO-IgG). AIMS: In this study, we evaluated a new multiparametric indirect immunofluorescence (IIF) assay for NMO serology. METHODS: Sera from 20 patients with NMO, 41 patients with multiple sclerosis (MS), 30 healthy subjects, and a commercial anti-AQP4 IgG antibody were tested in a commercial composite immunofluorescence assay ("Neurology Mosaic 17"; Euroimmun, Germany), consisting of five different diagnostic substrates (HEK cells transfected with AQP4, non-transfected HEK cells, primate cerebellum, cerebrum, and optic nerve tissue sections). RESULTS: We identified AQP4 specific and non-specific fluorescence staining patterns and established positivity criteria. Based on these criteria, this kit yielded a high sensitivity (95%) and specificity (100%) for NMO and had a significant positive and negative likelihood ratio (LR+ = ∞, LR- = 0.05). Moreover, a 100% inter- and intra-laboratory reproducibility was found. CONCLUSIONS: The biochip mosaic assay tested in this study is a powerful tool for NMO serology, fast to perform, highly sensitive and specific for NMO, reproducible, and suitable for inter-laboratory standardization as required for multi-centre clinical trials.

Classification of individuals based on ex-vivo glatiramer acetate-induced interferon-γ and interleukin-4 response.

BACKGROUND: Glatiramer acetate (GA) in multiple sclerosis acts through the induction of GA-specific T-helper 2 cells. Nevertheless, the phenomenon is not universal in patients, explaining individual differences in clinical response. OBJECTIVE: The objective of this article was to categorize GA-treated patients. METHOD: An enhanced quantitative PCR assay was used for measuring ex-vivo GA-induced IFNγ and IL4 mRNA responses in mononuclear cells from 23 healthy donors, 27 untreated patients, 33 short-term (≤6 months) and 77 long-term (>6 months) GA-treated patients. Thresholds for IFNγ and IL4 transcriptional response were calculated by ROC analysis and long-term treated patients were compared in terms of prognostic stratification. RESULTS: Thresholds for IFNγ and IL4 transcriptional response were calculated at 5.36 and 1.41 relative expression (RE). Finally, 67% of long-term treated patients scored above both response thresholds. These patients had a higher proportion of relapse-free subjects (74% vs 40% when compare to patients who scored below both thresholds) and a significantly better relapse-free survival rate (p=0.006; CI 0.29-0.75). The negative predictive value to predict adverse clinical outcome was 79% (CI 0.63-0.90), meaning that by a positive response, there is a 79% chance that the patient will not experience a negative outcome at 3 years. CONCLUSIONS: Our enhanced quantitative PCR assay produced clinically significant results for GA-treated patients. As such, if patients have a positive response, it means they have less chance of a relapse, while patients with a negative response have a greater probability of a worse outcome.

One-year evaluation of factors affecting the biological activity of interferon beta in multiple sclerosis patients.

MxA is an antiviral protein induced by type I interferons (IFN) and some viruses; MxA gene expression is an appropriate marker for measuring biologic activity of exogenous IFNß, as its induction indicates IFNAR receptor stimulation. A recent study has shown that measurement of MxA mRNA, after 1 year of treatment, predicts clinical responsiveness to IFNß therapy. Loss of IFNß bioactivity is mostly due to anti-IFNß antibodies (both neutralizing and binding), non-compliance and receptor saturation. The aim of this study was to evaluate all possible causes of loss of IFNß bioactivity after 1 year in treated patients. One hundred sixty-seven multiple sclerosis (MS) patients were included. One year after beginning IFNß therapy, each patient underwent a blood test; MxA gene expression was measured by real time PCR, antiviral CPE assay to detect neutralizing antibodies (NAbs), and capture-ELISA (cELISA) to measure binding antibodies (BAbs). For MxA an upper normal threshold of 87 (RE) was considered, 20 TRU/mL was the threshold for NAbs, and 1 U for BAbs positivity. Thirty-seven out of 167 patients (22%) were MxA-negative; of these, 22 were both BAbs and NAbs+, whereas 12 were BAbs+ but Nabs-, and three were both BAbs and NAbs-. The following conclusions were drawn from the study: (1) MxA mRNA should be measured after 1 year of IFNß therapy; (2) after 1 year of IFNß treatment, absence of IFNß bioactivity was detected in 22% of the patients; (3) different biological phenomena and reduced compliance explain this absence; (4) identification of the reason for absence of IFN bioactivity improves patients' management.

Western blot analysis for the detection of serum antibodies recognizing linear Aquaporin-4 epitopes in patients with Neuromyelitis Optica.

The current study presents a western blot assay showing good sensitivity (81%) and specificity (97%) for detecting serum antibodies to Aquaporin-4 (AQP4) in patients with Neuromyelitis Optica (NMO). By using western blot, we were able, for the first time, to distinguish serum immunoreactivity against either of the two AQP4 isoforms, showing that antibodies recognizing the linear AQP4-M1 isoform are specific for NMO. Moreover, the high sensitivity and specificity of the western blot assay in detecting serum anti-AQP4 antibodies in NMO patients suggest that such auto-antibodies may be of linear nature, thus recognizing epitopes that are displayed in denatured protein.

Anti-interferon-beta neutralising activity is not entirely mediated by antibodies.

Many multiple sclerosis (MS) patients treated with interferon-beta (IFNbeta) develop anti-IFNbeta antibodies (BAbs), which can interfere with both in vitro and in vivo bioactivity of the injected cytokine. Objective of this study was to correlate these measures. Among the 256 enrolled patients, 11 (4.3%) showed a significant inhibition of the IFNbeta activity in vitro, but no measurable BAbs. As a whole, in vivo bioactivity was inhibited in 9/11 (82%) of these patients. A minority of IFNbeta treated patients have a non-antibody mediated neutralising activity, which competitively inhibits the bioactivity both in vitro and in vivo.