Design, Synthesis and SAR in 2,4,7-Trisubstituted Pyrido[3,2-d]Pyrimidine Series as Novel PI3K/mTOR Inhibitors.

This work describes the synthesis, enzymatic activities on PI3K and mTOR, in silico docking and cellular activities of various uncommon 2,4,7 trisubstituted pyrido[3,2-d]pyrimidines. The series synthesized offers a chemical diversity in C-7 whereas C-2 (3-hydroxyphenyl) and C-4 groups (morpholine) remain unchanged, in order to provide a better understanding of the molecular determinants of PI3K selectivity or dual activity on PI3K and mTOR. Some C-7 substituents were shown to improve the efficiency on kinases compared to the 2,4-di-substituted pyrimidopyrimidine derivatives used as references. Six novel derivatives possess IC50 values on PI3Kα between 3 and 10 nM. The compounds with the best efficiencies on PI3K and mTOR induced micromolar cytotoxicity on cancer cell lines possessing an overactivated PI3K pathway.

Effect of decalcification protocols on immunohistochemistry and molecular analyses of bone samples.

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to >10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (<5 cycles of 6 h each) did not alter antigenicity and allowed for detection of gene mutations, amplifications or even fusion transcripts. EDTA showed superiority for in situ hybridization techniques. According to these results and our institutional experience, we propose recommendations for decalcification of bone samples, from biopsies to surgical specimens.

Ranolazine inhibits NaV1.5-mediated breast cancer cell invasiveness and lung colonization.

BACKGROUND: Na(V)1.5 voltage-gated sodium channels are abnormally expressed in breast tumours and their expression level is associated with metastatic occurrence and patients' death. In breast cancer cells, Na(V)1.5 activity promotes the proteolytic degradation of the extracellular matrix and enhances cell invasiveness. FINDINGS: In this study, we showed that the extinction of Na(V)1.5 expression in human breast cancer cells almost completely abrogated lung colonisation in immunodepressed mice (NMRI nude). Furthermore, we demonstrated that ranolazine (50 µM) inhibited Na(V)1.5 currents in breast cancer cells and reduced Na(V)1.5-related cancer cell invasiveness in vitro. In vivo, the injection of ranolazine (50 mg/kg/day) significantly reduced lung colonisation by Na(V)1.5-expressing human breast cancer cells. CONCLUSIONS: Taken together, our results demonstrate the importance of Na(V)1.5 in the metastatic colonisation of organs by breast cancer cells and indicate that small molecules interfering with Na(V) activity, such as ranolazine, may represent powerful pharmacological tools to inhibit metastatic development and improve cancer treatments.

Growth and survival of lung cancer cells: regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor.

The dysregulated expression of kallikrein-related peptidase 6 (KLK6) is involved in non-small cancer (NSCLC) cell growth. However, the mechanism that sustains KLK6 signaling remains unknown. We used an isogenic non-small cell lung cancer (NSCLC) cell model system to demonstrate that KLK6 promotes the proliferation of lung tumoral cells and restrains their apoptosis in vitro via ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of ß-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2). These observations support the concept of a role for KLK6 in the oncogenesis of NSCLC.

Proteomic demonstration of the recurrent presence of inter-alpha-inhibitor H4 heavy-chain during aspergillosis induced in an animal model.

Invasive pulmonary aspergillosis remains a matter of great concern in oncology/haematology, intensive care units and organ transplantation departments. Despite the availability of various diagnostic tools with attractive features, new markers of infection are required for better medical care. We therefore looked for potential pulmonary biomarkers of aspergillosis, by carrying out two-dimensional (2D) gel electrophoresis comparing the proteomes of bronchial-alveolar lavage fluids (BALF) from infected rats and from control rats presenting non-specific inflammation, both immunocompromised. A bioinformatic analysis of the 2D-maps revealed significant differences in the abundance of 20 protein spots (ANOVA P-value<0.01; q-value<0.03; power>0.8). One of these proteins, identified by mass spectrometry, was considered of potential interest: inter-alpha-inhibitor H4 heavy-chain (ITIH4), characterised for the first time in this infectious context. Western blotting confirmed its overabundance in all infected BALF, particularly at early stages of murine aspergillosis. Further investigations were carried on rat serum, and confirmed that ITIH4 levels increased during experimental aspergillosis. Preliminary results in human samples strengthened this trend. To our knowledge, this is the first description of the involvement of ITIH4 in aspergillosis.

The pig as a model for investigating the role of neutrophil serine proteases in human inflammatory lung diseases.

The serine proteases released by activated polymorphonuclear neutrophils [NSPs (neutrophil serine proteases)] contribute to a variety of inflammatory lung diseases, including CF (cystic fibrosis). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies [Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj and Gauthier (2009) J. Biol. Chem. 284, 34084-34091). Thus NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR-/- (CFTR is CF transmembrane conductance regulator) pig model is a promising alternative to the mouse model of CF [Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani et al. (2008) Science 321, 1837-1841]. We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and NETs (neutrophil extracellular traps). All of the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF.

Histopathogenesis of bone- and soft-tissue tumor spectrum with USP6 gene rearrangement: multiple partners involved in the tissue repair process.

Primary aneurysmal bone cyst, nodular fasciitis, myositis ossificans and related lesions as well as fibroma of tendon sheath are benign tumors that share common histological features and a chromosomal rearrangement involving the ubiquitin-specific peptidase 6 (USP6) gene. The tumorigenesis of this tumor spectrum has become complex with the identification of an increasing number of new partners involved in USP6 rearrangements. Because traumatic involvement has long been mentioned in the histogenesis of most lesions in the USP6 spectrum and they morphologically resemble granulation tissue or callus, we attempted to shed light on the function and role USP6 partners play in tissue remodelling and the repair process and, to a lesser extent, bone metabolism.

Validation of tumor-associated macrophage ferritin light chain as a prognostic biomarker in node-negative breast cancer tumors: A multicentric 2004 national PHRC study.

Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. Large screening of different biological compartments, such as the proteome, by means of high throughput techniques may greatly help scientists to find such markers. The present retrospective multicentric study included 268 node-negative breast cancer patients. We used a proteomic approach of SELDI-TOF-MS screening to identify differentially expressed cytosolic proteins with prognostic impact. The screening cohort was composed of 198 patients. Seventy supplementary patients were included for validation. Immunohistochemistry (IHC) and immunoassay (IA) were run to confirm the prognostic role of the marker identified by SELDI-TOF-MS screening. IHC was also used to explore links between selected marker and epithelial-mesenchymal transition (EMT)-like, proliferation and macrophage markers. Ferritin light chain (FTL) was identified as an independent prognostic marker (HR = 1.30-95% CI: 1.10-1.50, p = 0.001). Validation step by means of IHC and IA confirmed the prognostic value of FTL level. CD68 IHC showed that FTL was stored in tumor-associated macrophages (TAM), which exhibit an M2-like phenotype. We report here, first, the validation of FTL as a breast tumor prognostic biomarker in node-negative patients, and second, the fact that FTL is stored in TAM.

A substrate-based approach to convert SerpinB1 into a specific inhibitor of proteinase 3, the Wegener's granulomatosis autoantigen.

The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 × 107 M⁻¹ · s⁻¹, which is ∼100-fold higher than that observed with wt-SerpinB1 and compares with that of α1-protease inhibitor (α1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis.

Isolation of a new cell population in the glioblastoma microenvironment.

Glioblastoma (GB) is a highly infiltrative tumor recurring in 90% of cases within a few centimeters of the resection cavity, even in cases of complete tumor resection and adjuvant chemo/radiotherapy. This observation highlights the importance of understanding this special zone of brain tissue surrounding the tumor. It is becoming clear that the nonneoplastic stromal compartment of most solid cancers plays an active role in tumor proliferation, invasion, and metastasis. Very little information, other than that concerning angiogenesis and immune cells, has been collected for stromal cells from GB. As part of a translational research program, we have isolated a new stromal cell population surrounding GB by computer-guided stereotaxic biopsies and primary culture. We named these cells GB-associated stromal cells (GASCs). GASCs are diploid, do not display the genomic alterations typical of GB cells, and have phenotypic and functional properties in common with the cancer-associated fibroblasts (CAFs) described in the stroma of carcinomas. In particular, GASCs express markers associated with CAFs such as fibroblast surface protein, alpha-smooth muscle actin (α-SMA), and platelet-derived growth factor receptor-beta (PDGFRß). Furthermore, GASCs have a molecular expression profile different from that of control stromal cells derived from non-GB peripheral brain tissues. GASCs were also found to have tumor-promoting effects on glioma cells in vitro and in vivo. The isolation of GASCs in a tumor of neuroepithelial origin was unexpected, and further studies are required to determine their potential as a target for antiglioma treatment.

Accumulation of Arachidonic Acid, Precursor of Pro-Inflammatory Eicosanoids, in Adipose Tissue of Obese Women: Association with Breast Cancer Aggressiveness Indicators.

While obesity is linked to cancer risk, no studies have explored the consequences of body mass index (BMI) on fatty acid profiles in breast adipose tissue and on breast tumor aggressiveness indicators. Because of this, 261 breast adipose tissue samples of women with invasive breast carcinoma were analyzed. Fatty acid profile was established by gas chromatography. For normal-weight women, major changes in fatty acid profile occurs after menopause, with the enrichment of long-chain polyunsaturated fatty acids (LC-PUFAs) of both n-6 and n-3 series enrichment, but a stable LC-PUFAs n-6/n-3 ratio across age. BMI impact was analyzed by age subgroups to overcome the age effect. BMI increase is associated with LC-PUFAs n-6 accumulation, including arachidonic acid. Positive correlations between BMI and several LC-PUFAs n-6 were observed, as well as a strong imbalance in the LC-PUFAs n-6/n-3 ratio. Regarding cancer, axillary lymph nodes (p = 0.02) and inflammatory breast cancer (p = 0.08) are more frequently involved in obese women. Increased BMI induces an LC-PUFAs n-6 accumulation, including arachidonic acid, in adipose tissue. This may participate in the development of low-grade inflammation in obese women and breast tumor progression. These results suggest the value of lifestyle and LC-PUFAs n-3 potential, in the context of obesity and breast cancer secondary/tertiary prevention.

Total long-chain polyunsaturated n-3 fatty acids level is an independent predictive factor of breast cancer multifocality in women with positive hormone-receptors tumors.

In a previous pilot study, we showed that polyunsaturated n-3 fatty acids of breast adipose tissues were associated with breast cancer multifocality. In the present study, we investigated biochemical, clinical and histological factors associated with breast cancer focality in a large cohort of women with positive hormone-receptors tumors. One hundred sixty-one consecutive women presenting with positive hormone-receptors breast cancer underwent breast-imaging procedures including a Magnetic Resonance Imaging prior to treatment. Breast adipose tissue specimens were collected during surgery of tumors. A biochemical profile of breast adipose tissue fatty acids was established by gas chromatography. Clinicopathologic characteristics were correlated with multifocality. We assessed whether these factors were predictive of breast cancer focality. We found that tumor size (OR = 1.06 95%CI [1.02-1.09], p < 0.001) and decreased levels in breast adipose tissue of long-chain polyunsaturated n-3 fatty acids (OR = 0.11 95%CI [0.01-0.98], p = 0.03), were independent predictive factors of multifocality. Low levels of long chain polyunsaturated n-3 fatty acids in breast adipose tissue appear to contribute to breast cancer multifocality. The present results reinforce the link between dietary habits and breast cancer clinical presentation.

Novel partners of USP6 gene in a spectrum of bone and soft tissue lesions.

Nodular fasciitis, primary aneurysmal bone cyst, myositis ossificans, and their related lesions are benign tumors that share common histological features and a chromosomal rearrangement involving the ubiquitin-specific peptidase 6 (USP6) gene. The identification of an increasing number of new partners implicated in USP6 rearrangements demonstrates a complex tumorogenesis of this tumor spectrum. In this study on a series of 77 tumors (28 nodular fasciitis, 42 aneurysmal bone cysts, and 7 myositis ossificans) from the database of the French Sarcoma Group, we describe 7 new partners of the USP6 gene. For this purpose, rearrangements were first researched by multiplexed RT-qPCRs in the entire population. A targeted RNA sequencing was then used on samples selected according to a high USP6-transcription level expression estimated by RT-qPCR. Thanks to this multistep approach, besides the common USP6 fusions observed, we detected novel USP6 partners: PDLIM7 and MYL12A in nodular fasciitis and TPM4, DDX17, GTF2I, KLF3, and MEF2A in aneurysmal bone cysts. In order to try to bring to light the role played by the recently identified USP6 partners in this lesional spectrum, their functions are discussed. Taking into account that a traumatic participation has long been mentioned in the histogenesis of most of these lesions and because of their morphological resemblance to organizing granulation reparative tissue or callus, a focus is placed on their relationship with tissue remodeling and, to a lesser extent, with bone metabolism.

Inhibition of cervical cancer cell growth by human papillomavirus virus-like particles packaged with human papillomavirus oncoprotein short hairpin RNAs.

Overexpression of human papillomavirus (HPV E6 and HPV E7) oncogenes in human cervical cells results in the development of cancer, and E6 and E7 proteins are therefore targets for preventing cervical cancer progression. Here, we describe the silencing of E6 and E7 expression in cervical carcinoma cells by RNA interference. In order to increase the efficacy of the RNA interference, HPV pseudovirions coding for a short hairpin RNA (shRNA) sequence were produced. The results indicated the degradation of E6 and E7 mRNAs when shRNA against E6 or E7 were delivered by pseudovirions in HPV-positive cells (CaSki and TC1 cells). E6 silencing resulted in the accumulation of cellular p53 and reduced cell viability. More significant cell death was observed when E7 expression was suppressed. Silencing E6 and E7 and the consequences for cancer cell growth were also investigated in vivo in mice using the capacity of murine TC1 cells expressing HPV-16 E6 and E7 oncogenes to induce fast-growing tumors. Treatment with lentiviruses and HPV virus-like particle vectors coding for an E7 shRNA sequence both resulted in dramatic inhibition of tumor growth. These results show the ability of pseudovirion-delivered shRNA to produce specific gene suppression and provide an effective means of reducing HPV-positive tumor growth.

Outcome of patients with breast cancer in the oldest old (≥80 years).

PURPOSE: In the present study, we present a large institutional study to determine the influence of age≥ 80 years on breast cancer presentation and prognosis. METHODS: The study is a retrospective analysis of our prospectively maintained breast cancer database study using data from of women managed from January 2007 through December 2013. Clinicopathologic characteristics were correlated with outcomes according to age (<80 years and ≥ 80 years). RESULTS: During the study period, 2083 women with invasive breast cancer were included of which 160 women aged ≥ 80 years (7.7 %). Overall survival was lower in the oldest old than in younger counterparts (p < 0.0001) as was distant metastasis free survival (p = 0.004). Differences in management included more radical surgeries and less chemotherapy and radiotherapy in case of age≥ 80 years. By multivariate analysis, age ≥ 80 years was an independent predictive factor of poor overall survival. CONCLUSION: In the present study, age ≥ 80 years was an independent predictive factor of poor overall survival.

Low Levels of Omega-3 Long-Chain Polyunsaturated Fatty Acids Are Associated with Bone Metastasis Formation in Premenopausal Women with Breast Cancer: A Retrospective Study.

In the present study, we investigated various biochemical, clinical, and histological factors associated with bone metastases in a large cohort of pre- and postmenopausal women with breast cancer. Two hundred and sixty-one consecutive women with breast cancer were included in this study. Breast adipose tissue specimens were collected during surgery. After having established the fatty acid profile of breast adipose tissue by gas chromatography, we determined whether there were differences associated with the occurrence of bone metastases in these patients. Regarding the clinical and histological criteria, a majority of the patients with bone metastases (around 70%) had tumors with a luminal phenotype and 59% of them showed axillary lymph node involvement. Moreover, we found a negative association between the levels of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in breast adipose tissue and the development of bone metastases in premenopausal women. No significant association was observed in postmenopausal women. In addition to a luminal phenotype and axillary lymph node involvement, low levels of n-3 LC-PUFA in breast adipose tissue may constitute a risk factor that contributes to breast cancer bone metastases formation in premenopausal women.

Low eicosapentaenoic acid and gamma-linolenic acid levels in breast adipose tissue are associated with inflammatory breast cancer.

OBJECTIVE: Since it is thought that breast adipose tissue could influence breast cancer clinical presentation, we wanted to characterize specifically the relationship between breast adipose tissue fatty acid profile and Inflammatory Breast cancer (IBC). METHODS: Two hundred thirty-four women presenting with breast cancer were managed in our centre between January 2009 and December 2011. Breast adipose tissue specimens were collected during breast surgery. We established the biochemical profile of adipose tissue fatty acids (FA) by gas chromatography and assessed whether there were differences in function of the presence of breast inflammation or not. RESULTS: We found that IBC was associated with decreased levels in breast adipose tissue of eicosapentaenoic acid (EPA), one of the two main polyunsaturated n-3 fatty acids (n-3 PUFA) of marine origin, but also with decreased levels of Gamma Linolenic acid (GLA). Inversely, an increase in palmitic acid levels was associated with IBC. CONCLUSION: These differences in lipid content may contribute to the occurrence of breast cancer inflammation.

Sodium Channel Nav1.5 Controls Epithelial-to-Mesenchymal Transition and Invasiveness in Breast Cancer Cells Through its Regulation by the Salt-Inducible Kinase-1.

Loss of epithelial polarity and gain in invasiveness by carcinoma cells are critical events in the aggressive progression of cancers and depend on phenotypic transition programs such as the epithelial-to-mesenchymal transition (EMT). Many studies have reported the aberrant expression of voltage-gated sodium channels (NaV) in carcinomas and specifically the NaV1.5 isoform, encoded by the SCN5A gene, in breast cancer. NaV1.5 activity, through an entry of sodium ions, in breast cancer cells is associated with increased invasiveness, but its participation to the EMT has to be clarified. In this study, we show that reducing the expression of NaV1.5 in highly aggressive human MDA-MB-231 breast cancer cells reverted the mesenchymal phenotype, reduced cancer cell invasiveness and the expression of the EMT-promoting transcription factor SNAI1. The heterologous expression of NaV1.5 in weakly invasive MCF-7 breast cancer cells induced their expression of both SNAI1 and ZEB1 and increased their invasive capacities. In MCF-7 cells the stimulation with the EMT-activator signal TGF-ß1 increased the expression of SCN5A. Moreover, the reduction of the salt-inducible kinase 1 (SIK1) expression promoted NaV1.5-dependent invasiveness and expression of EMT-associated transcription factor SNAI1. Altogether, these results indicated a prominent role of SIK1 in regulating NaV1.5-dependent EMT and invasiveness.

Increased BRCA1 protein in mammary tumours of rats fed marine omega-3 fatty acids.

Any factor affecting BRCA gene regulation may be of interest in the prevention of breast tumourigenesis. We studied the influence of dietary docosahexaenoic acid (DHA), a major omega-3 fatty acid present in marine products, on rat autochthonous mammary tumourigenesis. DHA-supplementation significantly reduced the incidence of tumours (30%, P=0.007) and led to a 60% increase (P=0.02) in BRCA1 protein level. Since DHA influences the product of a major tumour suppressor gene, this finding may contribute to the observation that high-fish consumption reduces the risk of breast cancer.

Identification of SK3 channel as a new mediator of breast cancer cell migration.

Potassium channels have been involved in epithelial tumorigenesis but the role of small-conductance Ca(2+)-activated K(+) channels is unknown. We report here that small-conductance Ca(2+)-activated K(+) channels are expressed in a highly metastasizing mammary cancer cell line, MDA-MB-435s. Patch-clamp recordings showed typical small-conductance Ca(2+)-activated K(+) channel-mediated currents sensitive to apamin, 4-aminopyridine, and tetraethylammonium. Moreover, the cells displayed a high intracellular calcium concentration, which was decreased after 24 hours of apamin treatment. By regulating membrane potential and intracellular calcium concentration, these channels were involved in MDA-MB-435s cell migration, but not in proliferation. Only SK3 protein expression was observed in these cells in contrast to SK2, which was expressed both in cancer and noncancer cell lines. Whereas small interfering RNA directed against SK3 almost totally abolished MDA-MB-435s cell migration, transient expression of SK3 increased migration of the SK3-deficient cell lines, MCF-7 and 184A1. SK3 channel was solely expressed in tumor breast biopsies and not in nontumor breast tissues. Thus, SK3 protein channel seems to be a new mediator of breast cancer cell migration and represents a potential target for a new class of anticancer agents.