Anti-invasive effects of CXCR4 and FAK inhibitors in non-small cell lung carcinomas with mutually inactivated p53 and PTEN tumor suppressors

Non-small cell lung carcinoma (NSCLC) is the most common type of lung cancer. At the time of diagnosis, a large percentage of NSCLC patients have already developed metastasis, responsible for extremely high mortality rates. CXCR4 receptor and focal adhesion kinase (FAK) are known to regulate such invasive cancer behavior. Their expression is downregulated by p53 and PTEN tumor suppressors which are commonly co-inactivated in NSCLC patients and contribute to metastasis. Therefore, targeting CXCR4 or FAK seems to be a promising strategy in suppressing metastatic spread of p53/PTEN deficient NSCLCs. In this study, we first examined the invasive characteristics of NSCLC cells with suppressed p53 and PTEN activity using wound healing, gelatin degradation and invasion assays. Further, changes in the expression of CXCR4 and FAK were evaluated by RT-qPCR and Western Blot analysis. Finally, we tested the ability of CXCR4 and FAK inhibitors (WZ811 and PF-573228, respectively) to suppress the migratory and invasive potential of p53/PTEN deficient NSCLC cells, in vitro and in vivo using metastatic models of human NSCLC. Our results showed that cells with mutually inactive p53 and PTEN have significantly increased invasive potential associated with hyperactivation of CXCR4 and FAK signaling pathways. Treatments with WZ811 and PF-573228 inhibitors significantly reduced migratory and invasive capacity in vitro and showed a trend of improved survival in vivo. Accordingly, we demonstrated that p53/PTEN deficient NSCLCs have extremely invasive phenotype and provided a rationale for the use of CXCR4 or FAK inhibitors for the suppression of NSCLC dissemination.This is a post-peer-review, pre-copyedit version of an article published in Investigational New Drugs. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s10637-017-0494-4

Chimeric Stimuli-Responsive Liposomes as Nanocarriers for the Delivery of the Anti-Glioma Agent TRAM-34

Nanocarriers are delivery platforms of drugs, peptides, nucleic acids and other therapeutic molecules that are indicated for severe human diseases. Gliomas are the most frequent type of brain tumor, with glioblastoma being the most common and malignant type. The current state of glioma treatment requires innovative approaches that will lead to efficient and safe therapies. Advanced nanosystems and stimuli-responsive materials are available and well-studied technologies that may contribute to this effort. The present study deals with the development of functional chimeric nanocarriers composed of a phospholipid and a diblock copolymer, for the incorporation, delivery and pH-responsive release of the antiglioma agent TRAM-34 inside glioblastoma cells. Nanocarrier analysis included light scattering, protein incubation and electron microscopy, and fluorescence anisotropy and thermal analysis techniques were also applied. Biological assays were carried out in order to evaluate the nanocarrier nanotoxicity in vitro and in vivo, as well as to evaluate antiglioma activity. The nanosystems were able to successfully manifest functional properties under pH conditions, and their biocompatibility and cellular internalization were also evident. The chimeric nanoplatforms presented herein have shown promise for biomedical applications so far and should be further studied in terms of their ability to deliver TRAM-34 and other therapeutic molecules to glioblastoma cells

Sigma receptors: study of their role in cancer and the development of novel targeted anticancer therapies

Sigma receptors have attracted research interest as they have been proposed as therapeutic targets for the treatment of various diseases and pathological conditions, including some types of cancer. They consist of two subtypes, sigma-1 (σ1) and sigma-2 (σ2), however, the identification of σ2 is under investigation. Until recently it was related to the PGRMC1 receptor while a later theory identifies it with the TMEM97 receptor. Also, some ligands exhibit significant antiproliferative and cytotoxic activity in various types of cancer. However, the precise mechanism of action of both sigma receptors and their ligands remains unclear and remains to be elucidated.In the present doctoral thesis, the levels of σ receptor expression (σ1, σ2/PGRMC1, and σ2/TMEM97) in human established and primary cancer cell lines were investigated. For the first time in the literature, the simultaneous determination of the expression levels of the σ1 receptor and the σ2/TMEM97 and σ2/PGRMC1 receptors was performed simultaneously. Subsequently, the in vitro anticancer activity of some widely used σ1 and σ2 ligands against various types of cancer was studied. Their activity was compared under the same experimental conditions and their possible mechanism of action was investigated. Finally, the ligand that exhibited the best in vitro antiproliferative activity (Siramesine) was more extensively studied in primary patient-derived xenografts (PDX). The expression levels of the σ receptors were determined by Western blot. The antiproliferative activity of the ligands against 24 cancer cell lines was studied using the sulforhodamine B (SRB) assay. The COMPARE algorithm was used for the estimation of the mechanism of action of the ligands. The efficacy of Siramesine (monotherapy or in combination with Gemcitabine) was tested on cancer animal models derived from patients with pancreatic cancer (PDX) that were developed in our laboratory. From the present study, there is no direct correlation between the σ1 and σ2 receptors with cancer type and tumor aggression. In addition, it seems that the substances tested here, even though they have significant in vitro anticancer activity, exert this effect through mechanisms that do not involve the receptors. Among the tested σ ligands, Siramesine appeared to be the most promising. Studies of Siramesine, in tumor xenografts derived from patients (PDX) that were held for the first time under the present thesis, confirmed its good anticancer activity. Also, our experimental data show that Siramesine can be combined with other chemotherapeutics to improve the therapeutic effect. The major disadvantage of the substance appears to be some of its increased toxicity but it can be overcome, as the corresponding study using controlled release drug nanoparticles such as liposomes, has shown.Οι σίγμα υποδοχείς έχουν προσελκύσει το ερευνητικό ενδιαφέρον καθώς έχουν προταθεί ως θεραπευτικοί στόχοι για την αντιμετώπιση διαφόρων ασθενειών και παθολογικών καταστάσεων, μεταξύ των οποίων και ορισμένοι τύποι καρκίνου. Αποτελούνται από δύο υπότυπους, σίγμα-1 (σ1) και σίγμα-2 (σ2), ωστόσο, η ταυτοποίηση του σ2 βρίσκεται υπό διερεύνηση. Μέχρι πρόσφατα σχετίζονταν με τον υποδοχέα PGRMC1 ενώ μεταγενέστερη θεωρία τον ταυτίζει με τον υποδοχέα TMEM97. Επίσης, ορισμένοι σ προσδέτες επιδεικνύουν σημαντική αντιπολλαπλασιαστική και κυτταροτοξική δράση σε διάφορους τύπους καρκίνου. Ωστόσο, ο ακριβής μηχανισμός δράσης τόσο των σίγμα υποδοχέων όσο και των προσδετών τους παραμένει αδιευκρίνιστος και μένει να αποσαφηνιστεί.Στην παρούσα διδακτορική διατριβή, διερευνήθηκαν τα επίπεδα έκφρασης των σ υποδοχέων (σ1, σ2/PGRMC1 και σ2/TMEM97) σε ανθρώπινες εγκαθιδρυμένες και πρωτογενείς καρκινικές κυτταρικές σειρές. Για πρώτη φορά στη βιβλιογραφία, πραγματοποιήθηκε ο ταυτόχρονος προσδιορισμός των επιπέδων έκφρασης του υποδοχέα σ1 και των υποδοχέων σ2/TMEM97 και σ2/PGRMC1. Στη συνέχεια, μελετήθηκε η in vitro αντικαρκινική δράση κάποιων ευρέως χρησιμοποιούμενων σ1 και σ2 προσδετών, έναντι διαφόρων τύπων καρκίνου. Η δραστικότητά τους συγκρίθηκε υπό τις ίδιες πειραματικές συνθήκες και διερευνήθηκε ο πιθανός μηχανισμός δράσης τους. Τέλος, ο σ προσδέτης που επέδειξε την καλύτερη in vitro αντιπολλαπλασιαστική δράση (Siramesine), μελετήθηκε εκτενέστερα σε πρωτογενείς κυτταρικές σειρές και ξενομοσχεύματα προερχόμενα από ασθενείς (PDX). Ο προσδιορισμός των επιπέδων έκφρασης των σ υποδοχέων πραγματοποιήθηκε με ανοσοαποτύπωση κατά Western. Η αντιπολλαπλασιαστική δράση των σ προσδετών μελετήθηκε με τη χρήση της μεθόδου SRB. Ο αλγόριθμος COMPARE χρησιμοποιήθηκε για την εκτίμηση του μηχανισμού δράσης των σ προσδετών. Ο έλεγχος της δράσης της Siramesine (μονοθεραπεία ή συνδυαστικά με τη Gemcitabine) έγινε σε ζωικά πρότυπα καρκίνου προερχόμενα από ασθενείς με καρκίνο του παγκρέατος (PDX), που αναπτύχθηκαν στο εργαστήριό μας. Από την παρούσα μελέτη δεν προκύπτει κάποια άμεση συσχέτιση των υποδοχέων σ1 και σ2 με το είδος αλλά και την επιθετικότητα του όγκου. Φαίνεται ότι οι ουσίες που δοκιμάσθηκαν, ακόμη κι αν έχουν σημαντική in vitro αντικαρκινική δράση ασκούν τη δράση αυτή μέσω μηχανισμών στους οποίους δεν εμπλέκονται οι σ υποδοχείς. Από τις ουσίες που δοκιμάσθηκαν, η Siramesine φάνηκε να είναι η περισσότερη υποσχόμενη. Οι μελέτες της, σε ξενομοσχεύματα όγκων από ασθενείς, που για πρώτη φορά έγιναν στο πλαίσιο της παρούσας διατριβής (PDX), επιβεβαίωσαν την καλή αντικαρκινική της δράση. Επίσης, τα πειραματικά μας δεδομένα δείχνουν ότι η ουσία μπορεί να συνδυασθεί με άλλα χημειοθεραπευτικά ώστε να βελτιωθεί το θεραπευτικό αποτέλεσμα. Το βασικό μειονέκτημα της ουσίας φαίνεται να είναι η κάποια αυξημένη τοξικότητά της που όμως μπορεί να ξεπερασθεί, όπως έδειξε η αντίστοιχη μελέτη με τη χρήση νανοφορέων ελεγχόμενης αποδέσμευσης φαρμάκων, όπως είναι τα λιποσώματα

Pancreatic Cancer Organoids : An Emerging Platform for Precision Medicine?

Despite recent therapeutic advances, pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies, with remarkable resistance to treatment, poor prognosis, and poor clinical outcome. More efficient therapeutic approaches are urgently needed to improve patients’ survival. Recently, the development of organoid culture systems has gained substantial attention as an emerging preclinical research model. PDAC organoids have been developed to study pancreatic cancer biology, progression, and treatment response, filling the translational gap between in vitro and in vivo models. Here, we review the rapidly evolving field of PDAC organoids and their potential as powerful preclinical tools that could pave the way towards precision medicine for pancreatic cancer

Study of the relationship between sigma receptor expression levels and some common sigma ligand activity in cancer using human cancer cell lines of the nci-60 cell line panel

Sigma (σ) receptors have attracted great interest since they are implicated in various cellular functions and biological processes and diseases, including various types of cancer. The receptor family consists of two subtypes: sigma-1 (σ1) and sigma-2 (σ2). Both σ receptor subtypes have been proposed as therapeutic targets for various types of cancers, and many studies have provided evidence that their selective ligands (agonists and antagonists) exhibit antiproliferative and cytotoxic activity. Still, the precise mechanism of action of both σ receptors and their ligands remains unclear and needs to be elucidated. In this study, we aimed to simultaneously determine the expression levels of both σ receptor subtypes in several human cancer cell lines. Additionally, we investigated the in vitro antiproliferative activity of some widely used σ1 and σ2 ligands against those cell lines to study the relationship between σ receptor expression levels and σ ligand activity. Finally, we ran the NCI60 COMPARE algorithm to further elucidate the cytotoxic mechanism of action of the selected σ ligands studied herein

P90 ribosomal S6 kinases : A bona fide target for novel targeted anticancer therapies?

The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases. They are downstream effectors of the Ras/ERK/MAPK signaling cascade. ERK1/2 activation directly results in the phosphorylation of RSKs, which further, through interaction with a variety of different downstream substrates, activate various signaling events. In this context, they have been shown to mediate diverse cellular processes like cell survival, growth, proliferation, EMT, invasion, and metastasis. Interestingly, increased expression of RSKs has also been demonstrated in various cancers, such as breast, prostate, and lung cancer. This review aims to present the most recent advances in the field of RSK signaling that have occurred, such as biological insights, function, and mechanisms associated with carcinogenesis. We additionally present and discuss the recent advances but also the limitations in the development of pharmacological inhibitors of RSKs, in the context of the use of these kinases as putative, more efficient targets for novel anticancer therapeutic approaches

Potential of the dual mTOR kinase inhibitor AZD2014 to overcome paclitaxel resistance in anaplastic thyroid carcinoma.

PURPOSE Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-resistance is often associated with changes in activity of the RAS/MAPK/ERK and PI3K/AKT/mTOR pathways and/or a high expression of ATP binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). To assess the therapeutic efficacy in ATC of a combination of the dual mTOR kinase inhibitor vistusertib (AZD2014) and paclitaxel (PTX), we generated a new cell line (Rho-) via the selection of human thyroid carcinoma 8505C cells that exhibit a low accumulation of rhodamine 123, which serves as a P-gp and BCRP substrate. METHODS Immunohistochemistry was used for P-gp and BCRP expression analyses in primary ATC patient samples. Spheroid formation and immunodeficient NSG mice were used for performing in vitro and in vivo tumorigenicity assays, respectively. MTT, flow-cytometry, fluorescent microscopy, cell death and proliferation assays, as well as migration, invasion and gelatin degradation assays, were used to assess the potential of AZD2014 to enhance the effects of PTX. ATC xenografts in SCID mice were used for evaluating in vivo treatment efficacies. RESULTS Rho- cells were found to be 10-fold more resistant to PTX than 8505C cells and, in addition, to be more tumorigenic. We also found that AZD2014 sensitized Rho- cells to PTX by inhibiting proliferation and by inducing autophagy. The combined use of AZD2014 and PTX efficiently inhibited in vitro ATC cell migration and invasion. Subsequent in vivo xenograft studies indicated that the AZD2014 and PTX combination effectively suppressed ATC tumor growth. CONCLUSIONS Our data support results from recent phase I clinical trials using combinations of AZD2014 and PTX for the treatment of solid tumors. Such combinations may also be employed for the design of novel targeted ATC treatment strategies