Search

The immunoglobulin B cell receptor (IgBCR) expressed by chronic lymphocytic leukemia (CLL) B cells plays a pivotal role in tumorigenesis, supporting neoplastic transformation, survival, and expansion of tumor clones. We demonstrated that in the same patient, two or more CLL clones could coexist, recognized by the expression of different variable regions of the heavy chain of IgBCR, composing the antigen-binding site. In this regard, phage display screening could be considered the easier and most advantageous methodology for the identification of small peptide molecules able to mimic the natural antigen of the tumor IgBCRs. These molecules, properly functionalized, could be used as a probe to specifically identify and isolate single CLL subpopulations, for a deeper analysis in terms of drug resistance, phenotype, and gene expression. Furthermore, CLL cells express another surface membrane receptor, the CD5, which is commonly expressed by normal T cells. Piece of evidence supports a possible contribution of CD5 to the selection and maintenance of autoreactivity in B cells and the constitutive expression of CD5 on CLL cells could induce pro-survival stimuli. In this brief research report, we describe a peptide-based single-cell sorting using as bait the IgBCR of tumor cells; in the next step, we performed a quantitative analysis of CD5 expression by qRT-PCR related to the expressed IgBCR. Our approach could open a new perspective for the identification, isolation, and investigation of all subsets of IgBCR-related CLL clones, with particular attention to the more aggressive clones.

Ovarian cancer is the second most common gynecological malignancy worldwide. Paclitaxel is particularly important in the therapy of ovarian carcinomas, but the treatment efficacy is counteracted by the development of resistance to chemotherapy. The identification of target molecules that can prevent or control the development of chemoresistance might provide important tools for the management of patients affected by ovarian cancer. Serum- and glucocorticoid-regulated kinase 1 (SGK1) appears to be a key determinant of resistance to chemo- and radiotherapy. Specifically, SGK1 affects paclitaxel sensitivity in RKO colon carcinoma cells by modulating the specificity protein 1 (SP1)–dependent expression of Ran-specific GTPase-activating protein (RANBP1), a member of the GTP-binding nuclear protein Ran (RAN) network that is required for the organization and function of the mitotic spindle. SGK1 inhibition might thus be useful for counteracting the development of paclitaxel resistance. Here, we present in vitro data obtained using ovarian carcinoma cell lines that indicate that the SGK1 inhibitor SI113 inhibits cancer cell proliferation, potentiates the effects of paclitaxel-based chemotherapy, counteracts the development of paclitaxel resistance, and restores paclitaxel sensitivity in paclitaxel-resistant A2780 ovarian cancer cells. The results were corroborated by preclinical studies of xenografts generated in nude mice through the implantation of paclitaxel-resistant human ovarian cancer cells. The SGK1 inhibitor SI113 synergizes with paclitaxel in the treatment of xenografted ovarian cancer cells. Taken together, these data suggest that SGK1 inhibition should be investigated in clinical trials for the treatment of paclitaxel-resistant ovarian cancer.

BACKGROUND Inflammatory bowel disease (IBD) is due to the interaction of genetic and environmental factors that trigger an unbalanced immune response ultimately resulting in the peculiar inflammatory reaction. Experimental models of IBD point to a role of T-cell-derived cytokines (Th17) and to SGK1 as mediator of the Th17 switch. We hypothesize that SGK1, a salt inducible kinase, directs lymphocytic behavior and tissue damage. METHODS Eleven controls and 32 ulcerative colitis (UC) patients were randomized according to endoscopic Mayo score. Mucosal biopsies from different intestinal tracts were analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction to check the expression of disease markers including SGK1. Peripheral blood mononuclear cells (PBMCs) from patients and controls were analyzed by fluorescence-activated cell sorting. Finally, an in vitro cell model was developed to test the hypothesis. RESULTS SGK1 mRNA and protein expression in lesional areas of UC patients were lower than in normal peri-lesional areas of the same patients and in normal tissues of healthy controls. SGK1 expression was increased in PBMCs from UC patients, particularly in the CD4+ cell population, enriched in Th17 cells. IL17/IL13 was increased in patients and correlated with SGK1 expression. Genetically engineered Jurkat cells confirmed the effect of SGK1 overexpression on viability of RKO cells. CONCLUSIONS These observations suggest a pathogenic mechanism whereby SGK1 overexpression in CD4+ T cells induces the secretion of the inflammatory cytokines IL17 and IL13, which downregulate the expression of SGK1 in target tissues. Our data suggest a novel hypothesis in the pathogenesis of UC, integrating colonic epithelial cells and lymphocytes.

Glioblastoma multiforme (GBM) is the most aggressive CNS tumor and is characterized by a very high frequency of clinical relapse after therapy and thus by a dismal prognosis, which strongly compromises patients survival. We have recently identified the small molecule SI113, as a potent and selective inhibitor of SGK1, a serine/threonine protein kinase, that modulates several oncogenic signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation and perturbs cell cycle progression by modulating SGK1-related substrates. SI113 is also able to strongly and consistently block, in vitro and in vivo, growth and survival of human hepatocellular-carcinomas, either used as a single agent or in combination with ionizing radiations. In the present paper we aim to study the effect of SI113 on human GBM cell lines with variable p53 expression. Cell viability, cell death, caspase activation and cell cycle progression were then analyzed by FACS and WB-based assays, after exposure to SI113, with or without oxidative stress and ionizing radiations. Moreover, autophagy and related reticulum stress response were evaluated. We show here, that i) SGK1 is over-expressed in highly malignant gliomas and that the treatment with SI113 leads to ii) significant increase in caspase-mediated apoptotic cell death in GBM cell lines but not in normal fibroblasts; iii)enhancement of the effects of ionizing radiations; iv) modulation of the response to oxidative reticulum stress; v) induction of cytotoxic autophagy. Evidence reported here underlines the therapeutic potential of SI113 in GBM, suggesting a new therapeutic strategy either alone or in combination with radiotherapy.

Among the proteins putatively interacting with PTPRJ, a tyrosine phosphatase with tumor suppressor activity, we focused on the oncoprotein CD98hc as a very interesting candidate for the development of innovative targeted drugs. In fact, CD98hc, representing the heavy chain of a transmembrane aminoacid transporter, is overexpressed in several human cancers. Furthermore, CD98hc higher expression is associated with poor prognosis in lung cancer patients. CD98hc is linked to light chains (LATs, xCT) by disulfide bridge, polar and hydrophobic interactions. The light chain confers substrate specificity, and ERK, AKT, FAK and mTOR pathways are involved in CD98hc-LATs/xCT downstream signals. Moreover, CD98hc is a coreceptor of b-integrins and it is involved in cell proliferation, migration and invasion. We identified two peptides and 15 small molecules (putatively targeting the disulfide bridge) as candidate CD98hc inhibitors by phage display and in silico screenings, respectively, and validated in vitro the binding between peptides and transmembrane fraction of CD98hc by cytofluorimetric assay. Then, we tested the capability of both types of compounds to affect cell viability and proliferation through MTT and CFSE assays, respectively. We observed that the targeting of CD98hc through both peptides and small molecules reduced cell proliferation in A549 human lung cancer cells, strongly encouraging a deeper characterization of these candidate anticancer molecules. Indeed, our next goals will be the assessment of biochemical activity of CD98hc following to its inhibition, as well as the evaluation of compounds toxicity, both in vitro and in vivo. The final aim is to identify lead compounds inhibiting CD98hc, in order to develop novel molecules to be translated in the clinical setting and to be used as monotherapy and/or in a combinatorial approaches for the treatment of cancer patients. Citation Format: Delia Lanzillotta, Enrico Iaccino, Anna Artese, Selena Mimmi, Sabrina D'Agostino, Isabella Romeo, Patrizia Cantafio, Vincenzo Dattilo, Giosuè Costa, Carolina Brescia, Eugenio Gaudio, Stefano Alcaro, Francesco Trapasso. The CD98hc oncoprotein as a target of new anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B122. doi:10.1158/1535-7163.TARG-19-B122

PTPRJ, a receptor protein tyrosine phosphatase strongly downregulated in human cancer, displays tumor suppressor activity by negatively modulating several proteins involved in proliferating signals. Here, through a proteomic-based approach, we identified a list of potential PTPRJ-interacting proteins and among them we focused on CD98hc, a type II glycosylated integral membrane protein encoded by SLC3A2, corresponding to the heavy chain of a heterodimeric transmembrane amino-acid transporter, including LAT1. CD98hc is widely overexpressed in several types of cancers and contributes to the process of tumorigenesis by interfering with cell proliferation, adhesion, and migration. We first validated PTPRJ-CD98hc interaction, then demonstrated that PTPRJ overexpression dramatically reduces CD98hc protein levels in A549 lung cancer cells. In addition, following to the treatment of PTPRJ-transduced cells with MG132, a proteasome inhibitor, CD98hc levels did not decrease compared to controls, indicating that PTPRJ is involved in the regulation of CD98hc proteasomal degradation. Moreover, PTPRJ overexpression combined with CD98hc silencing consistently reduced cell proliferation and triggered apoptosis of lung cancer cells. Interestingly, by interrogating the can Evolve database, we observed an inverse correlation between PTPRJ and SLC3A2 gene expression. Indeed, the non-small cell lung cancers (NSCLCs) of patients showing a short survival rate express the lowest and the highest levels of PTPRJ and SLC3A2, respectively. Therefore, the results reported here contribute to shed lights on PTPRJ signaling in cancer cells: moreover, our findings also support the development of a novel anticancer therapeutic approach by targeting the pathway of PTPRJ that is usually downregulated in highly malignant human neoplasias.

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches.

Background/Aims: The importance of copper in the metabolism of cancer cells has been widely studied in the last 20 years and a clear-cut association between copper levels and cancer deregulation has been established. Copper-64, emitting positrons and β-radiations, is indicated for the labeling of a large number of molecules suitable for radionuclide imaging as well as radionuclide therapy. Glioblastoma multiforme (GBM) is the CNS tumor with the worse prognosis, characterized by high number of recurrences and strong resistance to chemo-radio therapy, strongly affecting patients survival. We have recently discovered and studied the small molecule SI113, as inhibitor of SGK1, a serine/threonine protein kinase, that affects several neoplastic phenotypes and signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation, perturbs cell cycle progression and restores chemo-radio sensibility by modulating SGK1-related substrates. In the present paper we aim to characterize the combined effects of 64CuCl2 and SI113 on human GBM cell lines with variable p53 expression. Methods: Cell viability, cell death and stress/authopagic related pathways were then analyzed by FACS and WB-based assays, after exposure to SI113 and/or 64CuCl2. Results: We demonstrate here, that i) 64CuCl2 is able to induce a time and dose dependent modulation of cell viability (with different IC50 values) in highly malignant gliomas and that the co-treatment with SI113 leads to ii) additive/synergistic effects in terms of cell death; iii) enhancement of the effects of ionizing radiations, probably by a TRC1 modulation; iv) modulation of the autophagic response. Conclusions: Evidence reported here underlines the therapeutic potential of the combined treatment with SI113 and 64CuCl2 in GBM cells.

The serum- and glucocorticoid-regulated kinase (SGK1) controls cell transformation and tumor progression. SGK1 affects mitotic stability by regulating the expression of RANBP1/RAN. Here, we demonstrate that SGK1 fluctuations indirectly modify the maturation of pre-miRNAs, by modulating the equilibrium of the RAN/RANBP1/RANGAP1 axis, the main regulator of nucleo-cytoplasmic transport. The levels of pre-miRNAs and mature miRNAs were assessed by qRT-PCR, in total extracts and after differential nuclear/cytoplasmic extraction. RANBP1 expression is the limiting step in the regulation of SGK1-SP1 dependent nuclear export. These results were validated in unrelated tumor models and primary human fibroblasts and corroborated in tumor-engrafted nude mice. The levels of pri-miRNAs, DROSHA, DICER and the compartmental distribution of XPO5 were documented. Experiments using RANGTP conformational antibodies confirmed that SGK1, through RANBP1, decreases the level of the GTP-bound state of RAN. This novel mechanism may play a role in the epigenomic regulation of cell physiology and fate.

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression. Electronic supplementary material The online version of this article (10.1186/s12943-017-0730-8) contains supplementary material, which is available to authorized users.

The serum- and glucocorticoid-regulated kinase (SGK) family consists of three members, SGK1, SGK2 and SGK3, all displaying serine/threonine kinase activity and sharing structural and functional similarities with the AKT family of kinases. SGK1 was originally described as a key enzyme in the hormonal regulation of several ion channels and pumps. Over time, growing and impressive evidence has been accumulated, linking SGK1 to the cell survival, de-differentiation, cell cycle control, regulation of caspases, response to chemical, mechanical and oxidative injury in cancer models as well as to the control of mitotic stability. Much evidence shows that SGK1 is over-expressed in a variety of epithelial tumors. More recently, many contributions to the published literature demonstrate that SGK1 can mediate chemo-and radio-resistance during the treatment of various human tumors, both in vitro and in vivo. SGK1 appears therefore as a dirty player in the stress response to chemical and radio-agents, responsible of a selective advantage that favors the uncontrolled tumor progression and the selection of the most aggressive clones. The purpose of this review is the analysis of the literature describing SGK1 as central node of the cell resistance, and a summary of the possible strategies in the pharmacological targeting of SGK1.

Expression of PTPRJ, which is a ubiquitous receptor-type protein tyrosine phosphatase, is significantly reduced in a vast majority of human epithelial cancers and cancer cell lines (i.e. colon, lung, thyroid, mammary and pancreatic tumours). A possible role for microRNAs (miRNAs) in the negative regulation of PTPRJ expression has never been investigated. In this study, we show that overexpression of microRNA-328 (miR-328) decreases PTPRJ expression in HeLa and SKBr3 cells. Further investigations demonstrate that miR-328 acts directly on the 3′UTR of PTPRJ, resulting in reduced mRNA levels. Luciferase assay and site-specific mutagenesis were used to identify a functional miRNA response element in the 3′UTR of PTPRJ. Expression of miR-328 significantly enhances cell proliferation in HeLa and SKBr3 cells, similar to the effects of downregulation of PTPRJ with small interfering RNA. Additionally, in HeLa cells, the proliferative effect of miR-328 was not observed when PTPRJ was silenced with small interfering RNA; conversely, restoration of PTPRJ expression in miR-328-overexpressing cells abolished the proliferative activity of miR-328. In conclusion, we report the identification of miR-328 as an important player in the regulation of PTPRJ expression, and we propose that the interaction of miR-328 with PTPRJ is responsible for miR-328-dependent increase of epithelial cell proliferation.

// Cristina Talarico 1, * , Lucia D’Antona 1, * , Domenica Scumaci 2 , Agnese Barone 2 , Francesco Gigliotti 1 , Claudia Vincenza Fiumara 2 , Vincenzo Dattilo 1 , Enzo Gallo 3 , Paolo Visca 3 , Francesco Ortuso 1 , Claudia Abbruzzese 4 , Lorenzo Botta 5 , Silvia Schenone 6 , Giovanni Cuda 2 , Stefano Alcaro 1 , Cataldo Bianco 2 , Patrizia Lavia 7 , Marco G. Paggi 4 , Nicola Perrotti 1, * , Rosario Amato 1, * 1 Department of “Scienze della Salute”, University “Magna Graecia” of Catanzaro, Viale Europa, Catanzaro, Italy 2 Department of “Medicina Sperimentale e Clinica”, University “Magna Graecia” of Catanzaro, Viale Europa, Catanzaro, Italy 3 Section of Pathology, Regina Elena National Cancer Institute, IRCCS, Rome, Italy 4 Experimental Oncology, Regina Elena National Cancer Institute, IRCCS, Rome, Italy 5 Department of Biotecnologie, Chimica e Farmacia, University of Siena, Siena, Italy 6 Department of Farmacia, University of Genova, Genova, Italy 7 Institute of Molecular Biology and Pathology (IBPM), National Research Council of Italy (CNR), c/o University “La Sapienza”, Rome, Italy * These authors have contributed equally to this work Correspondence to: Rosario Amato, e-mail: rosario.amato@unicz.it Nicola Perrotti, e-mail: perrotti@unicz.it Keywords: SGK1, HCC, kinase inhibitor, radiotherapy, apoptosis Received: June 04, 2015 Accepted: September 28, 2015 Published: October 08, 2015 ABSTRACT The SGK1 kinase is pivotal in signal transduction pathways operating in cell transformation and tumor progression. Here, we characterize in depth a novel potent and selective pyrazolo[3,4-d]pyrimidine-based SGK1 inhibitor. This compound, named SI113, active in vitro in the sub-micromolar range, inhibits SGK1-dependent signaling in cell lines in a dose- and time-dependent manner. We recently showed that SI113 slows down tumor growth and induces cell death in colon carcinoma cells, when used in monotherapy or in combination with paclitaxel. We now demonstrate for the first time that SI113 inhibits tumour growth in hepatocarcinoma models in vitro and in vivo . SI113-dependent tumor inhibition is dose- and time-dependent. In vitro and in vivo SI113-dependent SGK1 inhibition determined a dramatic increase in apotosis/necrosis, inhibited cell proliferation and altered the cell cycle profile of treated cells. Proteome-wide biochemical studies confirmed that SI113 down-regulates the abundance of proteins downstream of SGK1 with established roles in neoplastic transformation, e.g. MDM2, NDRG1 and RAN network members. Consistent with knock-down and over-expressing cellular models for SGK1, SI113 potentiated and synergized with radiotherapy in tumor killing. No short-term toxicity was observed in treated animals during in vivo SI113 administration. These data show that direct SGK1 inhibition can be effective in hepatic cancer therapy, either alone or in combination with radiotherapy.