STAT3 silencing by an aptamer-based strategy hampers the crosstalk between NSCLC cells and cancer-associated fibroblasts.

The identification of new effective therapeutic options for non-small-cell lung cancer (NSCLC) represents a crucial challenge in oncology. Recent studies indicate that cancer-associated fibroblasts (CAFs) participate in tumor progression by establishing a favorable microenvironment that promotes cancer progression. Therefore, the development of strategies inhibiting the interplay between CAFs and cancer cells is considered a winning approach for the development of effective anti-cancer drugs. Among other factors, the signal transducer and activator of transcription-3 (STAT3) has been reported as a key mediator of CAF oncogenic actions, representing a promising therapeutic target. Here, we applied an aptamer-based conjugate (named Gint4.T-STAT3), containing a STAT3 siRNA linked to an aptamer binding and inhibiting the platelet-derived growth factor receptor (PDGFR)ß, to obtain STAT3-specific silencing and interfere with CAF pro-tumorigenic functions. We demonstrated that this molecule effectively delivers the STAT3 siRNA in NSCLC cells, and blocks CAF-induced cancer cell growth and migration and reduced spheroid dimension. In addition, we found that Gint4.T-STAT3 alters CAF phenotype, thus functioning as a double-acting molecule able to inhibit the entire tumor bulk. Our data provide a proof of principle for the targeting of CAF pro-tumor functions through an aptamer-based drug, and can open innovative horizons in NSCLC therapy.

AmotL2, IQGAP1, and FKBP51 Scaffold Proteins in Glioblastoma Stem Cell Niches.

Glioma stem cells (GSCs) live in a continuous process of stemness reprogramming to achieve specific cell commitment within the so-called GSC niches, specifically located in periarteriolar regions. In this review, we analyze the expression levels, cellular and subcellular location, and role of three scaffold proteins (IQGAP1, FKBP51, and AmotL2) in GSC niches. Scaffold proteins contribute to cell differentiation, migration, and angiogenesis in glioblastoma. It could be of diagnostic interest for establishing stages, for therapeutic targets, and for improving glioblastoma prognosis, which is still at the experimental level.

Selection of a Nuclease-Resistant RNA Aptamer Targeting CD19.

The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell-specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as 'chemical antibodies', they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2'-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies.

Molecular-Morphological Relationships of the Scaffold Protein FKBP51 and Inflammatory Processes in Knee Osteoarthritis.

Knee osteoarthritis (OA) is one of the most prevalent chronic conditions affecting the adult population. OA is no longer thought to come from a purely biomechanical origin but rather one that has been increasingly recognized to include a persistent low-grade inflammatory component. Intra-articular corticosteroid injections (IACSI) have become a widely used method for treating pain in patients with OA as an effective symptomatic treatment. However, as the disease progresses, IACSI become ineffective. FKBP51 is a regulatory protein of the glucocorticoid receptor function and have been shown to be dysregulated in several pathological scenario's including chronic inflammation. Despite of these facts, to our knowledge, there are no previous studies of the expression and possible role of FKBP51 in OA. We investigated by double and triple immunofluorescence confocal microscopy the cellular and subcellular expression of FKBP51 and its relations with inflammation factors in osteoarthritic knee joint tissues: specifically, in the tibial plateau knee cartilage, Hoffa's fat pad and suprapatellar synovial tissue of the knee. Our results show co-expression of FKBP51 with TNF-α, IL-6, CD31 and CD34 in OA chondrocytes, synovial membrane cells and adipocytes in Hoffa's fat pad. FKBP51 is also abundant in nerve fibers within the fat pad. Co-expression of FKBP51 protein with these markers may be indicative of its contribution to inflammatory processes and associated chronic pain in OA.

Celastrol Prevents Oxidative Stress Effects on FSHR, PAPP, and CYP19A1 Gene Expression in Cultured Human Granulosa-Lutein Cells.

Regulation of oxidative stress (OS) is important to prevent damage to female reproductive physiology. While normal OS levels may have a regulatory role, high OS levels may negatively affect vital processes such as folliculogenesis or embryogenesis. The aim of this work was to study OS induced by glucose, a reactive oxygen species generator, or peroxynitrite, a reactive nitrogen species generator, in cultured human granulosa-lutein (hGL) cells from oocyte donors, analyzing expression of genes involved in oocyte maturation (FSHR, PAPP, and CYP19A1) and OS damage response (ALDH3A2). We also evaluated the effect of celastrol as an antioxidant. Our results showed that although both glucose and peroxynitrite produce OS increments in hGL cells, only peroxynitrite treatment increases ALDH3A2 and PAPP gene expression levels and decreases FSHR gene expression levels. Celastrol pre-treatment prevents this effect of peroxynitrite. Interestingly, when celastrol alone was added, we observed a reduction of the expression of all genes studied, which was independent of both OS inductors. In conclusion, regulation of OS imbalance by antioxidant substances such as celastrol may prevent negative effects of OS in female fertility. In addition to the antioxidant activity, celastrol may well have an independent role on regulation of gene expression in hGL cells.

Identification of a novel RNA aptamer that selectively targets breast cancer exosomes.

Breast cancer is a leading cause of cancer mortality in women. Despite advances in its management, the identification of new options for early-stage diagnosis and therapy of this tumor still represents a crucial challenge. Increasing evidence indicates that extracellular vesicles called exosomes may have great potential as early diagnostic biomarkers and regulators of many cancers, including breast cancer. Therefore, exploiting molecules able to selectively recognize them is of great interest. Here, we developed a novel differential SELEX strategy, called Exo-SELEX, to isolate nucleic acid aptamers against intact exosomes derived from primary breast cancer cells. Among the obtained sequences, we optimized a high-affinity aptamer (ex-50.T) able to specifically recognize exosomes from breast cancer cells or patient serum samples. Furthermore, we demonstrated that the ex.50.T is a functional inhibitor of exosome cellular uptake and antagonizes cancer exosome-induced cell migration in vitro. This molecule provides an innovative tool for the specific exosome detection and the development of new therapeutic approaches for breast cancer.

Advances in Oligonucleotide Aptamers for NSCLC Targeting.

Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer worldwide, with the highest incidence in developed countries. NSCLC patients often face resistance to currently available therapies, accounting for frequent relapses and poor prognosis. Indeed, despite great recent advancements in the field of NSCLC diagnosis and multimodal therapy, most patients are diagnosed at advanced metastatic stage, with a very low overall survival. Thus, the identification of new effective diagnostic and therapeutic options for NSCLC patients is a crucial challenge in oncology. A promising class of targeting molecules is represented by nucleic-acid aptamers, short single-stranded oligonucleotides that upon folding in particular three dimensional (3D) structures, serve as high affinity ligands towards disease-associated proteins. They are produced in vitro by SELEX (systematic evolution of ligands by exponential enrichment), a combinatorial chemistry procedure, representing an important tool for novel targetable biomarker discovery of both diagnostic and therapeutic interest. Aptamer-based approaches are promising options for NSCLC early diagnosis and targeted therapy and may overcome the key obstacles of currently used therapeutic modalities, such as the high toxicity and patients' resistance. In this review, we highlight the most important applications of SELEX technology and aptamers for NSCLC handling.

Emerging Therapeutic RNAs for the Targeting of Cancer Associated Fibroblasts.

Tumor mass consists of a complex ensemble of malignant cancer cells and a wide variety of resident and infiltrating cells, secreted factors, and extracellular matrix proteins that are referred as tumor microenvironment (TME). Cancer associated fibroblasts (CAFs) are key TME components that support tumor growth, generating a physical barrier against drugs and immune infiltration, and contributing to regulate malignant progression. Thus, it is largely accepted that therapeutic approaches aimed at hampering the interactions between tumor cells and CAFs can enhance the effectiveness of anti-cancer treatments. In this view, nucleic acid therapeutics have emerged as promising molecules. Here, we summarize recent knowledge about their role in the regulation of CAF transformation and tumor-promoting functions, highlighting their therapeutic utility and challenges.

IQGAP1, AmotL2, and FKBP51 Scaffoldins in the Glioblastoma Microenvironment.

Glioblastoma (GB) is the most frequently occurring and aggressive primary brain tumor. Glioma stem cells (GSCs) and astrocytoma cells are the predominant malignant cells occurring in GB besides a highly heterogeneous population of migrating, neovascularizing and infiltrating myeloid cells that forms a complex tumor microenvironment (TME). Cross talk between the TME cells is pivotal in the biology of this tumor and, consequently, adaptor proteins at critical junctions of signaling pathways may be crucial. Scaffold proteins (scaffolins or scaffoldins) integrate external and internal stimuli to regulate various signaling pathways, interacting simultaneously with multiple proteins involved. We investigated by double and triple immunofluorescence the localization of IQGAP1, AmotL2, and FKBP51, three closely related scaffoldins, in malignant cells and TME of human GB tumors. We found that IQGAP1 is preferentially expressed in astrocytoma cells, AmotL2 in GSCs, and FKBP51 in white blood cells in human GB tumors. As GSCs are specially the target for novel therapies, we will investigate in further studies whether AmotL2 inhibition is effective in the treatment of GB.

Granulosa-Lutein Cell Sirtuin Gene Expression Profiles Differ between Normal Donors and Infertile Women.

Sirtuins are a family of deacetylases that modify structural proteins, metabolic enzymes, and histones to change cellular protein localization and function. In mammals, there are seven sirtuins involved in processes like oxidative stress or metabolic homeostasis associated with aging, degeneration or cancer. We studied gene expression of sirtuins by qRT-PCR in human mural granulosa-lutein cells (hGL) from IVF patients in different infertility diagnostic groups and in oocyte donors (OD; control group). Study 1: sirtuins genes' expression levels and correlations with age and IVF parameters in women with no ovarian factor. We found significantly higher expression levels of SIRT1, SIRT2 and SIRT5 in patients ≥40 years old than in OD and in women between 27 and 39 years old with tubal or male factor, and no ovarian factor (NOF). Only SIRT2, SIRT5 and SIRT7 expression correlated with age. Study 2: sirtuin genes' expression in women poor responders (PR), endometriosis (EM) and polycystic ovarian syndrome. Compared to NOF controls, we found higher SIRT2 gene expression in all diagnostic groups while SIRT3, SIRT5, SIRT6 and SIRT7 expression were higher only in PR. Related to clinical parameters SIRT1, SIRT6 and SIRT7 correlate positively with FSH and LH doses administered in EM patients. The number of mature oocytes retrieved in PR is positively correlated with the expression levels of SIRT3, SIRT4 and SIRT5. These data suggest that cellular physiopathology in PR's follicle may be associated with cumulative DNA damage, indicating that further studies are necessary.

The Na, K-ATPase ß-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles.

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the ß-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase ß subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, ß1, ß2/AMOG (Adhesion Molecule On Glia) and ß3, were found to be expressed in GBM samples. Generally, ß1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. ß2/AMOG and ß3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, ß2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the ß3 subunit was more intense. These changes in ß subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (ß2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase ß subunits as adaptor proteins and transcription factors.

Alterations in IQGAP1 expression and localization in colorectal carcinoma and liver metastases following oxaliplatin-based chemotherapy.

IQGAP1 is a scaffolding protein that serves a key role in cell dynamics by integrating internal and external stimuli to distinct signal outputs. Previous studies have identified several genes that are significantly up- or downregulated in the peripheral white cells (PWCs) of patients with colorectal adenocarcinoma (CRC), who underwent oxaliplatin-based chemotherapy (CT). In addition, screening studies have reported that IQ-motif containing GTPase activating protein 1 (IQGAP1) transcriptional expression levels varied from 'off' to 'on' following oxaliplatin CT. In order to determine if variations previously described in PWCs are able to be observed at the protein level in tumors and in metastases following CT, the present study performed an immunohistochemical analysis of IQGAP1 in CRC and primary metastases. IQGAP1 expression was observed in the nuclear envelope and in lateral cell membranes and cytoplasm in normal colon tissue. However, in tumor tissue, cells exhibited a diffuse pattern, with variable expression levels of staining in the nuclear membrane and cytoplasm, with the highest expression intensity observed at the invasive front. In healthy and metastasized liver tissue and in the metastases themselves, expression levels varied from cell to cell from no expression to a high level. In the majority of cells, IQGAP1 co-localized with microtubules at the cytoplasmic face of the nuclear envelope. Strong positive expression was observed in areas of the lesion where cells were detaching from the lesion into the lumen. Despite the homogeneous IQGAP1 staining pattern observed in healthy colon tissue sections, CRC demonstrated heterogeneity in staining, which was more marked in metastasized liver tissue resected following CT. However, the most notable findings were the observed effects on the cellular and subcellular distribution and its implications for cancer biology. These results suggest that IQGAP1 may be a putative biomarker, a candidate for clinical diagnostics and a potential novel target for anti-cancer therapeutics.

Commitment of Scaffold Proteins in the Onco-Biology of Human Colorectal Cancer and Liver Metastases after Oxaliplatin-Based Chemotherapy.

Scaffold proteins play pivotal roles in the regulation of signaling pathways, integrating external and internal stimuli to various cellular outputs. We report the pattern of cellular and subcellular expression of scaffoldins angiomotin-like 2 (AmotL2), FK506 binding protein 5 (FKBP51) and IQ motif containing GTPase-activating protein 1 (IQGAP1) in colorectal cancer (CRC) and metastases in liver resected after oxaliplatin-based chemotherapy (CT). Positive immunostaining for the three scaffoldins was found in most cells in healthy colon, tumor, healthy liver and metastasized liver. The patterns of expression of AmotL2, FKBP51 and IQGAP1 show the greatest variability in immune system cells and neurons and glia cells and the least in blood vessel cells. The simultaneous subcellular localization in tumor cells and other cell types within the tumor suggest an involvement of these three scaffoldins in cancer biology, including a role in Epithelial Mesenchymal Transition. The display in differential localization and quantitative expression of AmotL2, FKBP51, and IQGAP1 could be used as biomarkers for more accurate tumor staging and as potential targets for anti-cancer therapeutics by blocking or slowing down their interconnecting functions. Tough further research needs to be done in order to improve these assessments.

IQGAP1 in Podosomes/Invadosomes Is Involved in the Progression of Glioblastoma Multiforme Depending on the Tumor Status.

Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor. GBM is formed by a very heterogeneous astrocyte population, neurons, neovascularization and infiltrating myeloid cells (microglia and monocyte derived macrophages). The IQGAP1 scaffold protein interacts with components of the cytoskeleton, cell adhesion molecules, and several signaling molecules to regulate cell morphology and motility, cell cycle and other cellular functions. IQGAP1 overexpression and delocalization has been observed in several tumors, suggesting a role for this protein in cell proliferation, transformation and invasion. IQGAP1 has been identified as a marker of amplifying cancer cells in GBMs. To determine the involvement of IQGAP1 in the onco-biology of GBM, we performed immunohistochemical confocal microscopic analysis of the IQGAP1 protein in human GBM tissue samples using cell type-specific markers. IQGAP1 immunostaining and subcellular localization was heterogeneous; the protein was located in the plasma membrane and, at variable levels, in nucleus and/or cytosol. Moreover, IQGAP1 positive staining was found in podosome/invadopodia-like structures. IQGAP1⁺ staining was observed in neurons (Map2⁺ cells), in cancer stem cells (CSC; nestin⁺) and in several macrophages (CD31⁺ or Iba1⁺). Our results indicate that the IQGAP1 protein is involved in normal cell physiology as well as oncologic processes.

Oxidative Stress in Granulosa-Lutein Cells From In Vitro Fertilization Patients.

Ovarian aging is associated with gradual follicular loss by atresia/apoptosis. Increased production of toxic metabolites such as reactive oxygen species (ROS) and reactive nitrogen species as well as external oxidant agents plays an important role in the process of ovarian senescence and in the pathogenesis of ovarian pathologies such as endometriosis and polycystic ovary syndrome (PCOS). This review provides a synthesis of available studies of oxidative stress (OS) in the ovary, focusing on the most recent evidence obtained in mural granulosa-lutein (GL) cells of in vitro fertilization patients. Synthesis of antioxidant enzymes such as peroxiredoxin 4, superoxide dismutase, and catalase and OS damage response proteins such as aldehyde dehydrogenase 3, member A2 decreases with aging in human GL cells, favoring an unbalance in ROS/antioxidants that mediates molecular damage and altered cellular function. The increase in OS in the granulosa cell correlates with diminished expression of follicle-stimulating hormone receptor (FSHR) and a dysregulation of the FSHR signaling pathway and may be implicated in disrupted steroidogenic function and poor response to FSH in women with aging. Women with endometriosis and PCOS have lower antioxidant production capacity that may contribute to abnormal follicular development and infertility. Further investigation of the signaling pathways involved in cellular response to OS could shed light into molecular characterization of these diseases and development of new treatment strategies to improve reproductive potential in these women.

Expression and localization of the immunophilin FKBP51 in colorectal carcinomas and primary metastases, and alterations following oxaliplatin-based chemotherapy.

The immunophilin FK506-binding protein 5 (FKBP51) is a scaffold protein that serves a pivotal role in the regulation of multiple signaling pathways, integrating external and internal stimuli into distinct signal outputs. In a previous study, we identified several genes that are significantly up- or downregulated in the peripheral white cells (PWCs) of colorectal adenocarcinoma (CRC) patients undergoing oxaliplatin-based chemotherapy. In our screening, FKBP51 gene expression was downregulated following chemotherapy. In order to determine whether this alteration in gene expression observed in PWCs may be detected at the protein level in tumors and metastases following the administration of adjuvant chemotherapy, an immunohistochemical analysis of FKBP51 in CRC and primary metastasis tissues was performed. The present study confirmed the downregulation of FKBP51 gene expression elicited by chemotherapy with folinic acid (leucovorin), fluorouracil and oxaliplatin in metastasized liver tissue that had been resected after the oxaliplatin-based chemotherapy, compared with tissue section samples of CRC from patients (prior to antineoplastic treatment). Furthermore, the results indicated that, in CRC tissue sections, the expression of FKBP51 protein is associated with an immature phenotype of stromal fibroblasts and with the epithelial-to-mesenchymal transition (EMT) phenotype, suggesting a role for this protein in the EMT process in CRC. Finally, the observation that only certain cells of the stroma express FKBP51 protein suggests a potential role for this immunophilin as a stroma cell subtype marker.

Na,K-ATPase Isozymes in Colorectal Cancer and Liver Metastases.

The goal of this study was to define Na,K-ATPase α and ß subunit isoform expression and isozyme composition in colorectal cancer cells and liver metastases. The α1, α3, and ß1 isoforms were the most highly expressed in tumor cells and metastases; in the plasma membrane of non-neoplastic cells and mainly in a cytoplasmic location in tumor cells. α1ß1 and α3ß1 isozymes found in tumor and metastatic cells exhibit the highest and lowest Na(+) affinity respectively and the highest K(+) affinity. Mesenchymal cell isozymes possess an intermediate Na(+) affinity and a low K(+) affinity. In cancer, these ions are likely to favor optimal conditions for the function of nuclear enzymes involved in mitosis, especially a high intra-nuclear K(+) concentration. A major and striking finding of this study was that in liver, metastasized CRC cells express the α3ß1 isozyme. Thus, the α3ß1 isozyme could potentially serve as a novel exploratory biomarker of CRC metastatic cells in liver.

Early and late events induced by polyQ-expanded proteins: identification of a common pathogenic property of polYQ-expanded proteins.

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24-36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.

Oogenesis in Drosophila melanogaster: a model system for studying cell differentiation and development.

Drosophila oogenesis is a complex developmental process involving the coordinated differentiation of germ line and somatic cells. Correct execution and timing of cell fate specification and patterning events is achieved during this process by the integration of different cell-cell signalling pathways, eventually leading to the generation of positional information inside the oocyte, that is instrumental for the establishment of embryonic polarity. The large body of data accumulated at both cellular and molecular levels in the last decade clearly demonstrated how Drosophila oogenesis is a genetically tractable system particularly suited for the investigation of key developmental biology questions. Our recent contribution to the field relies on the characterisation of three different mutants named tegamino (teg), hold hup (hup) and tulipano (tip), identifying novel gene functions required during oogenesis. Specifically, teg is implicated in the morphogenesis of the follicular epithelium surrounding the germ line cells in the egg chamber, hup is involved in the establishment of egg chamber polarity and tip in the regulation of the dynamic germ cell chromatin organisation.