NF-κB: Governing Macrophages in Cancer.

Tumor-associated macrophages (TAMs) are the major component of the tumor microenvironment (TME), where they sustain tumor progression and or-tumor immunity. Due to their plasticity, macrophages can exhibit anti- or pro-tumor functions through the expression of different gene sets leading to distinct macrophage phenotypes: M1-like or pro-inflammatory and M2-like or anti-inflammatory. NF-κB transcription factors are central regulators of TAMs in cancers, where they often drive macrophage polarization toward an M2-like phenotype. Therefore, the NF-κB pathway is an attractive therapeutic target for cancer immunotherapy in a wide range of human tumors. Hence, targeting NF-κB pathway in the myeloid compartment is a potential clinical strategy to overcome microenvironment-induced immunosuppression and increase anti-tumor immunity. In this review, we discuss the role of NF-κB as a key driver of macrophage functions in tumors as well as the principal strategies to overcome tumor immunosuppression by targeting the NF-κB pathway.

Molecular Mechanisms Underpinning Immunometabolic Reprogramming: How the Wind Changes during Cancer Progression.

Metabolism and the immunological state are intimately intertwined, as defense responses are bioenergetically expensive. Metabolic homeostasis is a key requirement for the proper function of immune cell subsets, and the perturbation of the immune-metabolic balance is a recurrent event in many human diseases, including cancer, due to nutrient fluctuation, hypoxia and additional metabolic changes occurring in the tumor microenvironment (TME). Although much remains to be understood in the field of immunometabolism, here, we report the current knowledge on both physiological and cancer-associated metabolic profiles of immune cells, and the main molecular circuits involved in their regulation, highlighting similarities and differences, and emphasizing immune metabolic liabilities that could be exploited in cancer therapy to overcome immune resistance.

Intranasal delivery of NGF rescues hearing impairment in aged SAMP8 mice.

Hearing loss impacts the quality of life and affects communication resulting in social isolation and reduced well-being. Despite its impact on society and economy, no therapies for age-related hearing loss are available so far. Loss of mechanosensory hair cells of the cochlea is a common event of hearing loss in humans. Studies performed in birds demonstrating that they can be replaced following the proliferation and transdifferentiation of supporting cells, strongly pointed out on HCs regeneration as the main focus of research aimed at hearing regeneration. Neurotrophins are growth factors involved in neuronal survival, development, differentiation, and plasticity. NGF has been involved in the interplay between auditory receptors and efferent innervation in the cochlea during development. During embryo development, both NGF and its receptors are highly expressed in the inner ears. It has been reported that NGF is implicated in the differentiation of auditory gangliar and hair cells. Thus, it has been proposed that NGF administration can decrease neuronal damage and prevent hearing loss. The main obstacle to the development of hearing impairment therapy is that efficient means of delivery for selected drugs to the cochlea are missing. Herein, in this study NGF was administered by the intranasal route. The first part of the study was focused on a biodistribution study, which showed the effective delivery in the cochlea; while the second part was focused on analyzing the potential therapeutic effect of NGF in senescence-accelerated prone strain 8 mice. Interestingly, intranasal administration of NGF resulted protective in counteracting hearing impairment in SAMP8 mice, ameliorating hearing performances (analyzed by auditory brainstem responses and distortion product otoacoustic emission) and hair cells morphology (analyzed by microscopy analysis). The results obtained were encouraging indicating that the neurotrophin NGF was efficiently delivered to the inner ear and that it was effective in counteracting hearing loss.

MiR-182-5p Is Upregulated in Hepatic Tissues from a Diet-Induced NAFLD/NASH/HCC C57BL/6J Mouse Model and Modulates Cyld and Foxo1 Expression.

Non-alcoholic fatty liver disease (NAFLD) is considered a relevant liver chronic disease. Variable percentages of NAFLD cases progress from steatosis to steatohepatitis (NASH), cirrhosis and, eventually, hepatocellular carcinoma (HCC). In this study, we aimed to deepen our understanding of expression levels and functional relationships between miR-182-5p and Cyld-Foxo1 in hepatic tissues from C57BL/6J mouse models of diet-induced NAFL/NASH/HCC progression. A miR-182-5p increase was detected early in livers as NAFLD damage progressed, and in tumors compared to peritumor normal tissues. An in vitro assay on HepG2 cells confirmed Cyld and Foxo1, both tumor-suppressor, as miR-182-5p target genes. According to miR-182-5p expression, decreased protein levels were observed in tumors compared to peritumor tissues. Analysis of miR-182-5p, Cyld and Foxo1 expression levels, based on datasets from human HCC samples, showed results consistent with those from our mouse models, and also highlighted the ability of miR-182-5p to distinguish between normal and tumor tissues (AUC 0.83). Overall, this study shows, for the first time, miR-182-5p overexpression and Cyld-Foxo1 downregulation in hepatic tissues and tumors from a diet-induced NAFLD/HCC mouse model. These data were confirmed by the analysis of datasets from human HCC samples, highlighting miR-182-5p diagnostic accuracy and demonstrating the need for further studies to assess its potential role as a biomarker or therapeutic target.

Comparative Analysis of Dasatinib Effect between 2D and 3D Tumor Cell Cultures.

Three-dimensional cell culture methods are able to confer new predictive relevance to in vitro tumor models. In particular, the 3D multicellular tumor spheroids model is considered to better resemble tumor complexity associated with drug resistance compared to the 2D monolayer model. Recent advances in 3D printing techniques and suitable biomaterials have offered new promises in developing 3D tissue cultures at increased reproducibility and with high-throughput characteristics. In our study, we compared the sensitivity to dasatinib treatment in two different cancer cell lines, prostate cancer cells DU145 and glioblastoma cells U87, cultured in the 3D spheroids model and in the 3D bioprinting model. DU145 and U87 cells were able to proliferate in 3D alginate/gelatin bioprinted structures for two weeks, forming spheroid aggregates. The treatment with dasatinib demonstrated that bioprinted cells were considerably more resistant to drug toxicity than corresponding cells cultured in monolayer, in a way that was comparable to behavior observed in the 3D spheroids model. Recovery and analysis of cells from 3D bioprinted structures led us to hypothesize that dasatinib resistance was dependent on a scarce penetrance of the drug, a phenomenon commonly reported also in spheroids. In conclusion, the 3D bioprinted model utilizing alginate/gelatin hydrogel was demonstrated to be a suitable model in drug screening when spheroid growth is required, offering advantages in feasibility, reproducibility, and scalability compared to the classical 3D spheroids model.

The NF-κB Pharmacopeia: Novel Strategies to Subdue an Intractable Target.

NF-κB transcription factors are major drivers of tumor initiation and progression. NF-κB signaling is constitutively activated by genetic alterations or environmental signals in many human cancers, where it contributes to almost all hallmarks of malignancy, including sustained proliferation, cell death resistance, tumor-promoting inflammation, metabolic reprogramming, tissue invasion, angiogenesis, and metastasis. As such, the NF-κB pathway is an attractive therapeutic target in a broad range of human cancers, as well as in numerous non-malignant diseases. Currently, however, there is no clinically useful NF-κB inhibitor to treat oncological patients, owing to the preclusive, on-target toxicities of systemic NF-κB blockade. In this review, we discuss the principal and most promising strategies being developed to circumvent the inherent limitations of conventional IκB kinase (IKK)/NF-κB-targeting drugs, focusing on new molecules that target upstream regulators or downstream effectors of oncogenic NF-κB signaling, as well as agents targeting individual NF-κB subunits.

EV-Mediated Chemoresistance in the Tumor Microenvironment: Is NF-κB a Player?

Drug resistance is a major impediment to patient survival and remains the primary cause of unsuccessful cancer therapy. Drug resistance occurs in many tumors and is frequently induced by chemotherapy which triggers a defensive response both in cancerous and cancer-associated cells that constitute the tumor microenvironment (TME). Cell to cell communication within the TME is often mediated by extracellular vesicles (EVs) which carry specific tumor-promoting factors able to activate survival pathways and immune escape mechanisms, thus sustaining tumor progression and therapy resistance. NF-κB has been recognized as a crucial player in this context. NF-κB activation is involved in EVs release and EVs, in turn, can trigger NF-κB pathway activation in specific contexts, based on secreting cytotype and their specific delivered cargo. In this review, we discuss the role of NF-κB/EVs interplay that sustain chemoresistance in the TME by focusing on the molecular mechanisms that underlie inflammation, EVs release, and acquired drug resistance.

NF-κB: A Druggable Target in Acute Myeloid Leukemia.

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy that relies on highly heterogeneous cytogenetic alterations. Although in the last few years new agents have been developed for AML treatment, the overall survival prospects for AML patients are still gloomy and new therapeutic options are still urgently needed. Constitutive NF-κB activation has been reported in around 40% of AML patients, where it sustains AML cell survival and chemoresistance. Given the central role of NF-κB in AML, targeting the NF-κB pathway represents an attractive strategy to treat AML. This review focuses on current knowledge of NF-κB's roles in AML pathogenesis and summarizes the main therapeutic approaches used to treat NF-κB-driven AML.

Elevated NF-κB/SHh/GLI1 Signature Denotes a Worse Prognosis and Represent a Novel Potential Therapeutic Target in Advanced Prostate Cancer.

Prostate cancer (PCa) is the second most frequent cancer in men worldwide. NF-κB seems to play a key role in cell survival, proliferation and invasion, sustaining the heterogeneous multifocal nature of PCa. In recent years, the Hedgehog (Hh) signaling pathway has attracted attention as a therapeutic target due to its implication in tumorigenesis and metastasis in several types of cancer, including PCa. Although it is well-known that Sonic Hedgehog (SHh) is a transcriptional target of NF-κB(p65), and that GLI1 is the effector of this crosstalk, the precise role played by this axis in PCa is still not completely clear. Here, we set out to explore the correlation between NF-κB activation and SHh pathways in PCa, investigating if the interplay between NF-κB(p65) and SHh-GLI1 in advanced PCa could be a prospective therapeutic target. Our findings demonstrate that a NF-κB-SHh-GLI1 gene signature is enriched in PCa patients featuring a higher Gleason score. Moreover, elevated levels of this signature are associated with worse prognosis, thus suggesting that this axis could provide a route to treat aggressive PCa.

MicroRNAs Expression in Response to rhNGF in Epithelial Corneal Cells: Focus on Neurotrophin Signaling Pathway.

PURPOSE: Nerve growth factor efficacy was demonstrated for corneal lesions treatment, and recombinant human NGF (rhNGF) was approved for neurotrophic keratitis therapy. However, NGF-induced molecular responses in cornea are still largely unknown. We analyzed microRNAs expression in human epithelial corneal cells after time-dependent rhNGF treatment. METHODS: Nearly 700 microRNAs were analyzed by qRT-PCR. MicroRNAs showing significant expression differences were examined by DIANA-miRpath v.3.0 to identify target genes and pathways. Immunoblots were performed to preliminarily assess the strength of the in silico results. RESULTS: Twenty-one microRNAs (miR-26a-1-3p, miR-30d-3p, miR-27b-5p, miR-146a-5p, miR-362-5p, mir-550a-5p, mir-34a-3p, mir-1227-3p, mir-27a-5p, mir-222-5p, mir-151a-5p, miR-449a, let7c-5p, miR-337-5p, mir-29b-3p, miR-200b-3p, miR-141-3p, miR-671-3p, miR-324-5p, mir-411-3p, and mir-425-3p) were significantly regulated in response to rhNGF. In silico analysis evidenced interesting target genes and pathways, including that of neurotrophin, when analyzed in depth. Almost 80 unique target genes (e.g., PI3K, AKT, MAPK, KRAS, BRAF, RhoA, Cdc42, Rac1, Bax, Bcl2, FasL) were identified as being among those most involved in neurotrophin signaling and in controlling cell proliferation, growth, and apoptosis. AKT and RhoA immunoblots demonstrated congruence with microRNA expression, providing preliminary validation of in silico data. CONCLUSIONS: MicroRNA levels in response to rhNGF were for the first time analyzed in corneal cells. Novel insights about microRNAs, target genes, pathways modulation, and possible biological responses were provided. Importantly, given the putative role of microRNAs as biomarkers or therapeutic targets, our results make available data which might be potentially exploitable for clinical applications.

A natural product, voacamine, sensitizes paclitaxel-resistant human ovarian cancer cells.

Most women with ovarian cancer are treated with chemotherapy before or after surgery. Unfortunately, chemotherapy treatment can cause negative side effects and the onset of multidrug resistance (MDR). The aim of this study is to evaluate the chemosensitizing effect of a natural compound, voacamine (VOA), in ovarian (A2780 DX) and colon (LoVo DX) cancer drug-resistant cell lines which overexpress P-glycoprotein (P-gp), in combination with paclitaxel (PTX), or doxorubicin (DOX) or 5-fluorouracil (5-FU). VOA, a bisindole alkaloid extracted from Peschiera fuchsiaefolia, has already been shown to be effective in enhancing the effect of doxorubicin, because it interferes with the P-gp function. Ovarian cancer cytotoxicity test shows that single treatments with VOA, DOX and PTX do not modify cell viability, while pretreatment with VOA, and then PTX or DOX for 72 h, induces a decrease. In colon cancer, since 5-FU is not a-substrate for P-gp, VOA has no sensitizing effect while in VOA + DOX there is a decrease in viability. Annexin V/PI test, cell cycle analysis, activation of cleaved PARP1 confirm that VOA plus PTX induce apoptotic cell death. Confocal microscopy observations show the different localization of NF-kB after treatment with VOA + PTX, confirming the inhibition of nuclear translocation induced by VOA pretreatment. Our data show the specific effect of VOA which only works on drugs known to be substrates of P-gp.

Ultrasound-Based Method for the Identification of Novel MicroRNA Biomarkers in Prostate Cancer.

The detection of circulating microRNA (miRNA)-based biomarkers represents an innovative, non-invasive method for the early detection of cancer. However, the low concentration of miRNAs released in body fluids and the difficult identification of the tumor site have limited their clinical use as effective cancer biomarkers. To evaluate if ultrasound treatment could amplify the release of extracellular cancer biomarkers, we treated a panel of prostate cancer (PCa) cell lines with an ultrasound-based prototype and profiled the release of miRNAs in the extracellular space, with the aim of identifying novel miRNA-based biomarkers that could be used for PCa diagnosis and the monitoring of tumor evolution. We provide evidence that US-mediated sonoporation amplifies the release of miRNAs from both androgen-dependent (AD) and -independent (AI) PCa cells. We identified four PCa-related miRNAs, whose levels in LNCaP and DU145 supernatants were significantly increased following ultrasound treatment: mir-629-5p, mir-374-5p, mir-194-5p, and let-7d-5p. We further analyzed a publicly available dataset of PCa, showing that the serum expression of these novel miRNAs was upregulated in PCa patients compared to controls, thus confirming their clinical relevance. Our findings highlight the potential of using ultrasound to identify novel cell-free miRNAs released from cancer cells, with the aim of developing new biomarkers with diagnostic and predictive value.

Emerging Role of isomiRs in Cancer: State of the Art and Recent Advances.

The advent of Next Generation Sequencing technologies brought with it the discovery of several microRNA (miRNA) variants of heterogeneous lengths and/or sequences. Initially ascribed to sequencing errors/artifacts, these isoforms, named isomiRs, are now considered non-canonical variants that originate from physiological processes affecting the canonical miRNA biogenesis. To date, accurate IsomiRs abundance, biological activity, and functions are not completely understood; however, the study of isomiR biology is an area of great interest due to their high frequency in the human miRNome, their putative functions in cooperating with the canonical miRNAs, and potential for exhibiting novel functional roles. The discovery of isomiRs highlighted the complexity of the small RNA transcriptional landscape in several diseases, including cancer. In this field, the study of isomiRs could provide further insights into the miRNA biology and its implication in oncogenesis, possibly providing putative new cancer diagnostic, prognostic, and predictive biomarkers as well. In this review, a comprehensive overview of the state of research on isomiRs in different cancer types, including the most common tumors such as breast cancer, colorectal cancer, melanoma, and prostate cancer, as well as in the less frequent tumors, as for example brain tumors and hematological malignancies, will be summarized and discussed.

Biochemical Methods to Analyze the Subcellular Localization of NF-κB Proteins Using Cell Fractionation.

Cell fractionation is a method used to study different cellular events like protein translocation and sequestration by disrupting cells and fractionating their contents, thus allowing an enrichment of the protein of interest. Using different concentrations of sucrose or detergent buffer formulations in combination with centrifugations, the cell fractions are separated based on their density and size.

Immunohistochemical Analysis of Expression, Phosphorylation, and Nuclear Translocation of NF-κB Proteins in Human Tissues.

Immunohistochemistry (IHC) is a technique aimed at detecting specific antigens on tissue sections by the use of targeting reagents labeled with reporter molecules. This technique allows a snapshot of the structure of tissue and determines the cellular and subcellular localization of a target antigen. This chapter describes how to identify and localize NF-κB proteins in human tissue using immunohistochemical staining on formalin-fixed paraffin-embedded and frozen tissue.

Extracellular Flux Analysis to Investigate the Impact of NF-κB on Mitochondrial Respiration in Colorectal Carcinoma (CRC).

The reprogramming of cell metabolism is a hallmark of cancer. NF-κB transcription factors coordinate the host defense responses to stress, injury, and infection. They also play a central role in oncogenesis, at least in part by regulating cell metabolism and the adaptation to energy stress conditions in various types of cancer, such as colorectal carcinoma (CRC). Here, we describe the XF Cell Mito Stress Test methodology aimed at characterizing the metabolic and bioenergetic profile of CRC cells following the silencing of the essential NF-κB subunit, RelA. This methodology may also be applied to other cancers to reveal novel core vulnerabilities of malignant cells.

The Screening of Combinatorial Peptide Libraries for Targeting Key Molecules or Protein-Protein Interactions in the NF-κB Pathway.

Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of cancer and metabolic and cardiovascular diseases, which are all areas of high interest to the pharmaceutical industry. The global market for peptide therapeutics was valued at about 25 billion USD in 2018 and is estimated to reach 57.2 billion USD by the end of 2027. Here, we describe a method for the screening and deconvolution of combinatorial peptide libraries to discover compounds that target discrete signaling components of the NF-κB pathway. Recently, we used this approach to specifically disrupt the interaction between the JNK-activating kinase, MKK7, and the NF-κB-regulated antiapoptotic factor, GADD45ß, in multiple myeloma (MM). We showed that the GADD45ß/MKK7 complex is a functionally critical survival module downstream of NF-κB in MM cells and as such provides an attractive therapeutic target to selectively inhibit NF-κB antiapoptotic signaling in cancer cells. By integrating the library screening and deconvolution methods described here with a rational chemical optimization strategy, we developed the first-in-class GADD45ß/MKK7 inhibitor, DTP3 (a D-tripeptide), which is now being trialed in MM and diffuse large B-cell lymphoma (DLBCL) patients. The same drug discovery approach may be generally applied to therapeutically target other key components of the NF-κB pathway in cancers beyond MM and DLBCL, as well as in non-malignant NF-κB-driven diseases.

Life, death, and autophagy in cancer: NF-κB turns up everywhere.

Escaping programmed cell death is a hallmark of cancer. NF-κB transcription factors are key regulator of cell survival and aberrant NF-κB signaling has been involved in the pathogenesis of most human malignancies. Although NF-κB is best known for its antiapoptotic role, other processes regulating the life/death balance, such as autophagy and necroptosis, seem to network with NF-κB. This review discusses how the reciprocal regulation of NF-κB, autophagy and programmed cell death affect cancer development and progression.

Low Radiation Environment Switches the Overgrowth-Induced Cell Apoptosis Toward Autophagy.

Low radiation doses can affect and modulate cell responses to various stress stimuli, resulting in perturbations leading to resistance or sensitivity to damage. To explore possible mechanisms taking place at an environmental radiation exposure, we set-up twin biological models, one growing in a low radiation environment (LRE) laboratory at the Gran Sasso National Laboratory, and one growing in a reference radiation environment (RRE) laboratory at the Italian National Health Institute (Istituto Superiore di Sanità, ISS). Studies were performed on pKZ1 A11 mouse hybridoma cells, which are derived from the pKZ1 transgenic mouse model used to study the effects of low dose radiation, and focused on the analysis of cellular/molecular end-points, such as proliferation and expression of key proteins involved in stress response, apoptosis, and autophagy. Cells cultured up to 4 weeks in LRE showed no significant differences in proliferation rate compared to cells cultured in RRE. However, caspase-3 activation and PARP1 cleavage were observed in cells entering to an overgrowth state in RRE, indicating a triggering of apoptosis due to growth-stress conditions. Notably, in LRE conditions, cells responded to growth stress by switching toward autophagy. Interestingly, autophagic signaling induced by overgrowth in LRE correlated with activation of p53. Finally, the gamma component of environmental radiation did not significantly influence these biological responses since cells grown in LRE either in incubators with or without an iron shield did not modify their responses. Overall, in vitro data presented here suggest the hypothesis that environmental radiation contributes to the development and maintenance of balance and defense response in organisms.

GADD45ß Loss Ablates Innate Immunosuppression in Cancer.

T-cell exclusion from the tumor microenvironment (TME) is a major barrier to overcoming immune escape. Here, we identify a myeloid-intrinsic mechanism governed by the NF-κB effector molecule GADD45ß that restricts tumor-associated inflammation and T-cell trafficking into tumors. In various models of solid cancers refractory to immunotherapies, including hepatocellular carcinoma and ovarian adenocarcinoma, Gadd45b inhibition in myeloid cells restored activation of proinflammatory tumor-associated macrophages (TAM) and intratumoral immune infiltration, thereby diminishing oncogenesis. Our results provide a basis to interpret clinical evidence that elevated expression of GADD45B confers poor clinical outcomes in most human cancers. Furthermore, they suggest a therapeutic target in GADD45ß for reprogramming TAM to overcome immunosuppression and T-cell exclusion from the TME.Significance: These findings define a myeloid-based immune checkpoint that restricts T-cell trafficking into tumors, with potentially important therapeutic implications to generally improve the efficacy of cancer immunotherapy. Cancer Res; 78(5); 1275-92. ©2017 AACR.