Extracellular vesicles (EVs), defined as intercellular messengers that carry their cargos between cells, are involved in several physiological and pathological processes. These small membranous vesicles are released by most cells and contain biological molecules, including nucleic acids, proteins and lipids, which can modulate signaling pathways of nearby or distant recipient cells. Exosomes, one the most characterized classes of EVs, include, among others, microRNAs (miRNAs), small non-coding RNAs able to regulate the expression of several genes at post-transcriptional level. In cancer, exosomal miRNAs have been shown to influence tumor behavior and reshape tumor microenvironment. Furthermore, their possible involvement in drug resistance mechanisms has become evident in recent years. Hepatocellular carcinoma (HCC) is the major type of liver cancer, accounting for 75-85% of all liver tumors. Although the improvement in HCC treatment approaches, low therapeutic efficacy in patients with intermediate-advanced HCC is mainly related to the development of tumor metastases, high risk of recurrence and drug resistance. Exosomes have been shown to be involved in pathogenesis and progression of HCC, as well as in drug resistance, by regulating processes such as cell proliferation, epithelial-mesenchymal transition and immune response. Herein, we summarize the current knowledge about the involvement of exosomal miRNAs in HCC therapy, highlighting their role as modulators of therapeutic response, particularly chemotherapy and immunotherapy, as well as possible therapeutic tools.

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.

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.

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.

HER2 is considered one of the most traditional prognostic and predictive biomarkers in breast cancer. Literature data confirmed that the addition of pertuzumab to a standard neoadjuvant chemotherapy backbone (either with or without anthracyclines), in patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer (EBC), leads to a higher pathological complete response (pCR) rate, which is known to correlate with a better prognosis. In this retrospective analysis, 47 consecutive patients with HER2-positive EBC received sequential anthracyclines and taxanes plus trastuzumab (ATH) or pertuzumab, trastuzumab and docetaxel (THP). Despite the limited sample size, this monocentric experience highlights the efficacy (in terms of pCR) and safety of THP in the neoadjuvant setting of HER2-positive EBC as an anthracycline-free approach. Given the role of PIK3CA as a prognostic and therapeutic target in breast cancer, tumors were also analyzed to assess the PIK3CA mutational status. Thirty-eight out of forty-seven patients were evaluated, and PIK3CA variants were identified in 21% of tumor samples: overall, one mutation was detected in exon 4 (2.6%), two in exon 9 (5.3%) and four in exon 20 (10.5%). Of note, one sample showed concurrent mutations in exons 9 (codon 545) and 20 (codon 1047). Among patients reaching pCR (n = 13), 38.5% were PIK3CA mutants; on the other hand, among those lacking pCR (n = 25), just 12% showed PIK3CA variants. Regarding THP-treated mutant patients (n = 5), 80% reached pCR (three hormone-receptor-negative, one hormone-receptor-positive). Interestingly, the only patient not achieving pCR had a tumor with two co-occurring PIK3CA mutations. In conclusion, this study provides new evidence about the efficacy and good safety profile of THP, compared to the ATH regimen, as an anthracycline-free neoadjuvant treatment of HER2-positive EBC. Further studies on larger/multicentric cohorts are planned for more in-depth analysis to confirm our molecular and clinical results.

The assessment of RAS and BRAF mutational status is one of the main steps in the diagnostic and therapeutic algorithm of metastatic colorectal cancer (mCRC). Multiple mutations in the BRAF and RAS pathway are described as a rare event, with concurrent variants in KRAS and BRAF genes observed in approximately 0.05% of mCRC cases. Here, we report data from a case series affected by high-risk stage III and stage IV CRC and tested for RAS and BRAF mutation, treated at our Medical Oncology Unit. The analysis of KRAS, NRAS (codons 12, 13, 59, 61, 117, 146), and BRAF (codon 600) hotspot variants was performed in 161 CRC tumors from August 2018 to September 2021 and revealed three (1.8%) patients showing mutations in both KRAS and BRAF (V600E), including two cases with earlier CRC and one with metastatic disease. We also identified one patient (0.6%) with a mutation in both KRAS and NRAS genes and another one (0.6%) with a double KRAS mutation. Notably, the latter was characterized by aggressive behavior and poor clinical outcome. The mutational status, pathological features, and clinical history of these five CRC cases are described. Overall, this study case series adds evidence to the limited available literature concerning both the epidemiological and clinical aspects of CRC cases characterized by the presence of concurrent RAS/BRAF variants. Future multicentric studies will be required to increase the sample size and provide additional value to results observed so far in order to improve clinical management of this subgroup of CRC patients.

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.

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.

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.

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.

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.

Breast cancer (BC) is a common and heterogeneous disease, of which six molecular subtypes, characterized by different biological features and clinical outcomes, were described. The identification of additional biomarkers able to further connote and distinguish the different BC subtypes is essential to improve the diagnostic, prognostic and therapeutic strategies in BC patients. MicroRNAs (miRNAs) are short non-coding RNA involved in several physiological and pathological processes, including cancer development and progression. In particular, circulating miRNAs, which can be found in an adequately stable structure in serum/plasma of cancer patients, are emerging as very promising non-invasive biomarkers. Several studies have analyzed the potential role of circulating miRNAs as prognostic and therapeutic markers in BC. In the present review we describe circulating miRNAs, identified as putative biomarker in BC, with special reference to different BC molecular subtypes.

Leptin is a hormone that plays a major role as mediator of long-term regulation of energy balance, suppressing food intake, and stimulating weight loss. More recently, important physiological roles other than controlling appetite and energy expenditure have been suggested for leptin, including neuroendocrine, reparative, reproductive, and immune functions. These emerging peripheral roles let hypothesize that leptin can modulate also cancer progression. Indeed, many studies have demonstrated that elevated chronic serum concentrations of leptin, frequently seen in obese subjects, represent a stimulatory signal for tumor growth. Current knowledge indicates that also different non-tumoral cells resident in tumor microenvironment may respond to leptin creating a favorable soil for cancer cells. In addition, leptin is produced also within the tumor microenvironment creating the possibility for paracrine and autocrine action. In this review, we describe the main mechanisms that regulate peripheral leptin availability and how leptin can shape tumor microenvironment.

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.

Next generation sequencing (NGS) provides a powerful tool in the field of medical genetics, allowing one to perform multi-gene analysis and to sequence entire exomes (WES), transcriptomes or genomes (WGS). The generated high-throughput data are particularly suitable for enhancing the understanding of the genetic bases of complex, multi-gene diseases, such as cancer. Among the various types of tumors, those with a familial predisposition are of great interest for the isolation of novel genes or gene variants, detectable at the germline level and involved in cancer pathogenesis. The identification of novel genetic factors would have great translational value, helping clinicians in defining risk and prevention strategies. In this regard, it is known that the majority of breast/ovarian cases with familial predisposition, lacking variants in the highly penetrant BRCA1 and BRCA2 genes (non-BRCA), remains unexplained, although several less penetrant genes (e.g., ATM, PALB2) have been identified. In this scenario, NGS technologies offer a powerful tool for the discovery of novel factors involved in familial breast/ovarian cancer. In this review, we summarize and discuss the state of the art applications of NGS gene panels, WES and WGS in the context of familial breast/ovarian cancer.

NF-κB plays a pivotal role in oncogenesis. This transcription factor is best known for promoting cancer cell survival and tumour-driving inflammation. However, several lines of evidence support a crucial role for NF-κB in governing energy homeostasis and mediating cancer metabolic reprogramming. Mitochondria are central players in many metabolic processes altered in cancer. Beyond their bioenergetic activity, several facets of mitochondria biology, including mitochondrial dynamics and oxidative stress, promote and sustain malignant transformation. Recent reports revealed an intimate connection between NF-κB pathway and the oncogenic mitochondrial functions. NF-κB can impact mitochondrial respiration and mitochondrial dynamics, and, reciprocally, mitochondria can sense stress signals and convert them into cell biological responses leading to NF-κB activation. In this review we discuss their emerging reciprocal regulation and the significance of this interplay for anticancer therapy.

Systemic Sclerosis (SSc) is a disease with limited therapeutic possibilities. Mesenchymal stem cells (MSCs)-therapy could be a promising therapeutic option, however the ideal MSCs source has not yet been found. To address this problem, we perform comparison between bone marrow (BM)-MSCs and adipose (A)-MSCs, by the miRs expression profile, to identify the gene modulation in these two MSCs source. MicroRNAs (miRs) are RNAs sequences, regulating gene expression and MSCs, derived from different tissues, may differently respond to the SSc microenvironment. The miRs array was used for the miRs profiling and by DIANA-mirPath tool we identified the biological functions of the dysregulated miRs. In SSc-BM-MSCs, 6 miRs were significantly down-regulated and 4 miRs up-regulated. In SSc-A-MSCs, 11 miRs were significantly down-regulated and 3 miRs up-regulated. Interestingly, in both the sources, the involved pathways included the senescence mechanisms and the pro-fibrotic behaviour. Furthermore, both the MSCs sources showed potential compensatory ability. A deeper knowledge of this miRs signature might give more information about some pathogenic steps of the disease and in the same time clarify the possible therapeutic role of autologous MSCs in the regenerative therapy in SSc.

We describe a metastatic colorectal cancer patient, treated with first-line 5-fluorouracil, irinotecan, bevacizumab, and oxaliplatin (FIr-BFOx) therapy, with aggressive and resistant disease. KRAS, NRAS, BRAF, and PI3KCA were analyzed in primary tumor and liver metastasis. KRAS c.34G>A mutation was detected in primary tumor and liver metastasis, which additionally revealed 2 rare PI3KCA mutations (c.1633G>C and c.1645G>C). The c.1645G>C was never reported in colorectal cancer. Akt/p-AktSer473, phosphatase and tensin homolog, mismatch repair, and epidermal growth factor receptor expression was evaluated. Normal mismatch repair and epidermal growth factor receptor expression was detected. Akt was shown by primary tumor and liver metastasis, whereas p-AktSer473 was identified only in the latter, despite positive phosphatase and tensin homolog expression. Patient showed 7 months of progression-free survival and 15 months of overall survival, lower than median values reported in KRAS exon 2-mutant patients treated with the same therapy. Results lead to the hypothesis of a putative role of these mutations in worsening of the disease and are open to further confirmatory studies.

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 GADD45β 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 GADD45β 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.

Since the establishment of the Sanger sequencing method, scientists around the world focused their efforts to progress in the field to produce the utmost technology. The introduction of next-generation sequencing (NGS) represents a revolutionary step and promises to lead to massive improvements in our understanding on the role of nucleic acids functions. Cancer research began to use this innovative and highly performing method, and interesting results started to appear in colorectal cancer (CRC) analysis. Several studies produced high-quality data in terms of mutation discovery, especially about actionable or less frequently mutated genes, epigenetics, transcriptomics. Analysis of results is unveiling relevant perspectives aiding to evaluate the response to therapies. Novel evidences have been presented also in other directions such as gut microbiota or CRC circulating tumor cells. However, despite its unquestioned potential, NGS poses some issues calling for additional studies. This review intends to offer a view of the state of the art of NGS applications to CRC through examination of the most important technologies and discussion of recent published results.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease. It can progress to nonalcoholic steatohepatitis (NASH) and, in a percentage of cases, to hepatocarcinogenesis. The strong incidence in western countries of obesity and metabolic syndrome, whose NAFLD is the hepatic expression, is thought to be correlated to consumption of diets characterized by processed food and sweet beverages. Previous studies described high-fat diet-induced liver tumors. Conversely, the involvement of low-fat/high-carbohydrate diet in the progression of liver disease or cancer initiation has not been described yet. Here we show for the first time hepatic cancer formation in low-fat/high-carbohydrate diet fed NAFLD/NASH mouse model. Animals were long term high-fat, low-fat/high-carbohydrate or standard diet fed. We observed progressive liver damage in low-fat/high-carbohydrate and high-fat animals after 12 and, more, 18 months. Tumors were detected in 20% and 50% of high-fat diet fed mice after 12 and 18 months and, interestingly, in 30% of low-fat/high-carbohydrate fed animals after 18 months. No tumors were detected in standard diet fed mice. Global increase of hepatic interleukin-1β, interleukin-6, tumor necrosis factor-α and hepatocyte growth factor was detected in low-fat/high-carbohydrate and high-fat with respect to standard diet fed mice as well as in tumor with respect to non-tumor bearing mice. A panel of 15 microRNAs was analyzed: some of them revealed differential expression in low-fat/high-carbohydrate with respect to high-fat diet fed groups and in tumors. Data here shown provide the first evidence of the involvement of low-fat/high-carbohydrate diet in hepatic damage leading to tumorigenesis.

Tumour promoting inflammation is widely recognized as a hallmark of cancer. The source of this chronic inflammation in cancer has been ascribed to the progressive activation over time of immune cells, mostly of the innate arm of the immune system. However, recent evidence has shown that chronic inflammation may also derive, at least in part, from senescent cells. Hence, due to the prominent role of inflammation in cancer, the cancer secretome definition includes all the secretory factors ensuing from the crosstalk between the cancer cell and the tumour microenvironment. The mechanistic basis underlying the paracrine signalling between the cancer cell and the surrounding tumour microenvironment in malignancy have been widely investigated by using in vivo models of cancers, thus identifying the NF-κB transcription factor as the molecular hub linking inflammation and cancer. In this review, we highlight the roles of NF-κB in regulating the inflammation-derived secretome emanating from immune and senescent cells, with a special focus on the bright and the dark sides of their pro-inflammatory signalling on tumorigenesis.

Natural background radiation of Earth and cosmic rays played a relevant role during the evolution of living organisms. However, how chronic low doses of radiation can affect biological processes is still unclear. Previous data have indicated that cells grown at the Gran Sasso Underground Laboratory (LNGS, L'Aquila) of National Institute of Nuclear Physics (INFN) of Italy, where the dose rate of cosmic rays and neutrons is significantly reduced with respect to the external environment, elicited an impaired response against endogenous damage as compared to cells grown outside LNGS. This suggests that environmental radiation contributes to the development of defense mechanisms at cellular level. To further understand how environmental radiation affects metabolism of living organisms, we have recently launched the FLYINGLOW program that aims at exploiting Drosophila melanogaster as a model for evaluating the effects of low doses/dose rates of radiation at the organismal level. Here, we will present a comparative data set on lifespan, motility and fertility from different Drosophila strains grown in parallel at LNGS and in a reference laboratory at the University of L'Aquila. Our data suggest the reduced radiation environment can influence Drosophila development and, depending on the genetic background, may affect viability for several generations even when flies are moved back to normal background radiation. As flies are considered a valuable model for human biology, our results might shed some light on understanding the effect of low dose radiation also in humans.

Angiogenesis plays an essential role in the growth and progression of breast cancer. This observational single center study evaluated the efficacy and safety of a new weekly schedule of bevacizumab/paclitaxel combination in the first-line treatment of unselected, HER2-negative, metastatic breast cancer (MBC) patients, in a real-life setting. Thirty-five patients (median age 56 years, range 40-81) with HER2-negative MBC were treated with paclitaxel (70 mg/m(2)) dd 1,8,15 q21 (60 mg/m(2) if ≥65 years or secondary Cumulative Illness Rating Scale) plus bevacizumab (10 mg/kg) every 2 weeks. Twenty-two patients (63%) had ≥2 metastatic sites and 15 (43%) visceral disease. Eleven patients (31%) had a triple-negative breast cancer (TNBC). A clinical complete response (cCR) was observed in 6 (17%) cases after a median of seven cycles, a partial response (PR) in 22 (63%), and a stable disease (SD) in 6 (17%) cases; the overall clinical benefit rate was 97%. In TNBC subgroup, cCR occurred in 1 (9%) case, PR in 8 (73%), and SD in 2 (18%). At a median follow-up of 13 months (range 1-79 months), the median progression-free survival was 11 months and the median overall survival was 36 months. No grade 4 adverse events occurred. The main grade 3 toxicities observed were neutropenia (11.4%), hypertension (5.7%), stomatitis (2.8%), diarrhea (2.8%), and vomiting (2.8%). The administration of weekly paclitaxel plus bevacizumab in this real-life experience shows similar efficacy than previously reported schedules, with a comparable dose intensity and a good toxicity profile.