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miR-29b overexpression and HDAC4-silencing affect MM cell autophagy

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In silico analysis of HDAC4-targeting by miR-29b

MicroRNA (miRNA) are constituted of approximately 22 nucleotides and play an important role in the regulation of many physiological functions and diseases. In the last 10 years, an increasing interest has been recorded in studying the expression profile of miRNAs in cancer. Real time-quantitative polymerase chain reaction (RT-qPCR), microarrays, and small RNA sequencing represent the gold standard techniques used in the last 30 years as detection methods. The advent of nanotechnology has allowed the fabrication of nanostructured biosensors which are widely exploited in the diagnostic field. Nanostructured biosensors offer many advantages: (i) their small size allows the construction of portable, wearable, and low-cost products; (ii) the large surface–volume ratio enables the loading of a great number of biorecognition elements (e.g., probes, receptors); and (iii) direct contact of the recognition element with the analyte increases the sensitivity and specificity inducing low limits of detection (LOD). In this review, the role of nanostructured biosensors in miRNA detection is explored, focusing on electrochemical and optical sensing. In particular, four types of nanomaterials (metallic nanoparticles, graphene oxide, quantum dots, and nanostructured polymers) are reported for both detection strategies with the aim to show their distinct properties and applications.

Background: Randomised phase III clinical trials represent a methodological milestone to select effective drugs against metastatic cancers. In this context, and particularly in the efficacy assessment of biologic drugs, the initial metastatic tumour burden is a strong prognostic factor. Methods: A systematic literature review of randomised, phase III, first-line, clinical trials in metastatic breast, colorectal, and lung cancers, published from 2016 to 2021, was performed. Three groups of variables were collected: identity-, method- (including tumour burden assessment) and outcome-related. Results: Seventy trials were selected. A large portion of studies (41.4%) focused on the effects of biologic agents (signal inhibitors and immuno-therapies). A definition of low-burden disease based predominantly on the number of involved organs was reported in 28.6% of studies. No explicit reference to oligo-metastatic disease was found either in inclusion/exclusion criteria or in final descriptive data analyses. Disease extent, heterogeneously defined, was a stratification factor for randomisation in only 25.7% of studies. In two studies, a significant imbalance between arms in patients with low-burden disease was revealed. Conclusions: Attention to initial tumour burden in designing future clinical trials (including the harmonisation of definitions and the reporting of eventual oligo-metastatic disease, complete estimates of tumour volume, and its consideration as a stratification factor) should be increased.

Laryngeal squamous cell cancer (LSCC) accounts for almost 25–30% of all head and neck squamous cell cancers and is clustered according to the affected districts, as this determines distinct tendency to recur and metastasize. A major role for numerous genetic alterations in driving the onset and progression of this neoplasm is emerging. However, major efforts are still required for the identification of molecular markers useful for both early diagnosis and prognostic definition of LSCC that is still characterized by significant morbidity and mortality. Non-coding RNAs appear the most promising as they circulate in all the biological fluids allowing liquid biopsy determination, as well as due to their quick and characteristic modulation useful for non-invasive detection and monitoring of cancer. Other critical aspects are related to recent progress in circulating tumor cells and DNA detection, in metastatic status and chemo-refractoriness prediction, and in the functional interaction of LSCC with chronic inflammation and innate immunity. We review all these aspects taking into account the progress of the technologies in the field of next generation sequencing.

Salivary gland cancer (SGC) is an uncommon and heterogeneous disease that accounts for around 8.5% of all head and neck cancers. MicroRNAs (miRNAs) consist of a class of highly conserved, short, single-stranded segments (18–25 nucleotides) of noncoding RNA that represent key gene-transcription regulators in physiological and pathological human conditions. However, their role in SGC development and progression is not completely clear. This review aims to compile and summarize the recent findings on the topic, focusing on the prognostic and diagnostic value of the major modulated and validated microRNAs in SGC. Their differential expression could possibly aid the clinician in delivering an early diagnosis, therapeutic strategy and precision medicine.

Based on compelling preclinical evidence concerning the progress of our novel ruthenium-based metallotherapeutics, we are focusing research efforts on challenging indications for the treatment of invasive neoplasms such as the triple-negative breast cancer (TNBC). This malignancy mainly afflicts younger women, who are black, or who have a BRCA1 mutation. Because of faster growing and spreading, TNBC differs from other invasive breast cancers having fewer treatment options and worse prognosis, where existing therapies are mostly ineffective, resulting in a large unmet biomedical need. In this context, we benefited from an experimental model of TNBC both in vitro and in vivo to explore the effects of a biocompatible cationic liposomal nanoformulation, named HoThyRu/DOTAP, able to effectively deliver the antiproliferative ruthenium(III) complex AziRu, thus resulting in a prospective candidate drug. As part of the multitargeting mechanisms featuring metal-based therapeutics other than platinum-containing agents, we herein validate the potential of HoThyRu/DOTAP liposomes to act as a multimodal anticancer agent through inhibition of TNBC cell growth and proliferation, as well as migration and invasion. The here-obtained preclinical findings suggest a potential targeting of the complex pathways network controlling invasive and migratory cancer phenotypes. Overall, in the field of alternative chemotherapy to platinum-based drugs, these outcomes suggest prospective brand-new settings for the nanostructured AziRu complex to get promising goals for the treatment of metastatic TNBC.

Neuropathic pain has long-term consequences in terms of affective and cognitive disturbances suggesting the involvement of supraspinal mechanisms. In the present study, we used the spared nerve injury (SNI) model to characterize the development of sensory and aversive components of neuropathic pain, and to determine their electrophysiological impact across PFC and limbic regions. Moreover, we evaluated the regulation of several genes involved in immune response and inflammation triggered by the SNI.We showed that SNI led to sensorial hypersensitivity (cold and mechanical stimuli) and depressive-like behavior lasting 12 months after nerve injury. Interestingly, changes in non-emotional cognitive tasks (novel object recognition and Y maze) showed in 1-month SNI mice, were not evident normal in 12 months-SNI animals. In vivo electrophysiology revealed an impaired the Long Term Potentiation (LTP) at prefrontal cortex (PFC)- nucleus accumbens core (NAcore) pathway in both 1 and 12 months SNI. On the other hand, a reduced neural activity was recorded in the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway in 1 month-, but not in 12 months- SNI mice. Finally, we observed the upregulation of specific genes involved in involved in immune response in the hippocampus of 1 month-, but not 12 months-SNI mice, suggesting a neuroinflammatory response which may contribute to the SNI phenotype.These data suggest that distinct brain circuits may drive the psychiatric components of neuropathic pain and pave the way for better investigate the long-term consequences of peripheral nerve injury in which most of the available drugs are to date unsatisfactory.

Simple Summary The availability of selective, effective, and safe anticancer agents is a major challenge in the field of cancer research. As part of a multidisciplinary research project, in recent years our group has proposed an original class of nanomaterials for the delivery of new anticancer drugs based on ruthenium(III) complexes. In cellular models, these nanosystems have been shown to be effective in counteracting growth and proliferation of human breast cancer cells. Compared to conventional metallochemotherapeutics such as platinum-based agents whose clinical practice is associated with serious undesirable effects, ruthenium complexes share improved biochemical profiles making them more selective towards cancer cells and less cytotoxic to healthy cells. Their combination with biocompatible nanocarriers further enhances these promising features, as here showcased by our research carried out in an animal model which underscores the efficacy and safety in vivo of one of our most promising ruthenium-based nanosystems. Abstract Selectivity and efficacy towards target cancer cells, as well as biocompatibility, are current challenges of advanced chemotherapy powering the discovery of unconventional metal-based drugs and the search for novel therapeutic approaches. Among second-generation metal-based chemotherapeutics, ruthenium complexes have demonstrated promising anticancer activity coupled to minimal toxicity profiles and peculiar biochemical features. In this context, our research group has recently focused on a bioactive Ru(III) complex—named AziRu—incorporated into a suite of ad hoc designed nucleolipid nanosystems to ensure its chemical stability and delivery. Indeed, we proved that the structure and properties of decorated nucleolipids can have a major impact on the anticancer activity of the ruthenium core. Moving in this direction, here we describe a preclinical study performed by a mouse xenograft model of human breast cancer to establish safety and efficacy in vivo of a cationic Ru(III)-based nucleolipid formulation, named HoThyRu/DOTAP, endowed with superior antiproliferative activity. The results show a remarkable reduction in tumour with no evidence of animal suffering. Blood diagnostics, as well as biochemical analysis in both acute and chronic treated animal groups, demonstrate a good tolerability profile at the therapeutic regimen, with 100% of mice survival and no indication of toxicity. In addition, ruthenium plasma concentration analysis and tissue bioaccumulation were determined via appropriate sampling and ICP-MS analysis. Overall, this study supports both the efficacy of our Ru-containing nanosystem versus a human breast cancer model and its safety in vivo through well-tolerated animal biological responses, envisaging a possible forthcoming use in clinical trials.

In this review we have showcased the preclinical development of original amphiphilic nanomaterials designed for ruthenium-based anticancer treatments, to be placed within the current metallodrugs approach leading over the past decade to advanced multitarget agents endowed with limited toxicity and resistance. This strategy could allow for new options for breast cancer (BC) interventions, including the triple-negative subtype (TNBC) with poor therapeutic alternatives. BC is currently the second most widespread cancer and the primary cause of cancer death in women. Hence, the availability of novel chemotherapeutic weapons is a basic requirement to fight BC subtypes. Anticancer drugs based on ruthenium are among the most explored and advanced next-generation metallotherapeutics, with NAMI-A and KP1019 as two iconic ruthenium complexes having undergone clinical trials. In addition, many nanomaterial Ru complexes have been recently conceived and developed into anticancer drugs demonstrating attractive properties. In this field, we focused on the evaluation of a Ru(III) complex—named AziRu—incorporated into a suite of both zwitterionic and cationic nucleolipid nanosystems, which proved to be very effective for the in vivo targeting of breast cancer cells (BBC). Mechanisms of action have been widely explored in the context of preclinical evaluations in vitro, highlighting a multitarget action on cell death pathways which are typically deregulated in neoplasms onset and progression. Moreover, being AziRu inspired by the well-known NAMI-A complex, information on non-nanostructured Ru-based anticancer agents have been included in a precise manner.

Head and neck squamous cell carcinoma (HNSCC), a heterogeneous disease arising from various anatomical locations including the larynx, is a leading cause of death worldwide. Despite advances in multimodality treatment, the overall survival rate of the disease is still largely dismal. Early and accurate diagnosis of HNSCC is urgently demanded in order to prevent cancer progression and to improve the quality of the patient’s life. Recently, microRNAs (miRNAs), a family of small non-coding RNAs, have been widely reported as new robust tools for prediction, diagnosis, prognosis, and therapeutic approaches of human diseases. Abnormally expressed miRNAs are strongly associated with cancer development, resistance to chemo-/radiotherapy, and metastatic potential through targeting a large variety of genes. In this review, we summarize on the recent reports that emphasize the pivotal biological roles of miRNAs in regulating carcinogenesis of HNSCC, particularly laryngeal cancer. In more detail, we report the characterized miRNAs with an evident either oncogenic or tumor suppressive role in the cancers. In addition, we also focus on the correlation between miRNA deregulation and clinical relevance in cancer patients. On the basis of intriguing findings, the study of miRNAs will provide a new great opportunity to access better clinical management of the malignancies.

Laryngeal cancer (LCa), a neoplasm of the head and neck region, is a leading cause of death worldwide. Surgical intervention remains the mainstay of LCa treatment, but a crucial point is represented by the possible nodal involvement. Therefore, it is urgently needed to develop biomarkers and therapeutic tools able to drive treatment approaches for LCa. In this study, we investigated deregulated microRNAs (miRNAs) in tissues from LCa patients with either lymph node metastases (N+) or not (N−). miRNA expression profiling was performed by a comprehensive PCR array and subsequent validation by RT-qPCR. Results showed a significant decrease of miR-449a expression in N+ compared to N− patients, and miR-133b down-modulation in LCa tissues compared to paired normal ones. Receiver operating characteristic (ROC) curve analysis revealed the potential diagnostic power of miR-133b for LCa detection. According to the validation results, we selected miR-449a for further in vitro studies. Ectopic miR-449a expression in the LCa cell line Hep-2 inhibited invasion and motility in vitro, slowed cell proliferation, and induced the downregulation of Notch1 and Notch2 as direct targets of miR-449a. Collectively, this study provides new promising biomarkers for LCa diagnosis and a new opportunity to use miR-449a for the treatment of nodal metastases in LCa patients., Graphical Abstract, The need to identify molecular markers for early detection of laryngeal cancer prompted Kawasaki et al. to define a miRNA signature of tumor transformation and spreading. They showed the diagnostic and predictive potential of miR-133b and miR-449a, respectively, and demonstrated miR-449a-mediated suppression of the metastatic factors Notch1 and Notch2.

The current knowledge about non-coding RNAs (ncRNAs) as important regulators of gene expression in both physiological and pathological conditions, has been the main engine for the design of innovative platforms to finalize the pharmacological application of ncRNAs as either therapeutic tools or as molecular biomarkers in cancer. Biochemical alterations of cancer cells are, in fact, largely supported by ncRNA disregulation in the tumor site, which, in turn, reflects the cancer-associated specific modification of circulating ncRNA expression pattern. The aim of this review is to describe the state of the art of pre-clinical and clinical studies that analyze the involvement of miRNAs and lncRNAs in cancer-related processes, such as proliferation, invasion and metastases, giving emphasis to their functional role. A central node of our work has been also the examination of advantages and criticisms correlated with the clinical use of ncRNAs, taking into account the pressing need to refine the profiling methods aimed at identify novel diagnostic and prognostic markers and the request to optimize the delivery of such nucleic acids for a therapeutic use in an imminent future.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies: novel therapeutic approaches beyond conventional chemotherapy are still lacking and prognosis remains poor, even for resectable patients (pts). Furthermore, there is an almost complete absence of validated predictive factors. Consequently, robust biomarkers for the early diagnosis and the prognostic stratification are urgently needed in clinical practice, especially in the context of neoadjuvant and adjuvant settings. In the last years, evidence revealed the crucial role of miRNAs in cancer initiation and progression, as well as in the chemo-resistance mechanisms, suggesting their use as clinical biomarkers. Material and methods: In this pilot study, we performed a microarray analysis to characterize global miRNA expression profile from surgical tissue samples collected from 20 resected PDAC pts pooled into 4 groups according to different clinico-pathological features: nodal metastases (N+/N-) and tumor grading (G2/G3). Results: According to expression patterns, we identified, among 384 miRNAs, a significant different modulation for 11 miRNAs associated to G2 vs G3 and for 7 miRNAs in N+ vs N- disease, suggesting a possible specific signature reflecting histological grade and nodal metastasis occurrence, respectively. We focused on 2 up-regulated (miR-138-5p and miR-518-3p) and 3 down-regulated (miR-215-5p, miR-519a-3p and miR-576-5p) miRNAs in N+ pts, and on 3 up-regulated (miR-1-3p, miR-31-5p and miR-205-5p) in G3 pts, in order to verify their possible implication in the molecular changes behind tumor differentiation and spread, as well as their potential use for prognostic and therapeutic purpose. A bio-informatic analysis was also performed, using different in silico tools, to study both high affinity miRNA targets and cross-regulated pathways among the upand down-regulated miRNAs. The results identified several associated targets involved in multiple signaling pathways commonly dysregulated in cancer. Finally, BRCA1/2 and RB1 miRNAs-mediated-modulation is actually ongoing, considering the pivotal role of these genes in some PDAC pts. Conclusion: These preliminary data provide a strong rationale to further investigate miRNAs expression in larger cohorts of PDAC pts, possibly integrating validated tissue miRNAs data with circulating miRNAs, in order to identify strong (and easily accessible) potential biomarker(s) with prognostic and/or predictive significance. Citation Format: Luca Pompella, Michela Falco, Carlo Caputo, Anna Grimaldi, Giuseppe Tirino, Severo Campione, Francesca Sparano, Maria Lucia Iacovino, Chiara Carmen Miceli, Carlo Molino, Marco Montella, Renato Franco, Gennaro Galizia, Giovanni Conzo, Vincenzo Napolitano, Annamaria Auricchio, Francesca Cardella, Fortunato Ciardiello, Michele Caraglia, Angela Lombardi, Gabriella Misso, Ferdinando De Vita. A pilot study of miRNA expression profile in surgically resected pancreatic ductal adenocarcinoma: Initial report from a bi-institutional cohort [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-052.

The discovery that miRNAs are frequently deregulated in tumours offers the opportunity to identify them as prognostic and diagnostic markers. The aim of this multicentric study is to identify a miRNA expression profile specific for laryngeal cancer. The secondary endpoint was to identify specific deregulated miRNAs with potential as prognostic biomarkers for tumour spread and nodal involvement, and specifically to search for a miRNA pattern pathognomonic for N+ laryngeal cancer and for N- tissues. We identified 20 miRNAs specific for laryngeal cancer and a tissue-specific miRNA signature that is predictive of lymph node metastases in laryngeal carcinoma characterised by 11 miRNAs, seven of which are overexpressed (upregulated) and four downregulated. These results allow the identification of a group of potential specific tumour biomarkers for laryngeal carcinoma that can be used to improve its diagnosis, particularly in early stages, as well as its prognosis.La scoperta che i microRNA sono frequentemente deregolati nei tumori consente di utilizzarli come marker prognostici e diagnostici. Lo scopo di questo studio multicentrico è stato stilare un profilo di espressione di miRNA specifico per il carcinoma della laringe. L’obiettivo secondario è stato identificare particolari miRNA deregolati da usare come potenziali biomarker predittivi di diffusione tumorale e di coinvolgimento linfonodale, nello specifico è stato ricercare un pattern di miRNA patognomonico per tessuto di carcinoma della laringe N+ e per N- rispettivamente. Gli Autori hanno identificato venti miRNA specifici per carcinoma della laringe ed inoltre una miRNA signature tessuto-specifica predittiva di metastasi linfonodali da carcinoma della laringe caratterizzata da 11 miRNA, sette dei quali over-espressi (up-regolati) e quattro down-regolati. Questi risultati permettono l’identificazione di un gruppo di potenziali biomarker tumore-specifici per il carcinoma della laringe che potrebbe essere usata per migliorare la sua diagnosi, in particolare negli stadi iniziali, e soprattutto per la sua prognosi.

Medullary thyroid cancer (MTC) is a tumor highly resistant to chemo-and radiotherapy. Drug resistance can be induced by epigenetic changes such as aberrant DNA methylation. To overcome drug resistance, we explored a promising approach based on the use of 5-aza-2'-deoxycytidine (AZA), a demethylating agent, in combination with the mTOR inhibitor everolimus in MTC cells (MZ-CRC-1 and TT). This combined treatment showed a strong synergistic antiproliferative activity through the induction of apoptosis. The effect of everolimus and/or AZA on genome-wide expression profiling was evaluated by Illumina BeadChip in MZ-CRC-1 cells. An innovative bioinformatic pipeline identified four potential molecular pathways implicated in the synergy between AZA and everolimus: PI3K-Akt signaling, the neurotrophin pathway, ECM/receptor interaction, and focal adhesion. Among these, the neurotrophin signaling pathway was most directly involved in apoptosis, through the overexpression of NGFR and Bax genes. The increased expression of genes involved in the NGFRMAPK10-TP53-Bax/Bcl2 pathway during incubation with AZA plus everolimus was validated by western blotting in MZ-CRC-1 cells. Interestingly, addition of a neutralizing anti-NGFR antibody inhibited the synergistic cytotoxic activity between AZA and everolimus. These results open a new therapeutic scenario for MTC and potentially other neuroendocrine tumors, where therapy with mTOR inhibitors is currently approved. This study was funded by the Italian Ministry of Education, University and Research (Grant Number: FIRB RBAP11884M), to GV and MC. The study was partially supported by funds of the Ricerca Corrente of Istituto Auxologico Italiano, Milan, Italy (code: 05C205_2012), to GV, and MIMOmics Grant of the European Union's Seventh Framework Programme (Grant Number: 305280).

Molecular profiling of a tumor allows the opportunity to design specific therapies which are able to interact only with cancer cells characterized by the accumulation of several genomic aberrations. This study investigates the usefulness of next-generation sequencing (NGS) and mutation-specific analysis methods for the detection of target genes for current therapies in non-small-cell lung cancer (NSCLC), metastatic colorectal cancer (mCRC), and melanoma patients. We focused our attention on EGFR, BRAF, KRAS, and BRAF genes for NSCLC, melanoma, and mCRC samples, respectively. Our study demonstrated that in about 2% of analyzed cases, the two techniques did not show the same or overlapping results. Two patients affected by mCRC resulted in wild-type (WT) for BRAF and two cases with NSCLC were WT for EGFR according to PGM analysis. In contrast, these samples were mutated for the evaluated genes using the therascreen test on Rotor-Gene Q. In conclusion, our experience suggests that it would be appropriate to confirm the WT status of the genes of interest with a more sensitive analysis method to avoid the presence of a small neoplastic clone and drive the clinician to correct patient monitoring.

Mitochondria are the key energy-producing organelles and cellular source of reactive species. They are responsible for managing cell life and death by a balanced homeostasis passing through a network of structures, regulated principally via fission and fusion. Herein we discuss about the most advanced findings considering mitochondria as dynamic biophysical systems playing compelling roles in the regulation of energy metabolism in both physiologic and pathologic processes controlling cell death and survival. Precisely, we focus on the mitochondrial commitment to the onset, maintenance and counteraction of apoptosis, autophagy and senescence in the bioenergetic reprogramming of cancer cells. In this context, looking for a pharmacological manipulation of cell death processes as a successful route for future targeted therapies, there is major biotechnological challenge in underlining the location, function and molecular mechanism of mitochondrial proteins. Based on the critical role of mitochondrial functions for cellular health, a better knowledge of the main molecular players in mitochondria disfunction could be decisive for the therapeutical control of degenerative diseases, including cancer.

According to WHO, breast cancer incidence is increasing so that the search for novel chemotherapeutic options is nowadays an essential requirement to fight neoplasm subtypes. By exploring new effective metal-based chemotherapeutic strategies, many ruthenium complexes have been recently proposed as antitumour drugs, showing ability to impact on diverse cellular targets. In the framework of different molecular pathways leading to cell death in human models of breast cancer, here we demonstrate autophagy involvement behind the antiproliferative action of a ruthenium(III)-complex incorporated into a cationic nanosystem (HoThyRu/DOTAP), proved to be hitherto one of the most effective within the suite of nucleolipidic formulations we have developed for the in vivo transport of anticancer ruthenium(III)-based drugs. Indeed, evidences are implicating autophagy in both cancer development and therapy, and anticancer interventions endowed with the ability to trigger this biological response are currently considered attractive oncotherapeutic approaches. Moreover, crosstalk between apoptosis and autophagy, regulated by finely tuned metallo-chemotherapeutics, may provide novel opportunities for future improvement of cancer treatment. Following this line, our in vitro and in vivo preclinical investigations suggest that an original strategy based on suitable formulations of ruthenium(III)-complexes, inducing sustained cell death, could open new opportunities for breast cancer treatment, including the highly aggressive triple-negative subtype.

miR-125b, ubiquitously expressed and frequently dysregulated in several tumors, has gained special interest in the field of cancer research, displaying either oncogenic or oncosuppressor potential based on tumor type. We have previously demonstrated its tumor-suppressive role in multiple myeloma (MM), but the analysis of molecular mechanisms needs additional investigation. The purpose of this study was to explore the effects of miR-125b and its chemically modified analogs in modulating cell viability and cancer-associated molecular pathways, also focusing on the functional aspects of stress adaptation (autophagy and senescence), as well as programmed cell death (apoptosis). Based on the well-known low microRNA (miRNA) stability in therapeutic application, we designed chemically modified miR-125b mimics, laying the bases for their subsequent investigation in in vivo models. Our study clearly confirmed an oncosuppressive function depending on the repression of multiple targets, and it allowed the identification, for the first time, of miR-125b-dependent miR-34a stimulation as a possible consequence of the inhibitory role on the interleukin-6 receptor (IL-6R)/signal transducer and activator of transcription 3 (STAT3)/miR-34a feedback loop. Moreover, we identified a pattern of miR-125b-co-regulated miRNAs, shedding light on possible new players of anti-MM activity. Finally, functional studies also revealed a sequential activation of senescence, autophagy, and apoptosis, thus indicating, for the first two processes, an early cytoprotective and inhibitory role from apoptosis activation. Keywords: miR-125b, multiple myeloma, apoptosis, autophagy, senescence, miRNome, miRNA therapeutics, miR-34a, next generation sequencing, signal transduction

Laryngeal neuroendocrine carcinomas (LNECs) are rare and highly heterogeneous malignancies presenting a wide range of pathological and clinical manifestations. Herein, we retrospectively characterize ten patients diagnosticated with LNEC, five of which were defined as well-moderately differentiated neuroendocrine carcinomas, and five that were defined as poorly differentiated neuroendocrine carcinomas, according to the latest WHO classification. Clinical features were analyzed and compared between the two subgroups together with a microRNA study which evidenced a peculiar signature likely related to poorly differentiated larynx neuroendocrine carcinomas. These findings may offer new useful insights for clinicians to improve diagnosis efficiency, therapy response, and patients’ outcome for this aggressive neoplasm.

Tumor immunologic microenvironment is strongly involved in tumor progression and the presence of tumor infiltrating lymphocytes (TIL) with different phenotypes has been demonstrated to be of prognostic relevance in different malignancies. We investigated whether TIL infiltration of tumor tissues could also predict the outcome of prostate cancer patients. To this end, we carried out a retrospective analysis correlating the outcome of locally advanced prostate cancer patients undergone salvage radiotherapy upon relapse after radical surgery with the infiltration by different TIL populations. Twenty-two patients with resectable prostate cancer, with a mean age of 67 (+/−3.93) years, who received salvage radiotherapy with a mean of 69.66 (+/− 3.178) Gy in 8 weeks, between June 1999 and January 2009 and with a median follow up of 123 (+/− 55.82) months, were enrolled in this study. We evaluated, by immunohistochemistry, the intratumoral (t) and peripheral stroma (p) infiltration by CD45, CD3, CD4, CD8, CCR7, FoxP3 or PD-1-positive cells on tumor samples taken at the diagnosis (d) and relapse times (R). We correlated these variables with patients' biochemical progression free survival (bPFS), post-radiotherapy progression free survival (PFS), and overall survival (OS). Substantial changes in the rate of TIL subsets were found between the first and the second biopsy with progressive increase in CD4, CCR7, FoxP3, PD-1+ cells. Our analysis revealed that higher CD8p,R+ and lower PD-1R+ TIL scores correlated to a longer bPFS. Higher CD8p,R+ and CCR7t,R+ TIL scores and lower CD45p,R+ and FoxP3p,R+ TIL scores correlated to a prolonged PFS and OS. These results suggest that the immunological microenvironment of primary tumor is strictly correlated with patient outcome and provide the rationale for immunological treatment of prostate cancer.

Solitary intraparotid facial nerve plexiform neurofibromas are extremely rare. These tumors arise from Schwann cells. The plexiform variant is recognized by tortuous and multinodular gross and microscopic lesions. It has a high risk of malignant transformation. We report a case of a solitary plexiform neurofibroma in a 5-year-old Italian male, who initially presented a right parotid mass of four-month duration. He had not pain, trismus, facial weakness or previous trauma. There was not familiar history of Von Recklinghausen's disease. TC and MRI scans revealed a mass in the su- perficial lobe of the parotid gland. A partial parotidectomy was performed. Histopatological ex- amination indicated plexiform neurofibroma. The incidence, presentation, diagnosis and surgical treatment of this lesion are discussed. In our case, the tumor could be readily separated from the main trunk, and facial movement was completely preserved owing to the rich neural network around the mass.

MiR-34a acts as tumor suppressor microRNA (miRNA) in several cancers, including multiple myeloma (MM), by controlling the expression of target proteins involved in cell cycle, differentiation and apoptosis. Here, we have investigated the combination between miR-34a and γ-secretase inhibitor (γSI), Sirtinol or zoledronic acid (ZOL) in order to enhance the inhibitory action of this miRNA on its canonical targets such as Notch1 and SIRT1, and on Ras/MAPK-dependent pathways. Our data demonstrate that miR-34a synthetic mimics significantly enhance the anti-tumor activity of all the above-mentioned anti-cancer agents in RPMI 8226 MM cells. We found that γSI enhanced miR-34a-dependent anti-tumor effects by activating the extrinsic apoptotic pathway which could overcome the cytoprotective autophagic mechanism. Moreover, the combination between miR-34a and γSI increased the cell surface calreticulin (CRT) expression, that is well known for triggering anti-tumor immunological response. The combination between miR-34a and Sirtinol induced the activation of an intrinsic apoptotic pathway along with increased surface expression of CRT. Regarding ZOL, we found a powerful growth inhibition after enforced miR-34a expression, which was not likely attributable to neither apoptosis nor autophagy modulation. Based on our data, the combination of miR-34a with other anti-cancer agents appears a promising anti-MM strategy deserving further investigation.

HCC is the third leading cause of cancer-related deaths worldwide, accounting for about 1 million deaths annually. The incidence of HCC is highest in Asia and Africa, where the endemic high prevalence of hepatitis B and hepatitis C strongly predisposes to the development of chronic liver disease and subsequent development of HCC. Patients with HCC generally present at an advanced stage due to compensated cirrhosis defined by the absence of pathognomonic symptoms, resulting in death within 6 to 20 months, suggesting an urgent need in treatment modalities that will dramatically decrease the mortality rate of HCC. The molecular hepatocarcinogenesis is, however, a gradual process during which genetic alterations progressively accumulate and lead to HCC through intermediate preneoplastic stages. With the advent of whole genome sequencing tools, various mutations associated with HCC have been identified, which have advanced our molecular understanding of HCC. However, the frequency of these mutations is rare, and these genetic mutations only partly explain the etiology of the disease. Better understanding and characterization of novel genetic and epigenetic alterations, which are important to hepatocarcinogenesis, may help understand the molecular pathogenesis of HCC, as well as providing novel therapeutic targets for HCC treatment. Further consideration should be given to developing more effective molecular diagnostic markers and targeted drug therapy.

Autophagy activation is characterized by the accumulation of double-membrane autophagic vesicles (autophagosomes) in the cytoplasm. The mere presence of autophagosomes in the cytoplasm does not necessarily indicate an increased level of autophagy, since the blockade of any step downstream of autophagosome formation increases the number of autophagosomes. Therefore, quantitative methods for the detection of cytoplasmic protein turnover should be employed in addition to autophagosome monitoring, to verify increased levels of autophagy. At the present, multiple methods are available for the quantification of autophagy and the identification of autophagosomes. Here, we detail the in vitro methods currently available to detect autophagic cell death by flow cytometry analysis.

Early detection of laryngeal cancer, essential in the initial stages of the disease to achieve a high survival rate, is unfortunately hampered by the lack of specific symptoms. In this regard, there is a pressing need to dispose of reliable and non-invasive diagnostic and prognostic markers for a real time monitoring availing of accurate and reproducible methods, in order ensure applicability for early diagnosis and primary and secondary prevention. This review focuses on the recent reports that emphasize the crucial role of miRNAs in regulating laryngeal cancer tumorigenesis. In detail, we have reported the most characterized miRNAs with an established oncogenic or oncosuppressive role in cell biology of laryngeal cancer, also describing, for each of them, the main molecular mechanisms responsible for their specific function. We have also defined the potential of miRNAs as novel diagnostic and prognostic markers in virtue of their differential expression between laryngeal carcinoma tissues and the adjacent normal counterpart. Moreover, their proven stability in systemic circulation and other body fluids, as well as their easy detection and quantization, makes the analysis of miRNA signatures an excellent tool for clinicians as hallmark for cancer classification and diagnosis. Moreover, an eventual similarity between deregulated miRNAs in tumor tissues and in body fluids would allow to provide a considerable advantage for patients’ compliance, replacing invasive tissue biopsies with simple assays on easily obtained blood products.

Transglutaminases are a family of Ca2+- dependent enzymes which catalyze posttranslational modifications of proteins. The main activity of these enzymes is the crosslinking of glutaminyl residues of a protein/peptide substrate to lysyl residues of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted/ crosslinked adducts) or -OH groups (to form ester linkages). In absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. Transglutaminase activity has been suggested to be involved in molecular mechanisms responsible for either physiological or pathological processes. In particular, transglutaminase activity has been shown to be responsible for human autoimmune diseases and Celiac Disease is just one of them. Interestingly, cancer and neurodegenerative diseases, such as Alzheimer’s Disease, Parkinson’s Disease, supranuclear palsy and Huntington’s Disease, are characterized in part by aberrant transglutaminase activity and by increased cross-linked proteins in affected tissues. This review describes the possible molecular mechanisms by which these enzymes could be responsible for such diseases and the possible use of transglutaminase inhibitors for patients with diseases characterized by aberrant transglutaminase activity.

Hematological malignancies, accounting for about 10% of all deaths for cancer, include various forms of leukemia, lymphoma and myeloma. At present, hematological malignancies are analyzed and classified on the basis of morphologic characteristics, cell surface markers, cytogenetic aberrations and molecular markers. Unfortunately, in most cases, standard criteria are not sufficient for both an early diagnosis and a complete classification. The latter issue hampers an optimal therapeutic choice for these patients that often display heterogeneous clinical outcomes or responses to therapy. This heterogeneity has determined a need for improved methods of analysis and novel markers for diagnosis and classification of these malignancies. Non coding RNAs act as master regulators of numerous biological processes including epigenetic response, apoptosis and cell cycle. The recent advances in cancer research have led to a spreading out in the clinical use of genomic information; in fact, several studies are investigating the prominent role of both miRNAs and lncRNAs in hematopoietic differentiation and proliferation, as well as in the development of various hematological malignancies. These investigations are mainly aimed at researching new therapeutic opportunities that could boost a reduced risk of adverse events in normal tissues. Moreover, not less important, there is also a growing interest in determining how ncRNAs are associated with clinical features. In this review we focus on the aberrant ncRNAs expression in the most common forms of blood cancers, each of which exhibits a unique signature in comparison to normal counterparts. In addition to their regulatory role and in virtue of the well known ncRNAs' capacity of modulating signal and pathway networks, herein we discuss both miRNAs' and lncRNAs' potential as new powerful biomarkers for efficient diagnosis and prediction of response for patients with hematological malignancies. The hematological malignancies include various forms of leukemia, lymphoma, and myeloma and account for about 10% of all deaths for cancer. At the present, hematological malignancies are analyzed and classified on the basis of morphologic characteristics, cell surface markers, cytogenetic aberrations, and molecular markers. Unfortunately, in most cases, standard criteria are not sufficient for both an early diagnosis, and for a complete classification. The latter hampers an optimal therapeutic choice for these patients that often display heterogeneous clinical outcomes or responses to therapy. This heterogeneity has determined a need for improved analysis methods and new markers for diagnosis and classification of these malignancies. Non coding RNAs act as master regulators of numerous biological processes including epigenetic response, apoptosis, and cell cycle. The recent advances in cancer research have led to a spreading out in the clinical use of genomic information and several studies have investigated the prominent role of both miRNAs and lncRNAs in haematopoietic differentiation and proliferation, as well as in the development of various haematological malignancies. These investigations have been mainly aimed at research new therapeutic opportunities that could boast a reduced risk of adverse events in normal tissues. Moreover, not less important, there is also a growing interest in determining how ncRNAs are associated with clinical features. In this review we focus on the aberrant ncRNAs expression in the most common forms of blood cancers, each of which exhibits a unique signature in comparison to normal counterparts. In addition to their regulatory role and in virtue of the well known ncRNAs' capacity of modulating signal and pathway networks, herein we discuss on both miRNAs' and lncRNAs' potential as new powerful biomarkers for efficient diagnosis and prediction of response for patients with hematological malignancies.

Background: Results from the BEVA2007 trial, suggest that the metronomic chemotherapy regimen with dose-fractioned cisplatin and oral etoposide (mPE) +/− bevacizumab, a monoclonal antibody to the vascular endothelial growth factor (VEGF), shows anti-angiogenic and immunological effects and is a safe and active treatment for metastatic non-small cell lung cancer (mNSCLC) patients. We carried out a retrospective analysis aimed to evaluate the antitumor effects of this treatment in a subset of patients with squamous histology. Methods: Retrospective analysis was carried out in a subset of 31 patients with squamous histology enrolled in the study between September 2007 and September 2015. All of the patients received chemotherapy with cisplatin (30 mg/sqm, days 1–3q21) and oral etoposide (50 mg, days 1–15q21) (mPE) and 14 of them also received bevacizumab 5 mg/kg on the day 3q21 (mPEBev regimen). Results: This treatment showed a disease control rate of 71% with a mean progression free survival (PFS) and overall survival (OS) of 13.6 and 17 months respectively. After 4 treatment courses, 6 patients showing a remarkable tumor shrinkage, underwent to radical surgery, attaining a significant advantage in term of survival (P=0.04). Kaplan-Meier and log-rank test identified the longest survival in patients presenting low baseline levels in neutrophil-to-lymphocyte ratio (NLR) (P=0.05), interleukin (IL) 17A (P=0.036), regulatory-T-cells (T reg s) (P=0.020), and activated CD83+ dendritic cells (DCs) (P=0.03). Conclusions: These results suggest that the mPE +/− bevacizumab regimen is feasible and should be tested in comparative trials in advanced squamous-NSCLC (sqNSCLC). Moreover, its immune-biological effects strongly suggest the investigation in sequential combinations with immune check-point inhibitors.

Looking for new metal-based anticancer treatments, in recent years many ruthenium complexes have been proposed as effective and safe potential drugs. In this context we have recently developed a novel approach for the in vivo delivery of Ru(III) complexes, preparing stable ruthenium-based nucleolipidic nanoaggregates endowed with significant antiproliferative activity. Herein we describe the cellular response to our ruthenium-containing formulations in selected models of human breast cancer. By in vitro bioscreens in the context of preclinical studies, we have focused on their ability to inhibit breast cancer cell proliferation by the activation of the intrinsic apoptotic pathway, possibly via mitochondrial perturbations involving Bcl-2 family members and predisposing to programmed cell death. In addition, the most efficient ruthenium-containing cationic nanoaggregates we have hitherto developed are able to elicit both extrinsic and intrinsic apoptosis, as well as autophagy. To limit chemoresistance and counteract uncontrolled proliferation, multiple cell death pathways activation by metal-based chemotherapeutics is a challenging, yet very promising strategy for targeted therapy development in aggressive cancer diseases, such as triple-negative breast cancer with limited treatment options. These outcomes provide valuable, original knowledge on ruthenium-based candidate drugs and new insights for future optimized cancer treatment protocols.

Autophagy activation is characterized by the accumulation of double-membrane autophagic vesicles (autophagosomes) in the cytoplasm. The mere presence of autophagosomes in the cytoplasm does not necessarily indicate an increased level of autophagy, since the blockade of any step downstream of autophagosome formation increases the number of autophagosomes. Therefore, quantitative methods for the detection of cytoplasmic protein turnover should be employed in addition to autophagosome monitoring, to verify increased levels of autophagy. At the present, multiple methods are available for the quantification of autophagy and the identification of autophagosomes. Here, we detail the in vitro methods currently available to detect autophagic cell death by flow cytometry analysis.

// Pierpaolo Pastina 1 , Valerio Nardone 1 , Cirino Botta 2 , Stefania Croci 1 , Paolo Tini 1 , Giuseppe Battaglia 1 , Veronica Ricci 3 , Maria Grazia Cusi 4 , Claudia Gandolfo 4 , Gabriella Misso 5 , Silvia Zappavigna 5 , Michele Caraglia 5,6 , Antonio Giordano 6,7 , Donatella Aldinucci 8 , Pierfrancesco Tassone 2,6 , Pierosandro Tagliaferri 2 , Luigi Pirtoli 1 and Pierpaolo Correale 1,9 1 Radiotherapy Unit, Department of Medicine, Surgery, and Neuroscience, Siena University Hospital, Siena, Italy 2 Medical Oncology Unit, AUO “Mater Domini”, “Magna Graecia” University, Catanzaro, Italy 3 Radiology Unit,Department of Medicine, Surgery, and Neuroscience, Siena University Hospital, Siena, Italy 4 Department of Medical Biotechnology, Microbiology and Virology Unit, University of Siena, Siena, Italy 5 Department of Biochemistry, Biophysics and General Pathology, University of Campania “L. Vanvitelli”, Naples, Italy 6 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple University, Philadelphia, PA, USA 7 Department of Medicine, Surgery, and Neuroscience, University of Siena and Istituto Toscano Tumori (ITT), Siena, Italy 8 Department of Experimental Oncology 2, CRO Aviano National Cancer Institute, Aviano, Italy 9 Medical Oncology Unit, Metropolitan Hospital “Bianchi-Melacrino-Morelli, Reggio Calabria, Italy Correspondence to: Pierpaolo Correale, email: // Michele Caraglia, email: // Keywords : immune-modulation, radiation therapy, metronomic chemotherapy, NSCLC, retrospective analysis Received : March 21, 2017 Accepted : June 20, 2017 Published : August 24, 2017 Abstract Radiotherapy (RT), together with a direct cytolytic effect on tumor tissue, also elicits systemic immunological events, which sometimes result in the regression of distant metastases (abscopal effect). We have shown the safety and anti-tumor activity of a novel metronomic chemotherapy (mCH) regimen with dose-fractioned cisplatin, oral etoposide and bevacizumab, a mAb against the vasculo-endothelial-growth-factor (mPEBev regimen), in metastatic non-small-cell-lung cancer (mNSCLC). This regimen, designed on the results of translational studies, showed immune-modulating effects that could trigger and empower the immunological effects associated with tumor irradiation. In order to assess this, we carried out a retrospective analysis in a subset of 69 consecutive patients who received the mPEBev regimen within the BEVA2007 trial. Forty-five of these patients, also received palliative RT of one or more metastatic sites. Statistical analysis (a Log-rank test) revealed a much longer median survival in the group of patients who received RT [mCH vs mCH + RT: 12.1 +/-2.5 (95%CI 3.35-8.6) vs 22.12 +/-4.3 (95%CI 11.9-26.087) months; P =0.015] with no difference in progression-free survival. In particular, their survival correlated with the mPEBev regimen ability to induce the percentage of activated dendritic cells (DCs) (CD3-CD11b+CD15-CD83+CD80+) [Fold to baseline value (FBV) ≤1 vs >1: 4+/-5.389 (95%CI,0- 14.56) vs 56+/-23.05 (95%CI,10.8-101.2) months; P :0.049)] and central-memory- T-cells (CD3+CD8+CD45RA-CCR7+) [FBV ≤ 1 vs >1: 8+/-5.96 (95%CI,0-19.68) vs 31+/-12.3 (95%CI,6.94-55.1) months; P :0.045]. These results suggest that tumor irradiation may prolong the survival of NSCLC patients undergone mPEBev regimen presumably by eliciting an immune-mediated effect and provide the rationale for further perspective clinical studies.

Head and neck carcinoma (HNC) is a heterogeneous disease encompassing a variety of tumors according to the origin. Laryngeal cancer (LC) represents one of the most frequent tumors in the head and neck region. Despite clinical studies and advance in treatment, satisfactory curative strategy has not yet been reached. Therefore, there is an urgent need for the identification of specific molecular signatures that better predict the clinical outcomes and markers that serve as suitable therapeutic targets. Long non-coding RNAs (lncRNA) are reported as important regulators of gene expression and represent an innovative pharmacological application as molecular biomarkers in cancer. The purpose of this review is to discuss the most relevant epigenetic and histological prognostic biomarkers in HNC, with particular focus on LC. We summarize the emerging roles of long non-coding RNAs in HNC and LC development and their possible use in early diagnosis.

"Context: To date no efficacious treatments are available for advanced medullary thyroid carcinoma (MTC). Objective: We investigated in vitro and in vivo a new strategy for the therapy of MTC, combining human recombinant IL-2 with lanreotide (LAN), a somatostatin analog. Methods: The in vitro effects of LAN on the sensitivity of TT cells, a MTC cell line, to IL-2-stimulated human peripheral blood mononuclear cells were determined by a lactate dehydrogenase release assay. In addition, we evaluated the toxicity, the effects on quality of life, and the antitumor activity of sc low-dose IL-2 in combination with LAN (90 mg every 28 days) in a series of 6 patients with symptomatic and advanced MTC. Results: The cytotoxicity of IL-2-activated peripheral blood mononuclear cells was significantly increased in TT cells treated with LAN or LAN plus IL-2 compared with that in TT cells without treatment. The therapy was well tolerated, and a statistically significant improvement of quality of life was observed in patients treated with the combination of LAN and IL-2. After 6 months of therapy, partial response and stable disease have been recorded in 2 and 3 patients, respectively, with a significant decrease in calcitonin levels in 3 patients. Conclusions: Both in vitro and in vivo evidence suggests that the combination of LAN and IL-2 may have a role in the management of advanced and symptomatic MTC. However, these preliminary data require further validation in larger randomized trials. . . " Context: To date no efficacious treatments are available for advanced medullary thyroid carcinoma (MTC). Objective: We investigated in vitro and in vivo a new strategy for the therapy of MTC, combining human recombinant IL-2 with lanreotide (LAN), a somatostatin analog. Methods: The in vitro effects of LAN on the sensitivity of TT cells, a MTC cell line, to IL-2-stimulated human peripheral blood mononuclear cells were determined by a lactate dehydrogenase release assay. In addition, we evaluated the toxicity, the effects on quality of life, and the antitumor activity of sc low-dose IL-2 in combination with LAN (90 mg every 28 days) in a series of 6 patients with symptomatic and advanced MTC. Results: The cytotoxicity of IL-2-activated peripheral blood mononuclear cells was significantly increased in TT cells treated with LAN or LAN plus IL-2 compared with that in TT cells without treatment. The therapy was well tolerated, and a statistically significant improvement of quality of life was observed in patients treated with the combination of LAN and IL-2. After 6 months of therapy, partial response and stable disease have been recorded in 2 and 3 patients, respectively, with a significant decrease in calcitonin levels in 3 patients. Conclusions: Both in vitro and in vivo evidence suggests that the combination of LAN and IL-2 may have a role in the management of advanced and symptomatic MTC. However, these preliminary data require further validation in larger randomized trials. Copyright © 2013 by The Endocrine Society.