MicroRNA-based signatures impacting clinical course and biology of ovarian cancer: a miRNOmics study.

BACKGROUND: In Western countries, ovarian cancer (OC) still represents the leading cause of gynecological cancer-related deaths, despite the remarkable gains in therapeutical options. Novel biomarkers of early diagnosis, prognosis definition and prediction of treatment outcomes are of pivotal importance. Prior studies have shown the potentials of micro-ribonucleic acids (miRNAs) as biomarkers for OC and other cancers. METHODS: We focused on the prognostic and/or predictive potential of miRNAs in OC by conducting a comprehensive array profiling of miRNA expression levels in ovarian tissue samples from 17 non-neoplastic controls, and 60 tumor samples from OC patients treated at the Regina Elena National Cancer Institute (IRE). A set of 54 miRNAs with differential expression in tumor versus normal samples (T/N-deregulated) was identified in the IRE cohort and validated against data from the Cancer Genoma Atlas (TCGA) related to 563 OC patients and 8 non-neoplastic controls. The prognostic/predictive role of the selected 54 biomarkers was tested in reference to survival endpoints and platinum resistance (P-res). RESULTS: In the IRE cohort, downregulation of the 2 miRNA-signature including miR-99a-5p and miR-320a held a negative prognostic relevance, while upregulation of miR-224-5p was predictive of less favorable event free survival (EFS) and P-res. Data from the TCGA showed that downregulation of 5 miRNAs, i.e., miR-150, miR-30d, miR-342, miR-424, and miR-502, was associated with more favorable EFS and overall survival outcomes, while miR-200a upregulation was predictive of P-res. The 9 miRNAs globally identified were all included into a single biologic signature, which was tested in enrichment analysis using predicted/validated miRNA target genes, followed by network representation of the miRNA-mRNA interactions. CONCLUSIONS: Specific dysregulated microRNA sets in tumor tissue showed predictive/prognostic value in OC, and resulted in a promising biological signature for this disease.

COVID-19 epidemic strongly affected cancer research in Italy: a survey of the Italian Cancer Society (SIC).

BACKGROUND: Italy was among the first countries hit by the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The application of strict lockdown measures disproportionately affected both cancer patient care as well as basic and translational cancer research. MATERIALS AND METHODS: The Italian Cancer Society (SIC) conducted a survey on the effect of lockdown on laboratories involved in cancer research in Italy. The survey was completed by 570 researchers at different stages of their career, working in cancer centers, research institutes and universities from 19 Italian regions. RESULTS: During the lockdown period, the impact of the COVID-19 pandemic emergency on face-to-face research activities was high, with a complete (47.7%) or partial (36.1%) shutdown of the laboratories. In the post-lockdown period, research activities were resumed in most of the respondents' institutions (80.4%), though with some restrictions (77.2%). COVID-19 testing was offered to research personnel only in ~50% of research institutions. Overall, the response to the pandemic was fragmented as in many cases institutions adopted different strategies often aimed at limiting possible infections without a clearly defined contingency plan. Nevertheless, research was able to provide the first answers and possible ways out of the pandemic, also with the contribution of many cancer researchers that sacrificed their research programs to help overcome the pandemic by offering their knowledge and technologies. CONCLUSIONS: Given the current persistence of an emergency situation in many European countries, a more adequate organization of research centers will be urgent and necessary to ensure the continuity of laboratory activities in a safe environment.

Are intraoral customized stents still necessary in the era of Highly Conformal Radiotherapy for Head & Neck cancer? Case series and literature review.

AIM: To evaluate the dose sparing efficacy of intraoral customized stents in combination with IGRT/VMAT in Head & Neck cancer patients. BACKGROUND: Despite advances in high-dose conformal radiotherapy (RT) techniques, adverse effects (such as oral mucositis) during and after RT often require temporary suspension of treatment and affect the quality of life in survivors. Intraoral customized stents can decrease radiation doses in healthy tissues and minimize damage from radiations. At the best of our knowledge the clinical impact of such devices in combination with VMAT (volumetric modulated arc therapy) is not reported in the literature. CASES DESCRIPTION: Three Head & Neck cancer patients were submitted to image guided (IG) RT/VMAT in their treatment protocol. Dose distribution with and without the use of an intraoral stent was compared in each patient. Mean radiation doses proved to be lower in all patients, especially in the subsite: oral cavity. CONCLUSIONS: There are several reports on the efficacy of IS during RT for Head & Neck cancer. Despite technological advances, the combination between high conformal RT and intraoral stents could still play a role in the management of this kind of patients. This strengthens the usefulness of the individualization of treatments and multidisciplinary approach.

Retinoic acid and arsenic trioxide sensitize acute promyelocytic leukemia cells to ER stress.

Retinoic acid (RA) in association with chemotherapy or with arsenic trioxide (ATO) results in high cure rates of acute promyelocytic leukemia (APL). We show that RA-induced differentiation of human leukemic cell lines and primary blasts dramatically increases their sensitivity to endoplasmic reticulum (ER) stress-inducing drugs at doses that are not toxic in the absence of RA. In addition, we demonstrate that the PERK pathway, triggered in response to ER stress, has a major protective role. Moreover, low amounts of pharmacologically induced ER stress are sufficient to strongly increase ATO toxicity. Indeed, in the presence of ER stress, ATO efficiently induced apoptosis in RA-sensitive and RA-resistant APL cell lines, at doses ineffective in the absence of ER stress. Our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML.

Growth and adhesion to HT-29 cells inhibition of Gram-negatives by Bifidobacterium longum BB536 e Lactobacillus rhamnosus HN001 alone and in combination.

OBJECTIVE: The aim of this study was to test the inhibitory effect of supernatants of broth cultures of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001, both individually and in combination, against Gram-negative strains (uropathogens, enteropathogens and a reference strain). Moreover, in vitro protection of B. longum BB536 and L. rhamnosus HN001, both individually and in combination, against pathogen adhesion to HT-29 cell line, was investigated. MATERIALS AND METHODS: The inhibitory activity was performed by the agar diffusion test and in vitro antagonistic activity against pathogen adhesion to human epithelial intestinal HT-29 cells was performed using standardized culture techniques. RESULTS: The study showed that B. longum BB536 and L. rhamnosus HN001, individually and in combination have inhibitory activity against the majority of the Gram negative strains tested. Furthermore, the results showed that both probiotic strains have a good capacity to inhibit pathogenic adhesion to HT-29 cells. Moreover, the ability of B. longum BB536 and L. rhamnosus HN001 to inhibit pathogenic adhesion increased when they were used in combination. DISCUSSION: The combination of B. longum BB536 and L. rhamnosus HN001 showed inhibitory activity against Gram-negatives and an improved ability to reduce their adhesion properties and to compete with them. CONCLUSIONS: The simultaneous presence of the two-probiotic strains could promote competitive mechanisms able to reduce the adhesion properties of pathogen strains and have an important ecological role within the highly competitive environment of the human gut.

Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth.

Mutations in the p53 tumor-suppressor gene are prevalent in human cancers. The majority of p53 mutations are missense, which can be classified into contact mutations (that directly disrupts the DNA-binding activity of p53) and structural mutations (that disrupts the conformation of p53). Both of the mutations can disable the normal wild-type (WT) p53 activities. Nevertheless, it has been amply documented that small molecules can rescue activity from mutant p53 by restoring WT tumor-suppressive functions. These compounds hold promise for cancer therapy and have now entered clinical trials. In this study, we show that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutant under in vitro and in vivo conditions, revealing a new mechanism of action for a dietary-related compound. PEITC exhibits growth-inhibitory activity in cells expressing p53 mutants with preferential activity toward p53(R175), one of the most frequent 'hotspot' mutations within the p53 sequence. Mechanistic studies revealed that PEITC induces apoptosis in a p53(R175) mutant-dependent manner by restoring p53 WT conformation and transactivation functions. Accordingly, in PEITC-treated cells the reactivated p53(R175) mutant induces apoptosis by activating canonical WT p53 targets, inducing a delay in S and G2/M phase, and by phosphorylating ATM/CHK2. Interestingly, the growth-inhibitory effects of PEITC depend on the redox state of the cell. Further, PEITC treatments render the p53(R175) mutant sensitive to degradation by the proteasome and autophagy in a concentration-dependent manner. PEITC-induced reactivation of p53(R175) and its subsequent sensitivity to the degradation pathways likely contribute to its anticancer activities. We further show that dietary supplementation of PEITC is able to reactivate WT activity in vivo as well, inhibiting tumor growth in xenograft mouse model. These findings provide the first example of mutant p53 reactivation by a dietary compound and have important implications for cancer prevention and therapy.

Impact of gut microbiota on diabetes mellitus.

Various functions of the gut are regulated by sophisticated interactions among its functional elements, including the gut microbiota. These microorganisms play a crucial role in gastrointestinal mucosa permeability. They control the fermentation and absorption of dietary polysaccharides to produce short-chain fatty acids, which may explain their importance in the regulation of fat accumulation and the subsequent development of obesity-related diseases, suggesting that they are a crucial mediator of obesity and its consequences. In addition, gut bacteria play a crucial role in the host immune system, modulation of inflammatory processes, extraction of energy from the host diet and alterations of human gene expression. Dietary modulation of the human colonic microbiota has been shown to confer a number of health benefits to the host. Simple therapeutic strategies targeted at attenuating the progression of chronic low-grade inflammation and insulin resistance are urgently required to prevent or slow the development of diabetes in susceptible individuals. The main objective of this review is to address the pathogenic association between gut microbiota and diabetes, and to explore any novel related therapeutic targets. New insights into the role of the gut microbiota in diabetes could lead to the development of integrated strategies using probiotics to prevent and treat these metabolic disorders.

Microbiological characteristics of the probiotic strains B. longum BB536 and L. rhamnosus HN001 used in combination.

AIM: The aim of this study was to investigate the antagonistic activity between the probiotic strains Bifidobacterium longum (B. longum) BB536 and Lactobacillus rhamnosus (L. rhamnosus) HN001 (ZirCombi, Alfa Wassermann S.p.A., Italy) and to evaluate for the strains tested alone and in combination the resistance in simulated gastrointestinal conditions, the ability to adhere to epithelial intestinal cells and their competition for adhesion. METHODS: The antagonism between B. longum BB536 and L. rhamnosus HN001 was tested modifying the agar diffusion method. The in vitro resistance to gastrointestinal condition of the two strains used alone or in combination was tested using low pH (3.0, 2.5 and 2.0) and different concentrations of bile salts (0.3%, 0.5% and 0.7%). The adhesion ability and the competition for adhesivity on human colon cancer HT-29 cells were tested modifying quantitative methods described in literature. RESULTS: The results demonstrated that B. longum BB536 and L. rhamnosus HN001 showed no in vitro inhibition effect each other and a good resistance to low pH and to different concentrations of bile salts, that was enhanced when they were tested in combination. Moreover, the two strains tested alone and in combination showed a good adhesion on HT-29 cells and no mechanism of competition. CONCLUSION: The study suggests that B. longum BB536 and L. rhamnosus HN001 used in combination show no antagonism and could have functional endosymbiotic effects on intestinal host-microbiota.

Che-1 modulates the decision between cell cycle arrest and apoptosis by its binding to p53.

The tumor suppressor p53 is mainly involved in the transcriptional regulation of a large number of growth-arrest- and apoptosis-related genes. However, a clear understanding of which factor/s influences the choice between these two opposing p53-dependent outcomes remains largely elusive. We have previously described that in response to DNA damage, the RNA polymerase II-binding protein Che-1/AATF transcriptionally activates p53. Here, we show that Che-1 binds directly to p53. This interaction essentially occurs in the first hours of DNA damage, whereas it is lost when cells undergo apoptosis in response to posttranscriptional modifications. Moreover, Che-1 sits in a ternary complex with p53 and the oncosuppressor Brca1. Accordingly, our analysis of genome-wide chromatin occupancy by p53 revealed that p53/Che1 interaction results in preferential transactivation of growth arrest p53 target genes over its pro-apoptotic target genes. Notably, exposure of Che-1(+/-) mice to ionizing radiations resulted in enhanced apoptosis of thymocytes, compared with WT mice. These results confirm Che-1 as an important regulator of p53 activity and suggest Che-1 to be a promising yet attractive drug target for cancer therapy.

Reactivation of epigenetically silenced miR-512 and miR-373 sensitizes lung cancer cells to cisplatin and restricts tumor growth.

MicroRNAs (miRs) regulate a variety of cellular processes, and their impaired expression is involved in cancer. Silencing of tumor-suppressive miRs in cancer can occur through epigenetic modifications, including DNA methylation and histone deacetylation. We performed comparative miR profiling on cultured lung cancer cells before and after treatment with 5'aza-deoxycytidine plus Trichostatin A to reverse DNA methylation and histone deacetylation, respectively. Several tens of miRs were strongly induced by such 'epigenetic therapy'. Two representatives, miR-512-5p (miR-512) and miR-373, were selected for further analysis. Both miRs were secreted in exosomes. Re-expression of both miRs augmented cisplatin-induced apoptosis and inhibited cell migration; miR-512 also reduced cell proliferation. TEAD4 mRNA was confirmed as a direct target of miR-512; likewise, miR-373 was found to target RelA and PIK3CA mRNA directly. Our results imply that miR-512 and miR-373 exert cell-autonomous and non-autonomous tumor-suppressive effects in lung cancer cells, where their re-expression may benefit epigenetic cancer therapy.

Helical Tomotherapy of the breast: can thermoplastic immobilization improve the reproducibility of the treatment setup and the accuracy of the delivered dose?

PURPOSE: To evaluate the impact of thermoplastic mask immobilization in the setup reproducibility and delivered dose for Helical Tomotherapy (HT) of the breast/chest wall. METHODS: 16 patients treated with Accuray Hi-Art HT for breast-cancer were considered. Patients were positioned supine with arms extended above the head using Civco Wing Board (WB) system. In 50% of patients an Orfit thermoplastic mask was added in order to improve immobilization. Before each treatment fraction a megavoltage CT (MVCT) scan was taken and registered to the planning CT by experienced medical staff. The impact of thermoplastic mask was investigated analysing MVCT shift-roll data and MVCT dose distribution using Planned Adaptive software. RESULTS: In the analysed cases, the addition of thermoplastic mask had minor impact on the lateral, longitudinal and roll data distribution. Variance of vertical shifts was significantly reduced in the WB + Orfit group. Van Herk's margins were not affected by addition of thermoplastic immobilization. In both groups, target coverage (V95) and maximum dose (D1) were almost identical to planned values. D1 of organs at risk were not significantly different in the two groups. CONCLUSIONS: Analysis of shift-roll data shows no improvement in the group of patients immobilized with the addition of thermoplastic mask. Van Herk's margin is quite large (7-10 mm) in both groups evidencing the need to perform daily setup correction. The adapted dose distribution complies well with the planned one, showing that if MVCT is used before each treatment fraction, a 3 mm margin (setup component) for CTVs expansion could be adequate.

Gain-of-function mutant p53 downregulates miR-223 contributing to chemoresistance of cultured tumor cells.

Mutant p53 proteins are expressed at high frequency in human tumors and are associated with poor clinical prognosis and resistance to chemotherapeutic treatments. Here we show that mutant p53 proteins downregulate micro-RNA (miR)-223 expression in breast and colon cancer cell lines. Mutant p53 binds the miR-223 promoter and reduces its transcriptional activity. This requires the transcriptional repressor ZEB-1. We found that miR-223 exogenous expression sensitizes breast and colon cancer cell lines expressing mutant p53 to treatment with DNA-damaging drugs. Among the putative miR-223 targets, we focused on stathmin-1 (STMN-1), an oncoprotein known to confer resistance to chemotherapeutic drugs associated with poor clinical prognosis. Mutant p53 silencing or miR-223 exogenous expression lowers the levels of STMN-1 and knockdown of STMN-1 by small interfering RNA increases cell death of mutant p53-expressing cell lines. On the basis of these findings, we propose that one of the pathways affected by mutant p53 to increase cellular resistance to chemotherapeutic agents involves miR-223 downregulation and the consequent upregulation of STMN-1.

Protumorigenic effects of mir-145 loss in malignant pleural mesothelioma.

We identified a discrete number of microRNAs differentially expressed in benign or malignant mesothelial tissues. We focused on mir-145 whose levels were significantly downregulated in malignant mesothelial tissues and malignant pleural mesothelioma (MPM) cell lines as compared to benign tissues (pleura, peritoneum or cysts). We show that promoter hyper-methylation caused very low levels in MPM cell lines and specimens. Treatment of MPM cell lines with mir-145 agonists negatively modulated some protumorigenic properties of MPM cells, such as clonogenicity, cell migration and resistance to pemetrexed treatment. The main effector mechanism of the clonogenic death induced by mir-145 was that of accelerated senescence. We found that mir-145 targeted OCT4 via specific binding to its 3'-UTR. Increased intracellular levels of mir-145 decreased the levels of OCT4 and its target gene ZEB1, thereby counteracting the increase of OCT4 induced by pemetrexed treatment which is known to favor the development of chemoresistant cells. In line with this, reintroduction of OCT4 into mimic-145 treated cells counteracted the effects on clonogenicity and replicative senescence. This further supports the relevance of the mir-145-OCT4 interaction for the survival of MPM cells. The potential use of mir-145 expression levels to classify benign vs malignant mesothelial tissues and the differences between pemetrexed-induced senescence and that induced by the re-expression of mir-145 are discussed.

Expression of TP53 mutation-associated microRNAs predicts clinical outcome in head and neck squamous cell carcinoma patients.

BACKGROUND: TP53 mutation is associated with decreased survival rate in head and neck squamous cell carcinoma (HNSCC) patients. We set out to identify microRNAs (miRNAs) whose expression associates with TP53 mutation and survival in HNSCC. PATIENTS AND METHODS: We analyzed TP53 status by direct sequencing of exons 2 through 11 of a prospective series of 121 HNSCC samples and assessed its association with outcome in 109 followed-up patients. We carried out miRNA expression profiling on 121 HNSCC samples and 66 normal counterparts. miRNA associations with TP53 mutations and outcome were evaluated. RESULTS: A TP53 mutation was present in 58% of the tumors and TP53 mutations were significantly associated with a shorter recurrence-free survival. This association was stronger in the clinical subgroup of patients subjected to adjuvant therapy after surgery. The expression of 49 miRNAs was significantly associated with TP53 status. Among these 49, we identified a group of 12 miRNAs whose expression correlates with recurrence-free survival and a group of 4 miRNAs that correlates with cancer-specific survival. The two groups share three miRNAs. Importantly, miRNAs that correlate with survival are independent prognostic factors either when considered individually or as signatures. CONCLUSIONS: miRNAs expression associates with TP53 status and with reduced survival after surgical treatment of squamous cell carcinoma of the head and neck.

USP2a alters chemotherapeutic response by modulating redox.

Cancer cells are characterized by altered ubiquitination of many proteins. The ubiquitin-specific protease 2a (USP2a) is a deubiquitinating enzyme overexpressed in prostate adenocarcinomas, where it exhibits oncogenic behavior in a variety of ways including targeting c-Myc via the miR-34b/c cluster. Here we demonstrate that USP2a induces drug resistance in both immortalized and transformed prostate cells. Specifically, it confers resistance to typically pro-oxidant agents, such as cisplatin (CDDP) and doxorubicin (Doxo), and to taxanes. USP2a overexpression protects from drug-induced oxidative stress by reducing reactive oxygen species (ROS) production and stabilizing the mitochondrial membrane potential (ΔΨ), thus impairing downstream p38 activation and triggering of apoptosis. The molecular mediator of the USP2a protective function is the glutathione (GSH). Through miR-34b/c-driven c-Myc regulation, USP2a increases intracellular GSH content, thus interfering with the oxidative cascade triggered by chemotherapeutic agents. In light of these findings, targeting Myc and/or miR-34b/c might revert chemo-resistance.

ATM kinase enables the functional axis of YAP, PML and p53 to ameliorate loss of Werner protein-mediated oncogenic senescence.

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.

miR-204 targets Bcl-2 expression and enhances responsiveness of gastric cancer.

Micro RNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Here, we aim to identify miRs whose deregulated expression leads to the activation of oncogenic pathways in human gastric cancers (GCs). Thirty nine out of 123 tumoral and matched uninvolved peritumoral gastric specimens from three independent European subsets of patients were analyzed for the expression of 851 human miRs using Agilent Platform. The remaining 84 samples were used to validate miRs differentially expressed between tumoral and matched peritumoral specimens by qPCR. miR-204 falls into a group of eight miRs differentially expressed between tumoral and peritumoral samples. Downregulation of miR-204 has prognostic value and correlates with increased staining of Bcl-2 protein in tumoral specimens. Ectopic expression of miR-204 inhibited colony forming ability, migration and tumor engraftment of GC cells. miR-204 targeted Bcl-2 messenger RNA and increased responsiveness of GC cells to 5-fluorouracil and oxaliplatin treatment. Ectopic expression of Bcl-2 protein counteracted miR-204 pro-apoptotic activity in response to 5-fluorouracil. Altogether, these findings suggest that modulation of aberrant expression of miR-204, which in turn releases oncogenic Bcl-2 protein activity might hold promise for preventive and therapeutic strategies of GC.

A novel δ-hemolysis screening method for detecting heteroresistant vancomycin-intermediate Staphylococcus aureus and vancomycin-intermediate S. aureus.

We assessed a new screening method, based on δ-hemolysin production in the presence of 6 mg/liter vancomycin, to distinguish heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) from vancomycin-susceptible S. aureus (VSSA). On 37 clinical methicillin-resistant S. aureus (MRSA) isolates, hVISA and VISA displayed no δ-hemolysis whereas VSSA displayed strong δ-hemolysis, showing 91.6% sensitivity. These data, supported by real-time reverse transcription PCR (real-time RT-PCR) highlighting an hld downregulation, i.e., VSSA>hVISA>VISA, define this new assay as a valid screening method.

Melatonin triggers p53Ser phosphorylation and prevents DNA damage accumulation.

Several epidemiological studies have shown that high levels of melatonin, an indolic hormone secreted mainly by the pineal gland, reduce the risks of developing cancer, thus suggesting that melatonin triggers the activation of tumor-suppressor pathways that lead to the prevention of malignant transformation. This paper illustrates that melatonin induces phosphorylation of p53 at Ser-15 inhibiting cell proliferation and preventing DNA damage accumulation of both normal and transformed cells. This activity requires p53 and promyelocytic leukemia (PML) expression and efficient phosphorylation of p53 at Ser-15 residue. Melatonin-induced p53 phosphorylation at Ser-15 residue does not require ataxia telangiectasia-mutated activity, whereas it is severely impaired upon chemical inhibition of p38 mitogen-activated protein kinase activity. By and large, these findings imply that the activation of the p53 tumor-suppressor pathway is a critical mediator of melatonin and its anticancer effects. Therefore, it provides molecular insights into increasing observational evidence for the role that melatonin has in cancer prevention.