MicroRNA profiling in women with migraine: effects of CGRP-targeting treatment.

BACKGROUND: Migraine lacks biomarkers that can trace the biological pathways of the disease and predict the effectiveness of treatments. Monoclonal antibodies targeting calcitonin gene-related peptide pathway - including erenumab - offer the opportunity of investigating potential migraine biomarkers due to their specific mechanism of action in preventing both episodic (EM) and chronic (CM) migraine. Our study aims at evaluating the expression levels of circulating microRNAs (miRNAs) according to migraine type, before and after treatment with erenumab and based on treatment response, in order to identify miRNAs with potential role as epigenetic biomarkers. METHODS: The study included women aged 25-50 years with EM or CM treated with erenumab according to clinical indications. MiRNAs expression levels were assessed before (baseline) and after a 16-week treatment with erenumab, 140 mg every four weeks (post-treatment). An extensive miRNAs profiling was performed by qRT-PCR in small, pooled groups of ≤ 8 women each, classified according to migraine frequency (EM and CM) and the degree of response to erenumab. The expression levels of selected miRNAs were also validated using single miRNA assays in each woman with EM and CM. RESULTS: During the study, 36 women with migraine (19 with EM and 17 with CM) out of 40 who were initially screened, performed the assessment of miRNA expression at baseline and post-treatment, Erenumab treatment significantly improved migraine burden in both EM and CM. MiRNA profiling revealed differential expression levels of a wide set of miRNAs (hsa-let-7d-3p, hsa-miR-106b-3p, hsa-miR-122-5p, hsa-miR-143-3p, hsa-miR-144-3p, hsa-miR-16-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-25-3p, hsa-miR-29b-2-5p, hsa-miR-326, miR-363-3p, hsa-miR-424-5p, hsa-miR-485-3p, hsa-miR-532-5p, hsa-miR-543, hsa-miR-629-5p, hsa-miR-660-5p, hsa-miR-92a-3p) depending on treatment response. Among them, single miRNA assays confirmed the progressive decrease of hsa-miR-143-3p expression levels in relation to increasing response to erenumab in women with EM (7 with low, 6 with medium, and 6 with high response; p = 0.02). Additionally, single assays showed higher hsa-miR-34a-5p and hsa-miR-382-5p expression levels at baseline in women with CM compared with those with EM (p = 0.0002 and p = 0.0007, respectively), as well as their expression level decrease in women with CM from baseline to follow-up (p = 0.04 and p = 0.02, respectively). CONCLUSIONS: Our study suggests that targeting the CGRP pathway in migraine changes the expression levels of certain miRNAs. These miRNA levels are linked to the levels of response to CGRP receptor blockage. Future research challenges include assigning specific functions to the modulated miRNAs to unravel pathways modulated by the disease and the treatment. TRIAL REGISTRATION: The study was registered in clinicaltrials.gov with code NCT04659226 and in the Novartis database with code CAMG334AIT05T.

Classification of tumor types using XGBoost machine learning model: a vector space transformation of genomic alterations.

BACKGROUND: Machine learning (ML) represents a powerful tool to capture relationships between molecular alterations and cancer types and to extract biological information. Here, we developed a plain ML model aimed at distinguishing cancer types based on genetic lesions, providing an additional tool to improve cancer diagnosis, particularly for tumors of unknown origin. METHODS: TCGA data from 9,927 samples spanning 32 different cancer types were downloaded from cBioportal. A vector space model type data transformation technique was designed to build consistently homogeneous new datasets containing, as predictive features, calls for somatic point mutations and copy number variations at chromosome arm-level, thus allowing the use of the XGBoost classifier models. Considering the imbalance in the dataset, due to large difference in the number of cases for each tumor, two preprocessing strategies were considered: i) setting a percentage cut-off threshold to remove less represented cancer types, ii) dividing cancer types into different groups based on biological criteria and training a specific XGBoost model for each of them. The performance of all trained models was mainly assessed by the out-of-sample balanced accuracy (BACC) and the AUC scores. RESULTS: The XGBoost classifier achieved the best performance (BACC 77%; AUC 97%) on a dataset containing the 10 most represented tumor types. Moreover, dividing the 18 most represented cancers into three different groups (endocrine-related carcinomas, other carcinomas and other cancers),such analysis models achieved 78%, 71% and 86% BACC, respectively, with AUC scores greater than 96%. In addition, the model capable of linking each group to a specific cancer type reached 81% BACC and 94% AUC. Overall, the diagnostic potential of our model was comparable/higher with respect to others already described in literature and based on similar molecular data and ML approaches. CONCLUSIONS: A boosted ML approach able to accurately discriminate different cancer types was developed. The methodology builds datasets simpler and more interpretable than the original data, while keeping enough information to accurately train standard ML models without resorting to sophisticated Deep Learning architectures. In combination with histopathological examinations, this approach could improve cancer diagnosis by using specific DNA alterations, processed by a replicable and easy-to-use automated technology. The study encourages new investigations which could further increase the classifier's performance, for example by considering more features and dividing tumors into their main molecular subtypes.

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

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

Doxorubicin-Induced TrkAIII Activation: A Selection Mechanism for Resistant Dormant Neuroblastoma Cells.

Patients with advanced neuroblastoma (NB) receive multimodal clinical therapy, including the potent anthracycline chemotherapy drug doxorubicin (Dox). The acquisition of Dox resistance, however, is a major barrier to a sustained response and leads to a poor prognosis in advanced disease states, reinforcing the need to identify and inhibit Dox resistance mechanisms. In this context, we report on the identification and inhibition of a novel Dox resistance mechanism. This mechanism is characterized by the Dox-induced activation of the oncogenic TrkAIII alternative splice variant, resulting in increased Dox resistance, and is blocked by lestaurtinib, entrectinib, and crizotinib tyrosine kinase and LY294002 IP3-K inhibitors. Using time lapse live cell imaging, conventional and co-immunoprecipitation Western blots, RT-PCR, and inhibitor studies, we report that the Dox-induced TrkAIII activation correlates with proliferation inhibition and is CDK1- and Ca2+-uniporter-independent. It is mediated by ryanodine receptors; involves Ca2+-dependent interactions between TrkAIII, calmodulin and Hsp90; requires oxygen and oxidation; occurs within assembled ERGICs; and does not occur with fully spliced TrkA. The inhibitory effects of lestaurtinib, entrectinib, crizotinib, and LY294002 on the Dox-induced TrkAIII and Akt phosphorylation and resistance confirm roles for TrkAIII and IP3-K consistent with Dox-induced, TrkAIII-mediated pro-survival IP3K/Akt signaling. This mechanism has the potential to select resistant dormant TrkAIII-expressing NB cells, supporting the use of Trk inhibitors during Dox therapy in TrkAIII-expressing NBs.

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

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

Multidisciplinary Treatment, Including Locoregional Chemotherapy, for Merkel-Polyomavirus-Positive Merkel Cell Carcinomas: Perspectives for Patients Exhibiting Oncogenic Alternative Δ exon 6-7 TrkAIII Splicing of Neurotrophin Receptor Tropomyosin-Related Kinase A.

Merkel cell carcinomas (MCCs) are rare, aggressive, cutaneous neuroendocrine tumours, approximately 80% of which are caused by the genomic integration of Merkel cell polyomavirus (MCPyV). MCPyV-positive MCCs carry poor prognosis in approximately 70% of cases, highlighting the need for greater understanding of the oncogenic mechanisms involved in pathogenesis, progression and post-therapeutic relapse, and translation into novel therapeutic strategies. In a previous pilot study, we reported a potential relationship between MCPyV gene expression and oncogenic alternative Δ exon 6-7 TrkAIII splicing in formalin-fixed paraffin-embedded (FFPE) MCC tissues from a 12-patient cohort of >90% MCPyV-positive MCCs, diagnosed at San Salvatore Hospital, L'Aquila, Italy, characterising a new MCC subgroup and unveiling a novel potential MCPyV oncogenic mechanism and therapeutic target. This, however, could not be fully verified due to poor RNA quality and difficulty in protein extraction from FFPE tissues. Here, therefore, we extend our previous observations to confirm the relationship between MCPyV and oncogenic alternative Δ exon 6-7 TrkAIII splicing in fresh, nonfixed, MCPyV-positive MCC metastasis by detecting sequence-verified RT-PCR products, including full-length Δ exon 6-7 TrkAIII, and by Western blot detection of a 100 kDa TrkA protein isoform of identical size to 100 kDa Δ exon 6-7 TrkAIII expressed by stable transfected SH-SY5Y cells. We also report that in three MCC patients submitted for multidisciplinary treatment, including locoregional chemotherapy, MCPyV large T-antigen mRNA expression, Δ exon 6-7 TrkAIII mRNA expression and intracellular indirect immunofluorescence (IF) TrkA and phosphorylation protein isoform(s) immunoreactivity in FFPE tissues were not reduced in postchemotherapeutic-relapsed MCCs compared to pretherapeutic MCCs, extending the possible roles of this novel potential MCPyV oncogenic mechanism from MCC pathogenesis to post-therapeutic relapse and progression. Detection of alternative Δ exon 6-7 TrkAIII splicing in MCC, therefore, not only characterises a new MCPyV-positive MCC subgroup and unveils a novel potential MCPyV oncogenic mechanism but also identifies patients who may benefit from inhibitors of MCPyV T-antigen and/or TrkAIII expression or clinically approved Trk kinase inhibitors such as larotrectinib or entrectinib, which are known to inhibit activated TrkA oncogenes and to elicit durable responses in TrkA-fusion oncogene-driven cancers, supporting the call for a large-scale multicentre clinical study.

Insight into genetic susceptibility to male breast cancer by multigene panel testing: Results from a multicenter study in Italy.

Breast cancer (BC) in men is rare and genetic predisposition is likely to play a relevant role in its etiology. Inherited mutations in BRCA1/2 account for about 13% of all cases and additional genes that may contribute to the missing heritability need to be investigated. In our study, a well-characterized series of 523 male BC (MBC) patients from the Italian multicenter study on MBC, enriched for non-BRCA1/2 MBC cases, was screened by a multigene custom panel of 50 cancer-associated genes. The main clinical-pathologic characteristics of MBC in pathogenic variant carriers and non-carriers were also compared. BRCA1/2 pathogenic variants were detected in twenty patients, thus, a total of 503 non-BRCA1/2 MBC patients were examined in our study. Twenty-seven of the non-BRCA1/2 MBC patients were carriers of germline pathogenic variants in other genes, including two APC p.Ile1307Lys variant carriers and one MUTYH biallelic variant carrier. PALB2 was the most frequently altered gene (1.2%) and PALB2 pathogenic variants were significantly associated with high risk of MBC. Non-BRCA1/2 pathogenic variant carriers were more likely to have personal (p = 0.0005) and family (p = 0.007) history of cancer. Results of our study support a central role of PALB2 in MBC susceptibility and show a low impact of CHEK2 on MBC predisposition in the Italian population. Overall, our data indicate that a multigene testing approach may benefit from appropriately selected patients with implications for clinical management and counseling of MBC patients and their family members.

The antiquity of hydrocephalus: the first full palaeo-neuropathological description.

The Pathology Museum of the University of Florence houses a rich collection of anatomical specimens and over a hundred waxworks portraying pathological conditions occurring in the nineteenth century, when the museum was established. Clinical and autopsy findings of these cases can still be retrieved from the original museum catalogue, offering a rare opportunity for retrospective palaeo-pathological diagnostics. We present a historical case of severe hydrocephalus backed by modern-day anthropological, radiological and molecular analyses conducted on the skeleton of an 18-month-old male infant deceased in 1831. Luigi Calamai (1796-1851), a wax craftsman of La Specola workshop in Florence, was commissioned to create a life-sized wax model of the child's head, neck and upper thorax. This artwork allows us to appreciate the cranial and facial alterations determined by 30 lb of cerebrospinal fluid (CSF) accumulated within the cerebral ventricular system. Based on the autopsy report, gross malformations of the neural tube, tumours and haemorrhage could be excluded. A molecular approach proved helpful in confirming sex. We present this case as the so-far most compelling case of hydrocephalus in palaeo-pathological research.

Whole-exome sequencing and targeted gene sequencing provide insights into the role of PALB2 as a male breast cancer susceptibility gene.

BACKGROUND: Male breast cancer (MBC) is a rare disease whose etiology appears to be largely associated with genetic factors. BRCA1 and BRCA2 mutations account for about 10% of all MBC cases. Thus, a fraction of MBC cases are expected to be due to genetic factors not yet identified. To further explain the genetic susceptibility for MBC, whole-exome sequencing (WES) and targeted gene sequencing were applied to high-risk, BRCA1/2 mutation-negative MBC cases. METHODS: Germ-line DNA of 1 male and 2 female BRCA1/2 mutation-negative breast cancer (BC) cases from a pedigree showing a first-degree family history of MBC was analyzed with WES. Targeted gene sequencing for the validation of WES results was performed for 48 high-risk, BRCA1/2 mutation-negative MBC cases from an Italian multicenter study of MBC. A case-control series of 433 BRCA1/2 mutation-negative MBC and female breast cancer (FBC) cases and 849 male and female controls was included in the study. RESULTS: WES in the family identified the partner and localizer of BRCA2 (PALB2) c.419delA truncating mutation carried by the proband, her father, and her paternal uncle (all affected with BC) and the N-acetyltransferase 1 (NAT1) c.97C>T nonsense mutation carried by the proband's maternal aunt. Targeted PALB2 sequencing detected the c.1984A>T nonsense mutation in 1 of the 48 BRCA1/2 mutation-negative MBC cases. NAT1 c.97C>T was not found in the case-control series. CONCLUSIONS: These results add strength to the evidence showing that PALB2 is involved in BC risk for both sexes and indicate that consideration should be given to clinical testing of PALB2 for BRCA1/2 mutation-negative families with multiple MBC and FBC cases. Cancer 2017;123:210-218. © 2016 American Cancer Society.

EMSY copy number variation in male breast cancers characterized for BRCA1 and BRCA2 mutations.

PURPOSE: Male breast cancer (MBC) is a rare disease that shares some similarities with female breast cancer (FBC). Like FBC, genetic susceptibility to MBC can be referred to mutations in BRCA1 and, particularly, BRCA2 genes. However, only about 10 % of MBCs are caused by BRCA1/2 germ-line mutations, while the largest part are sporadic cancers and may derive from somatic alterations. EMSY, a BRCA2 inactivating gene, emerged as a candidate gene involved in the pathogenesis of sporadic FBC, and its amplification was suggested to be the somatic counterpart of BRCA2 mutations. Considering the relevant role of BRCA2 in MBC, we aimed at investigating the role of EMSY gene copy number variations in male breast tumors. METHODS: EMSY copy number variations were analyzed by quantitative real-time PCR with TaqMan probes in a selected series of 75 MBCs, characterized for BRCA1/2 mutations. RESULTS: We reported EMSY amplification in 34.7 % of MBCs. A significant association emerged between EMSY amplification and BRCA1/2 mutations (p = 0.03). We identified two amplification subgroups characterized by low and high amplification levels, with BRCA2-related tumors mostly showing low EMSY amplification. CONCLUSIONS: Our results show a high frequency of EMSY amplification in MBC, thus pointing to a role of EMSY in the pathogenesis of this disease. EMSY amplification may be a new feature that might uncover underlying molecular pathways of MBCs and may allow for the identification of MBC subgroups with potential clinical implication for targeted therapeutic approaches.

General Direct Anticancer Effects of Deer Growing Antler Extract in Several Tumour Cell Lines, and Immune System-Mediated Effects in Xenograft Glioblastoma.

Deer antlers are the fastest growing tissue. Because they are based on proto-oncogenes, to avoid the risk of cancer, antlers evolved strong anticancer mechanisms, and thus their extract (DVA) is effective also against the few human tumours studied so far. We assessed whether DVA is a general anticancer compound by testing the direct effects in cells of different tumours: glioblastoma (GBM; lines U87MG and U251), colorectal (CRC; lines DLD-1, HT-29, SW480, and SW620), breast cancer (BRCA; lines MCF7, SKBR3, and PA00), and leukaemia (THP-1). DVA reduced the viability of tumours but not healthy cells (NHC; lines 293T and HaCaT). Mobility decreased at least for the longest test (72 h). Intraperitoneal/oral 200 mg DVA/kg administration in GBM xenograft mice for 28 d reduced tumour weight by 66.3% and 61.4% respectively, and it also reduced spleen weight (43.8%). In addition, tumours treated with DVA showed symptoms of liquefactive necrosis. Serum cytokines showed DVA up-regulated factors related to tumour fighting and down-regulated those related to inducing immune tolerance to the tumour. DVA shows general anticancer effects in the lines tested and, in GBM mice, also strong indirect effects apparently mediated by the immune system. DVA may contain a future anticancer medicine without secondary effects.

A method to comprehensively identify germline SNVs, INDELs and CNVs from whole exome sequencing data of BRCA1/2 negative breast cancer patients.

In the rapidly evolving field of genomics, understanding the genetic basis of complex diseases like breast cancer, particularly its familial/hereditary forms, is crucial. Current methods often examine genomic variants-such as Single Nucleotide Variants (SNVs), insertions/deletions (Indels), and Copy Number Variations (CNVs)-separately, lacking an integrated approach. Here, we introduced a robust, flexible methodology for a comprehensive variants' analysis using Whole Exome Sequencing (WES) data. Our approach uniquely combines meticulous validation with an effective variant filtering strategy. By reanalyzing two germline WES datasets from BRCA1/2 negative breast cancer patients, we demonstrated our tool's efficiency and adaptability, uncovering both known and novel variants. This contributed new insights for potential diagnostic, preventive, and therapeutic strategies. Our method stands out for its comprehensive inclusion of key genomic variants in a unified analysis, and its practical resolution of technical challenges, offering a pioneering solution in genomic research. This tool presents a breakthrough in providing detailed insights into the genetic alterations in genomes, with significant implications for understanding and managing hereditary breast cancer.

A Study of Alternative TrkA Splicing Identifies TrkAIII as a Novel Potentially Targetable Participant in PitNET Progression.

Pituitary neuroendocrine tumors (PitNETs) are generally benign but comprise an aggressive, invasive, therapy-resistant, metastatic subset, underpinning a need for novel therapeutic targets. PitNETs exhibit low mutation rates but are associated with conditions linked to alternative splicing, an alternative oncogene pathway activation mechanism. PitNETs express the neurotrophin receptor TrkA, which exhibits oncogenic alternative TrkAIII splicing in other neuroendocrine tumors. We, therefore, assessed whether TrkAIII splicing represents a potential oncogenic participant in PitNETs. TrkAIII splicing was RT-PCR assessed in 53 PitNETs and TrkA isoform(s) expression and activation were assessed by confocal immunofluorescence. TrkAIII splicing was also compared to HIF1α, HIF2α, SF3B1, SRSF2, U2AF1, and JCPyV large T antigen mRNA expression, Xbp1 splicing, and SF3B1 mutation. TrkAIII splicing was detected in all invasive and most non-invasive PitNETs and was significantly elevated in invasive cases. In PitNET lineages, TrkAIII splicing was significantly elevated in invasive PIT1 PitNETs and high in invasive and non-invasive SF1 and TPIT lineages. Immunoreactivity consistent with TrkAIII activation characterized PitNET expressing TrkAIII mRNA, and invasive Pit1 PitNETs exhibited elevated HIF2α expression. TrkAIII splicing did not associate with SF3B1 mutations, altered SF3B1, SRSF2, and U2AF1 or JCPyV large T antigen expression, or Xbp1 splicing. Therefore, TrkAIII splicing is common in PitNETs, is elevated in invasive, especially PIT1 tumors, can result in intracellular TrkAIII activation, and may involve hypoxia. The data support a role for TrkAIII splicing in PitNET pathogenesis and progression and identify TrkAIII as a novel potential target in refractory PitNETs.

Mitotic phosphorylation of Tau/MAPT modulates cell cycle progression in prostate cancer cells.

PURPOSE: Tau/MAPT (microtubule associated protein tau) protein is actively studied for the pathologic consequences of its aberrant proteostasis in central nervous system leading to neurodegenerative diseases. Besides its ability to generate insoluble toxic oligomers, Tau homeostasis has attracted attention for its involvement in the formation of the mitotic spindle. This evidence, in association with the description of Tau expression in extra-neuronal tissues, and mainly in cancer tissues, constitutes the rationale for a more in-depth investigation of Tau role also in neoplastic diseases. METHODS: In our study, we investigated the expression of phosphorylated Tau in prostate cancer cell lines with particular focus on the residue Thr231 present in microtubule binding domain. RESULTS: The analysis of prostate cancer cells synchronized with nocodazole demonstrated that the expression of Tau protein phosphorylated at residue Thr231 is restricted to G2/M cell cycle phase. The phosphorylated form was unable to bind tubulin and it does not localize on mitotic spindle. As demonstrated by the use of specific inhibitors, the phosphorylation status of Tau is under the direct control of cdk5 and PP2A, while cdk1 activation was able to exert an indirect control. These mechanisms were also active in cells treated with docetaxel, where counteracting the expression of the dephosphorylated form, by kinase inhibition or protein silencing, determined resistance to drug toxicity. CONCLUSIONS: We hypothesize that phosphorylation status of Tau is a key marker for G2/M phase in prostate cancer cells and that the forced modulation of Tau phosphorylation can interfere with the capacity of cell to efficiently progress through G2/M phase.

The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma.

Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E)-mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM.

Concurrent RAS and RAS/BRAF V600E Variants in Colorectal Cancer: More Frequent Than Expected? A Case Report.

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.

Role of exosomal microRNAs in cancer therapy and drug resistance mechanisms: focus on hepatocellular carcinoma.

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.

Nutraceutical Preventative and Therapeutic Potential in Neuroblastoma: From Pregnancy to Early Childhood.

Neuroblastoma (NB) is a highly malignant embryonic extracranial solid tumor that arises from sympathoadrenal neuroblasts of neural crest origin. In addition to genetic factors, NB has been linked to maternal exposure to a variety of substances during pregnancy. Recent interest in the potential of nutrients to prevent cancer and reduce malignancy has resulted in the identification of several nutraceuticals including resveratrol, curcumin, and molecular components of garlic, which together with certain vitamins may help to prevent NB development. As NBs arise during fetal development and progress during early childhood, specific NB inhibiting nutraceuticals and vitamins could enhance the preventative influence of maternal nutrition and breast feeding on the development and early progression of NB. In this article, we review NB inhibitory nutraceuticals and vitamins, their mechanisms of action and expound their potential as maternal nutritional supplements to reduce NB development and progression during fetal growth and early childhood, whilst at the same time enhancing maternal, fetal, and infant health.

Anthracycline-Free Neoadjuvant Treatment in Patients with HER2-Positive Breast Cancer: Real-Life Use of Pertuzumab, Trastuzumab and Taxanes Association with an Exploratory Analysis of PIK3CA Mutational Status.

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.

Mechanisms involved in selecting and maintaining neuroblastoma cancer stem cell populations, and perspectives for therapeutic targeting.

Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumours that originate from cells of neural crest (NC) origin and in particular neuroblasts committed to the sympathoadrenal progenitor cell lineage. Therapeutic resistance, post-therapeutic relapse and subsequent metastatic NB progression are driven primarily by cancer stem cell (CSC)-like subpopulations, which through their self-renewing capacity, intermittent and slow cell cycles, drug-resistant and reversibly adaptive plastic phenotypes, represent the most important obstacle to improving therapeutic outcomes in unfavourable NBs. In this review, dedicated to NB CSCs and the prospects for their therapeutic eradication, we initiate with brief descriptions of the unique transient vertebrate embryonic NC structure and salient molecular protagonists involved NC induction, specification, epithelial to mesenchymal transition and migratory behaviour, in order to familiarise the reader with the embryonic cellular and molecular origins and background to NB. We follow this by introducing NB and the potential NC-derived stem/progenitor cell origins of NBs, before providing a comprehensive review of the salient molecules, signalling pathways, mechanisms, tumour microenvironmental and therapeutic conditions involved in promoting, selecting and maintaining NB CSC subpopulations, and that underpin their therapy-resistant, self-renewing metastatic behaviour. Finally, we review potential therapeutic strategies and future prospects for targeting and eradication of these bastions of NB therapeutic resistance, post-therapeutic relapse and metastatic progression.