CARATTERIZZAZIONE BIOMOLECOLARE DEL CARCINOMA DELLA MAMMELLA

Il carcinoma della mammella rimane ad oggi la principale causa di morte per cancro nelle donne nonostante i notevoli progressi ottenuti sia nella diagnosi che nella terapia. Nonostante lo studio dei profili di espressione genica, che ha contribuito ad identificare le alterazioni molecolari caratteristiche dei tumori umani, abbia fornito la maggior parte dei nuovi bio-marcatori con possibili applicazioni nell’ambito diagnostico, di sviluppo di nuovi farmaci e di nuove terapie mirate, non ha ancora elucidato in modo esaustivo tutti i meccanismi all’origine dei tumori, incluso il tumore della mammella. Questo suggerisce come il processo di tumorigenesi possa avvenire attraverso meccanismi nuovi non ancora del tutto caratterizzati. Il tumore della mammella è una malattia eterogenea caratterizzata da un ampio spettro di variabilità cliniche, patologiche e molecolari che può rendere conto delle diverse risposte alle terapie. Per molti anni la pratica clinica è stata guidata dai risultati delle metanalisi e da una classificazione esclusivamente morfologica del carcinoma della mammella. Le pazienti affette da carcinoma mammario HER2 positivo hanno in genere una peggiore prognosi e un andamento della malattia maggiormente aggressivo rispetto alle pazienti i cui tumori non esprimono la proteina di membrana. Attualmente il trattamento con trastuzumab, in associazione con la chemioterapia, rappresenta il trattamento standard di prima linea nelle pazienti con carcinoma mammario metastatico che iperesprime HER2. Tuttavia il 40% delle pazienti con diagnosi di tumore metastatico HER2 positivo, trattate con trastuzumab, non presenta alcun beneficio per lo sviluppo di una resistenza primaria o secondaria al farmaco. Lapatinib in associazione alla capecitabina rappresenterebbe una valida opzione terapeutica nel caso di resistenza a trastuzumab. Ad oggi sono stati individuati diversi determinanti molecolari che sembrano essere implicati nei meccanismi di resistenza al trastuzumab, sebbene nessuno sia stato ancora validato nella pratica clinica. Analisi precliniche e retrospettive hanno dimostrato come la forma recettoriale tronca di HER2, la perdita di espressione di PTEN e lo stato mutazionale di PI3K/AKT risultino essere associate ad una resistenza al trastuzumab. Ricerche di “gene espression profiling” hanno permesso di individuare alcuni sottogruppi molecolari di carcinoma della mammella con implicazioni clinico-prognostiche e terapeutiche. In particolari i tumori tripli negativi rappresentano un tipo particolarmente aggressivo di carcinoma della mammella, tipicamente caratterizzato da una prognosi infausta. Queste neoplasie rappresentano il 15-20% di tutti i tumori della mammella e si distinguono in quanto a differenza delle forme più comuni non esprimono il recettore per gli estrogeni, per il progesterone e non presentano amplificazione del gene codificante per il fattore di crescita epidermico. La loro crescita è pertanto guidata da meccanismi molecolari distinti e per questo sono resistenti ai trattamenti standard. Questi tumori sono caratterizzati da un’estrema variabilità genotipica e presentano un carattere fortemente evolutivo legato principalmente ad una forte instabilità del loro genoma. L’unica arma oggi a disposizione per questo tipo di tumori rimane la chemioterapia. Numerosi target terapeutici stanno emergendo come potenziali opzioni nel trattamento di questo gruppo di neoplasie fra cui inibitori di EGFFR, PARP-inibitori, inibitori di c-KIT ed inibitori della via di segnale PI3K/AKT. Recentemente una nuova classe di molecole di RNA non codificanti, noti come microRNA (miRNA), capaci di regolare l’espressione genica attraverso il legame con sequenze regolatorie (3’UTR) su mRNA, è stata associata a diverse malattie umane incluso il tumore della mammella. Un crescente numero di evidenze sperimentali ha mostrato come i miRNA abbiano un ruolo causale nel processo di tumorigenesi, agendo come una nuova classe di oncogeni od oncosoppressori. Dopo le iniziali osservazioni dell’associazione fra miRNA e tumori umani, questo campo di ricerca sta crescendo incredibilmente. Attualmente si sta cercando di elucidare i meccanismi molecolari in cui i miRNA sono coinvolti e di validare il loro potenziale ruolo sia come bio-marcatori che come strumenti o bersagli di terapie innovative. Tra i miRNA potenzialmente rilevanti nel carcinoma della mammella è stato identificato miR-205 capace di interferire con la proliferazione cellulare mediata dai recettori della famiglia HER ed in grado di aumentare la responsività al trattamento con inibitori tirosin-chinasici (TKI). I miRNA potrebbero fornire informazioni biologiche necessarie per spiegare il comportamento dei tumori triplo negativi e rappresentare un possibile strumento o bersaglio per la terapia specifica. Pertanto la caratterizzazione genotipica dei tumori della mammella è indispensabile per comprendere i meccanismi biologici che ne guidano la crescita e che determinano la risposta ai trattamenti. Tale procedura si rende ancora più necessaria per i tumori tripli negativi per i quali gli approcci terapeutici standard si rivelano spesso inefficaci e altre strategie devono essere sviluppate e sperimentate

The metastatic potential of grade I breast carcinoma of no special type: A deep insight into putative molecular mechanisms

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Pro-apoptotic and size-reducing effects of protein corona-modulating nano-architectures enclosing platinum prodrug in in vivo oral carcinoma

: The selective and localized delivery of active agents to neoplasms is crucial to enhance the chemotherapeutic efficacy while reducing the associated side effects. The encapsulation of chemotherapeutics in nanoparticles decorated with targeting agents is a strategy of special interest to improve drug delivery. However, serum protein adsorption often compromises the in vivo efficiency of targeting agents, leading to protein corona formation that interferes with the targeting process. Here, the enhanced efficacy of hybrid nano-architectures enclosing a platinum prodrug and decorated with a customized peptide (NAs-cisPt-Tf2) is demonstrated by employing alternative in vivo models of oral carcinoma. The peptide binds to transferrin and modulates the protein corona formation on NAs-cisPt-Tf2, supporting the identification of its receptor. Optimized chorioallantoic membrane cancer models (CAMs) enabled a thorough assessment of the tumor-suppressing effect of NAs-cisPt-Tf2 as well as the quantitative evaluation of angiogenesis and cell cycle associated mechanisms. The treatment strategy resulted in a significant tumor volume reduction coupled with anti-angiogenic and pro-apoptotic effects inferred from the downregulation of the vascular endothelial growth factor gene and increased expression of cleaved caspase-3. Overall, this study provides a potentially effective tumor-targeted approach for a non-invasive management of oral carcinoma

CXCL12/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axonterminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by -latrotoxin. CXCL12 acts via binding to the neuronal CXCR4 receptor. A CXCL12-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration invivo. Recombinant CXCL12 invivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons invitro. These findings indicate that the CXCL12-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

Mouse mammary tumour virus-like env nucleotide and p14 signal peptide are present in feline mammary carcinomas, but not in neoplastic or dysplastic canine mammary lesions

Mouse mammary tumour virus-like (MMTV-like) is suspected to be involved in human breast cancer and it has been hypothesized that companion animals might have a role in viral transmission. The aim of our study was to investigate the presence of MMTV-like nucleotide sequences and viral protein in a larger number of feline (FMCs) and canine mammary carcinomas (CMCs) by nested PCR and immunohistochemistry. Results showed that the presence of MMTV-like env sequence in FMCs was 7% (6/86), while all the CMCs and canine dysplastic lesions scored negative. All PCR-positive FMCs scored positive for the MMTV p14 signal peptide of the envelope precursor protein of the virus. In contrast, all PCR-negative FMCs and canine mammary lesions were also negative for immunohistochemistry analysis. Canine and feline normal mammary gland tissues scored negative for both PCR and MMTV-p14 protein. Multiple nucleotide alignment of MMTV-like env gene sequences isolated from cat showed 97% and 99% similarity with HMTV and MMTV, respectively, while the others two presented some polimorphisms. Particularly the sequences of one of these two tumors showed a polymorphism (c.7575 A> G), that causes a previously unreported amino acid substitution (Thr > Ala). In conclusion, the results of our study showed the presence of MMTV-like sequences and viral protein in some FMCs. Further studies are needed to understand whether this virus does play a role in the development of FMCs, if MMTV-like is an exogenous virus as these data suggest and, in such a case, how and from whom this virus was acquired

Molecular changes underlying decay of sensory responses and enhanced seizure propensity in peritumoral neurons

Background: Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target. Methods: We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection we analysed layer II-III pyramidal neurons molecular profile and with Local Field Potentials (LFP) recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice. Results: We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP-25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (i.e., AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signalling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in two mouse models of tumor-bearing mice. Conclusions: These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients' quality of life

Hepatitis C Virus Infection and Human Pancreatic β-Cell Dysfunction

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Micro‑RNA‑21 (biomarker) and global longitudinal strain (functional marker) in detection of myocardial fibrotic burden in severe aortic valve stenosis: a pilot study

Aims: Myocardial fibrosis (MF) is a deleterious consequence of aortic valve stenosis (AVS). Global longitudinal strain (GLS) is a novel left ventricular (LV) functional parameter potentially useful to non-invasively estimate MF. MicroRNAs (miRNAs) are non-coding small ribonucleic acids (RNA) modulating genes function, mainly through RNA degradation. miRNA-21 is a biomarker associated with MF in pressure overload. The aim of the present study was to find an integrated algorithm for detection of MF using a combined approach with both bio- and functional markers. Methods: Thirty-six patients (75.2±8 y.o.; 63% Female) with severe AVS and preserved LV ejection fraction (EF), candidate to surgical aortic valve replacement (sAVR) were enrolled. Clinical, bio-humoral evaluation (including plasmatic miRNA-21 collected using specific tubes, PAXgene, for stabilization of peripheral RNA) and a complete echocardiographic study, including GLS and septal strain, were performed before sAVR. Twenty-eight of those patients underwent sAVR and, in 23 of them, an inter-ventricular septum biopsy was performed. Tissues were fixed in formalin and embedded in paraffin. Sections were stained with Hematoxylin and Eosin for histological evaluation and with histochemical Masson trichrome for collagen fibers. The different components were calculated and expressed as micrometers2. To evaluate tissue miRNA components, sections2-μm thick were cut using a microtome blade for each slide. Regression analysis was performed to test association between dependent variable and various predictors included in the model. Results: Despite a preserved EF (66±11%), patients presented altered myocardial deformation parameters (GLS -14,02±3.8%; septal longitudinal strain, SSL -9.63±2.9%; septal longitudinal strain rate, SL-Sr -0.58±0.17 1/s; Septal Longitudinal early-diastolic strain rate, SL-SrE 0.62±0.32 1/s). The extent of MF showed an inverse association with both GLS and septal longitudinal deformation indices (GLS: R2=0.30; p=0.02; SSL: R2=0.36; p=0.01; SL-Sr: R2=0.39; p<0.001; SL-SrE: R2=0.35; p=0.001). miRNA-21 was mainly expressed in fibrous tissue (p<0.0001). A significant association between MF and plasmatic miRNA-21, alone and weighted for measures of structural (LVMi R2=0.50; p=0.0005) and functional (SSL R2=0.35; p=0.006) remodeling, was found. Conclusions: In AVS, MF is associated with alterations of regional and global strain. Plasmatic miRNA-21 is directly related to MF and associated with LV structural and functional impairment. © 2016 The Author(s)

Cancer astrocytes have a more conserved molecular status in long recurrence free survival (RFS) IDH1 wild-type glioblastoma patients: New emerging cancer players

Glioblastoma is a devastating disease that despite all the information gathered so far, its optimal management remains elusive due to the absence of validated targets from clinical studies. A better clarification of the molecular mechanisms is needed. In this study, having access to IDH1 wild-type glioblastoma of patients with exceptionally long recurrence free survival (RFS), we decided to compare their mutational and gene expression profile to groups of IDH1 wild-type glioblastoma of patients with shorter RFS, by using NGS technology. The exome analysis revealed that Long-RFS tumors have a lower mutational rate compared to the other groups. A total of 158 genes were found differentially expressed among the groups, 112 of which distinguished the two RFS extreme groups. Overall, the exome data suggests that shorter RFS tumors could be, chronologically, in a more advanced state in the muli-step tumor process of sequential accumulation of mutations. New players in this kind of cancer emerge from the analysis, confirmed at the RNA/DNA level, identifying, therefore, possible oncodrivers or tumor suppressor genes

Presence of a mouse mammary tumour virus-like in feline lymphomas: a preliminary study

Abstract: The mouse mammary tumour virus (MMTV) is implicated in the aetiology of murine mammary carcinomas and a variant of it, the type B leukemogenic virus, can cause murine thymic lymphomas. Interestingly, a MMTV-like virus is suspected to be involved in human breast cancer and feline mammary carcinomas. However, to date, no cases of MMTV-like sequence amplifications have been described in lymphoid neoplasms in veterinary literature. The aim of this study was to investigate the presence of env nucleotide sequences and protein 14 (p14) of a MMTV-like virus in fifty-three feline lymphoma samples. Our results show that MMTV-like sequences were detected in 5/53 tumours (9.4%): three gastrointestinal lymphomas (one B-type diffuse large, one B-type small non-cleaved, and one T-type diffuse mixed lymphoma); and two nasal lymphomas (one B-type diffuse small cleaved lymphoma and one B-type diffuse mixed lymphoma). P14 expression was detected in the cytoplasm, and rarely in nuclei, exclusively of neoplastic cells from PCR-positive tumours. The correlation between the presence of the MMTV-env like sequences (MMTVels) and p14 antigen was statistically significant in nasal lymphomas. All cats with MMTVels-positive lymphoma had a history of contact with the outdoor environment and/or catteries, and two deceased subjects shared their environment with cats that also died of lymphoma. In conclusion, this study succeeds in demonstrating the presence of MMTVels and p14 in feline lymphomas. The characterization of the immunophenotype of MMTVels-positive lymphomas could contribute to the understanding of a possible role of a MMTV-like virus in feline tumour aetiology. The significant association between the presence of the viral sequences in lymphoid tumours and their nasal localization, together with the data collected through supplementary anamnesis, should be further analysed in order to understand the epidemiology of the virus