The role of HOTAIR in the modulation of resistance to anticancer therapy.

Leading anti-tumour therapeutic strategies typically involve surgery and radiotherapy for locally advanced (non-metastatic) cancers, while hormone therapy, chemotherapy, and molecular targeted therapy are the current treatment options for metastatic cancer. Despite the initially high sensitivity rate to anticancer therapies, a large number of patients develop resistance, leading to a poor prognosis. The mechanisms related to drug resistance are highly complex, and long non-coding RNAs appear to play a crucial role in these processes. Among these, the lncRNA homeobox transcript antisense intergenic RNA (HOTAIR), widely implicated in cancer initiation and progression, likewise plays a significant role in anticancer drug resistance. It can modulate cell activities such as proliferation, apoptosis, hypoxia, autophagy, as well as epithelial-mesenchymal transition, thereby contributing to the development of resistant tumour cells. In this manuscript, we describe different mechanisms of antitumor drug resistance in which HOTAIR is involved and suggest its potential as a therapeutic predictive biomarker for the management of cancer patients.

Bispecific aptamer-decorated and light-triggered nanoparticles targeting tumor and stromal cells in breast cancer derived organoids: implications for precision phototherapies.

BACKGROUND: Based on the established role of cancer-stroma cross-talk in tumor growth, progression and chemoresistance, targeting interactions between tumor cells and their stroma provides new therapeutic approaches. Dual-targeted nanotherapeutics selectively acting on both tumor and stromal cells may overcome the limits of tumor cell-targeting single-ligand nanomedicine due to the complexity of the tumor microenvironment. METHODS: Gold-core/silica-shell nanoparticles embedding a water-soluble iridium(III) complex as photosensitizer and luminescent probe (Iren-AuSiO2_COOH) were efficiently decorated with amino-terminated EGFR (CL4) and PDGFRß (Gint4.T) aptamers (Iren-AuSiO2_Aptamer). The targeting specificity, and the synergistic photodynamic and photothermal effects of either single- and dual-aptamer-decorated nanoparticles have been assessed by confocal microscopy and cell viability assays, respectively, on different human cell types including mesenchymal subtype triple-negative breast cancer (MES-TNBC) MDA-MB-231 and BT-549 cell lines (both EGFR and PDGFRß positive), luminal/HER2-positive breast cancer BT-474 and epidermoid carcinoma A431 cells (only EGFR positive) and adipose-derived mesenchymal stromal/stem cells (MSCs) (only PDGFRß positive). Cells lacking expression of both receptors were used as negative controls. To take into account the tumor-stroma interplay, fluorescence imaging and cytotoxicity were evaluated in preclinical three-dimensional (3D) stroma-rich breast cancer models. RESULTS: We show efficient capability of Iren-AuSiO2_Aptamer nanoplatforms to selectively enter into target cells, and kill them, through EGFR and/or PDGFRß recognition. Importantly, by targeting EGFR+ tumor/PDGFRß+ stromal cells in the entire tumor bulk, the dual-aptamer-engineered nanoparticles resulted more effective than unconjugated or single-aptamer-conjugated nanoparticles in either 3D spheroids cocultures of tumor cells and MSCs, and in breast cancer organoids derived from pathologically and molecularly well-characterized tumors. CONCLUSIONS: Our study proposes smart, novel and safe multifunctional nanoplatforms simultaneously addressing cancer-stroma within the tumor microenvironment, which are: (i) actively delivered to the targeted cells through highly specific aptamers; (ii) localized by means of their luminescence, and (iii) activated via minimally invasive light, launching efficient tumor death, thus providing innovative precision therapeutics. Given the unique features, the proposed dual targeted nanoformulations may open a new door to precision cancer treatment.

Endocrine nuclear receptors and long non­coding RNAs reciprocal regulation in cancer (Review).

Nuclear receptors (NRs) are transcriptional regulators involved in different aspects of normal cell physiology. Their deregulation is associated with aberrant expression, gene mutations and/or epigenetic alterations that can be related to the pathogenesis of various human diseases, and especially in cancer. In particular, a complex genomic network involved in the development and progression of NR­mediated cancer has been highlighted. Advanced genomic technologies have made it possible to understand that the expression of any particular NR in a given cancer subtype is only one component of a larger transcriptional machinery that is controlled by multiple associated NRs and transcription factors. Additionally, their ability to regulate and to be regulated by molecules of non­coding RNAs, microRNAs as well as long non­coding RNAs, is opening new scenarios for understanding the role of NRs in cancer initiation and progression. In the present review, the authors aimed to outline the reciprocal interactions that exist between the main NRs and long non­coding RNAs in different tumor diseases, to suggest new diagnostic biomarkers as well as therapeutic strategies for these tumors.

Identification of functional pathways and molecular signatures in neuroendocrine neoplasms by multi-omics analysis.

BACKGROUND: Neuroendocrine neoplasms (NENs) represent a heterogeneous class of rare tumors with increasing incidence. They are characterized by the ability to secrete peptide hormones and biogenic amines but other reliable biomarkers are lacking, making diagnosis and identification of the primary site very challenging. While in some NENs, such as the pancreatic ones, next generation sequencing technologies allowed the identification of new molecular hallmarks, our knowledge of the molecular profile of NENs from other anatomical sites is still poor. METHODS: Starting from the concept that NENs from different organs may be clinically and genetically correlated, we applied a multi-omics approach by combining multigene panel testing, CGH-array, transcriptome and miRNome profiling and computational analyses, with the aim to highlight common molecular and functional signatures of gastroenteropancreatic (GEP)-NENs and medullary thyroid carcinomas (MTCs) that could aid diagnosis, prognosis and therapy. RESULTS: By comparing genomic and transcriptional profiles, ATM-dependent signaling emerged among the most significant pathways at multiple levels, involving gene variations and miRNA-mediated regulation, thus representing a novel putative druggable pathway in these cancer types. Moreover, a set of circulating miRNAs was also selected as possible diagnostic/prognostic biomarkers useful for clinical management of NENs. CONCLUSIONS: These findings depict a complex molecular and functional landscape of NENs, shedding light on novel therapeutic targets and disease biomarkers to be exploited.

Epicardial Adipose Tissue-Derived IL-1ß Triggers Postoperative Atrial Fibrillation.

Background and aims: Post-operative atrial fibrillation (POAF), defined as new-onset AF in the immediate period after surgery, is associated with poor adverse cardiovascular events and a higher risk of permanent AF. Mechanisms leading to POAF are not completely understood and epicardial adipose tissue (EAT) inflammation could be a potent trigger. Here, we aim at exploring the link between EAT-secreted interleukin (IL)-1ß, atrial remodeling, and POAF in a population of coronary artery disease (CAD) patients. Methods: We collected EAT and atrial biopsies from 40 CAD patients undergoing cardiac surgery. Serum samples and EAT-conditioned media were screened for IL-1ß and IL-1ra. Atrial fibrosis was evaluated at histology. The potential role of NLRP3 inflammasome activation in promoting fibrosis was explored in vitro by exposing human atrial fibroblasts to IL-1ß and IL-18. Results: 40% of patients developed POAF. Patients with and without POAF were homogeneous for clinical and echocardiographic parameters, including left atrial volume and EAT thickness. POAF was not associated with atrial fibrosis at histology. No significant difference was observed in serum IL-1ß and IL-1ra levels between POAF and no-POAF patients. EAT-mediated IL-1ß secretion and expression were significantly higher in the POAF group compared to the no-POAF group. The in vitro study showed that both IL-1ß and IL-18 increase fibroblasts' proliferation and collagen production. Moreover, the stimulated cells perpetuated inflammation and fibrosis by producing IL-1ß and transforming growth factor (TGF)-ß. Conclusion: EAT could exert a relevant role both in POAF occurrence and in atrial fibrotic remodeling.

TLR4 Expression in Ex-Lichenoid Lesions-Oral Squamous Cell Carcinomas and Its Surrounding Epithelium: The Role of Tumor Inflammatory Microenvironment.

Toll-like receptors (TLRs) regulate innate and adaptive immune responses. Moreover, TLRs can induce a pro-survival and pro-proliferation response in tumor cells. This study aims to investigate the expression of TLR4 in the epithelium surrounding oral squamous cell carcinomas (OSCC) in relation to its inflammatory microenvironment. This study included 150 human samples: 30 normal oral control (NOC), 38 non-lichenoid epithelium surrounding OSCC (NLE-OSCC), 28 lichenoid epithelium surrounding OSCC (LE-OSCC), 30 OSCC ex-non oral lichenoid lesion (OSCC Ex-NOLL), and 24 OSCC ex-oral lichenoid lesion (OSCC Ex-OLL). TLR4 expression was investigated by immunohistochemistry and the percentage of positive cells was quantified. In addition, a semiquantitative analysis of staining intensity was performed. Immunohistochemical analysis revealed that TLR4 is strongly upregulated in LE-OSCC as compared to normal control epithelium and NLE-OSCC. TLR4 expression was associated with the inflammatory environment, since the percentage of positive cells increases from NOC and NLE-OSCC to LE-OSCC, reaching the highest value in OSCC Ex-OLL. TLR4 was detected in the basal third of the epithelium in NLE-OSCC, while in LE-OSCC, TLR4 expression reached the intermediate layer. These results demonstrated that an inflammatory microenvironment can upregulate TLR4, which may boost tumor development.

The Role of lncRNAs in Rare Tumors with a Focus on HOX Transcript Antisense RNA (HOTAIR).

Rare cancers are identified as those with an annual incidence of fewer than 6 per 100,000 persons and includes both epithelial and stromal tumors from different anatomical areas. The advancement of analytical methods has produced an accurate molecular characterization of most human cancers, suggesting a "molecular classification" that has allowed the establishment of increasingly personalized therapeutic strategies. However, the limited availability of rare cancer samples has resulted in very few therapeutic options for these tumors, often leading to poor prognosis. Long non coding RNAs (lncRNAs) are a class of non-coding RNAs mostly involved in tumor progression and drug response. In particular, the lncRNA HOX transcript antisense RNA (HOTAIR) represents an emergent diagnostic, prognostic and predictive biomarker in many human cancers. The aim of this review is to highlight the role of HOTAIR in rare cancers, proposing it as a new biomarker usable in the management of these tumors.

Aberrant Expression of Long Non Coding RNA HOTAIR and De-Regulation of the Paralogous 13 HOX Genes Are Strongly Associated with Aggressive Behavior of Gastro-Entero-Pancreatic Neuroendocrine Tumors.

Gastro-entero-pancreatic neuroendocrine neoplasms (GEP-NENs) are rare diseases occurring in the gastrointestinal tract and pancreas. They are characterized by the loss of epithelial tubular gland elements, and by the increased expression of neuroendocrine markers. GEP-NENs are subdivided into two histo-pathological types, gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) and gastro-entero-pancreatic neuroendocrine carcinomas (GEP-NECs). According to WHO 2017 and 2019 classification criteria are graded and staged in four categories, NET-G1, NET-G2, NET-G3, and NEC-G3. The molecular characterization of these tumors can be fundamental for the identification of new diagnostic, prognostic and predictive biomarkers. The main purpose of this study was to analyze the expression of the paralogous 13 HOX genes, normally involved in embryogenic development and frequently deregulated in human cancers, and of the HOX regulating lncRNA HOTAIR in GEP-NENs. The expression of HOX genes is gradually lost in the transition from GEP NET G1 to NET/NEC G3 tumors, while HOTAIR expression, inversely correlated with HOX genes expression and weakly expressed in low-grade GEP NENs, becomes aberrant in NET G3 and NEC G3 categories. Our data highlights their potential role in the molecular stratification of GEP-NENs by suggesting new prognostic markers and potential therapeutic targets.

The Ratio of GrzB+ - FoxP3+ over CD3+ T Cells as a Potential Predictor of Response to Nivolumab in Patients with Metastatic Melanoma.

The understanding of the molecular pathways involved in the dynamic modulation of the tumor microenvironment (TME) has led to the development of innovative treatments for advanced melanoma, including immune checkpoint blockade therapies. These approaches have revolutionized the treatment of melanoma, but are not effective in all patients, resulting in responder and non-responder populations. Physical interactions among immune cells, tumor cells and all the other components of the TME (i.e., cancer-associated fibroblasts, keratinocytes, adipocytes, extracellular matrix, etc.) are essential for effective antitumor immunotherapy, suggesting the need to define an immune score model which can help to predict an efficient immunotherapeutic response. In this study, we performed a multiplex immunostaining of CD3, FOXP3 and GRZB on both primary and unmatched in-transit metastatic melanoma lesions and defined a novel ratio between different lymphocyte subpopulations, demonstrating its potential prognostic role for cancer immunotherapy. The application of the suggested ratio can be useful for the stratification of melanoma patients that may or may not benefit from anti-PD-1 treatment.

Organoid biobanks as a new tool for pre-clinical validation of candidate drug efficacy and safety.

The growing need for personalized medicine for cancer patients has enhanced and optimized the production of living tumor organoids that have become optimal preclinical models for the discovery and screening of anticancer drugs. The systematic collection and storage of tumor organoids through the establishment of dedicated biobanks will represent a fundamental tool for cancer research and clinical trials.

Functional Interaction among lncRNA HOTAIR and MicroRNAs in Cancer and Other Human Diseases.

LncRNAs are a class of non-coding RNAs mostly involved in regulation of cancer initiation, metastatic progression, and drug resistance, through participation in post-transcription regulatory processes by interacting with different miRNAs. LncRNAs are able to compete with endogenous RNAs by binding and sequestering miRNAs and thereby regulating the expression of their target genes, often represented by oncogenes. The lncRNA HOX transcript antisense RNA (HOTAIR) represents a diagnostic, prognostic, and predictive biomarker in many human cancers, and its functional interaction with miRNAs has been described as crucial in the modulation of different cellular processes during cancer development. The aim of this review is to highlight the relation between lncRNA HOTAIR and different microRNAs in human diseases, discussing the contribution of these functional interactions, especially in cancer development and progression.

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer.

M2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC). Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-ß enhancement, IL-17F secretion, and extracellular vesicle protein content modulation. We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Detection of SARS-CoV-2 infection in a pediatric population from south Italy without symptoms of Coronavirus Disease 2019.

BACKGROUND: SARS-CoV-2 has had dramatic consequences on the world population in morbidity and mortality and socially. Clinical manifestations range from common cold-like to more severe disease such as bronchitis, pneumonia, severe acute respiratory distress syndrome, multi-organ failure and even death. The pediatric population may be infected with SARS-CoV-2, but is less likely to be symptomatic or develop severe symptoms. METHODS: We analyzed a cohort of pediatric subjects from Campania Region, south Italy, without symptoms of SARS-CoV2, to evaluate the distribution of infection in relation to gender and age. Detection on nasopharyngeal swabs was performed with two different RT-PCR methods, a qualitative rapid test (VitaPCRTM SARS-CoV-2 assay) and a quantitative test (SARS-CoV-2 ELITe MGB® assay). RESULTS: Positive subjects were 52.63% male and 47.36% female. Regarding age distribution, we described a consistent increase of detection rate (82.45%) in 0-2 year-old patients. CONCLUSION: The importance of children in transmitting the virus remains uncertain; however our analysis of the distribution of the infection in these subjects may help monitor SARS-CoV2 spread in the general population.

Immunohistochemical detection of paralogous 13 HOX genes in phyllodes tumor of the breast as a useful diagnostic tool.

Phyllodes tumor of the breast is a rare fibroepithelial lesion characterized by a propensity for local recurrence and distant metastasis. Its histologic classification, based on morphology, mitotic index and tumor margin, includes malignant, borderline, and benign. These tumors show similar cellular morphology, which may contribute to difficulty in classifying them histologically and in prediction of their clinical behavior. Thus, the identification of new biomarkers detectable also by in situ methods has become indispensable. Paralogous HOX13 genes (HOX A13, HOX B13, HOX C13 and HOX D13) play a relevant role in tumor development and progression in particular in breast cancer. In this study we analyzed the immunohistochemical expression of paralogous HOX13 homeoproteins on a phyllodes tumor case series to validate their usefulness in histologic classification.

Aptamer targeted therapy potentiates immune checkpoint blockade in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a uniquely aggressive cancer with high rates of relapse due to resistance to chemotherapy. TNBC expresses higher levels of programmed cell death-ligand 1 (PD-L1) compared to other breast cancers, providing the rationale for the recently approved immunotherapy with anti-PD-L1 monoclonal antibodies (mAbs). A huge effort is dedicated to identify actionable biomarkers allowing for combination therapies with immune-checkpoint blockade. Platelet-derived growth factor receptor ß (PDGFRß) is highly expressed in invasive TNBC, both on tumor cells and tumor microenvironment. We recently proved that tumor growth and lung metastases are impaired in mouse models of human TNBC by a high efficacious PDGFRß aptamer. Hence, we aimed at investigating the effectiveness of a novel combination treatment with the PDGFRß aptamer and anti-PD-L1 mAbs in TNBC. METHODS: The targeting ability of the anti-human PDGFRß aptamer toward the murine receptor was verified by streptavidin-biotin assays and confocal microscopy, and its inhibitory function by transwell migration assays. The anti-proliferative effects of the PDGFRß aptamer/anti-PD-L1 mAbs combination was assessed in human MDA-MB-231 and murine 4 T1 TNBC cells, both grown as monolayer or co-cultured with lymphocytes. Tumor cell lysis and cytokines secretion by lymphocytes were analyzed by LDH quantification and ELISA, respectively. Orthotopic 4 T1 xenografts in syngeneic mice were used for dissecting the effect of aptamer/mAb combination on tumor growth, metastasis and lymphocytes infiltration. Ex vivo analyses through immunohistochemistry, RT-qPCR and immunoblotting were performed. RESULTS: We show that the PDGFRß aptamer potentiates the anti-proliferative activity of anti-PD-L1 mAbs on both human and murine TNBC cells, according to its human/mouse cross-reactivity. Further, by binding to activated human and mouse lymphocytes, the aptamer enhances the anti-PD-L1 mAb-induced cytotoxicity of lymphocytes against tumor cells. Importantly, the aptamer heightens the antibody efficacy in inhibiting tumor growth and lung metastases in mice. It acts on both tumor cells, inhibiting Akt and ERK1/2 signaling pathways, and immune populations, increasing intratumoral CD8 + T cells and reducing FOXP3 + Treg cells. CONCLUSION: Co-treatment of PDGFRß aptamer with anti-PD-L1 mAbs is a viable strategy, thus providing for the first time an evidence of the efficacy of PDGFRß/PD-L1 co-targeting combination therapy in TNBC.

Molecular characterization of a bladder pleomorphic rhabdomyosarcoma in an adult patient.

Pleomorphic rhabdomyosarcoma (PRMS) is a rare but highly aggressive soft tissue tumor, accounting for 3% of soft tissue sarcomas. PRMS is the most frequent subtype of RMS in adulthood and it is mainly located in the large muscles of the extremities, particularly the lower limbs and the trunk, more rarely in other locations especially in the bladder. At our knowledge, only six cases of adult pleomorphic rhabdomyosarcoma of the bladder have been reported in the literature. In this study, we report a case of PRMS of bladder with a very poor prognosis. In fact, the patient died a month after surgery. The tumor was characterized by poorly differentiated, medium-sized sometimes rhabdoid cells, mixed with large-sized and pleomorphic elements with evident anisonucleosis, and with large areas of necrosis. We used an extensive immunohistochemical panel to exclude other tumors much more frequently reported at this site. The positivity for myogenic markers such as actin, desmin, myogenin and MyoD1 allowed the correct diagnosis. Furthermore, since preliminary studies highlighted a series of specific molecular alterations in PMRS cell lines, we analyzed a panel of specific mutations and gene rearrangements by RT-PCR and FISH methods. We showed a copy gains of CCND1 and MALT genes in our samples, suggesting an accurate molecular characterization of PRMS to establish a better management of patients and new therapeutic opportunities.

Long Non-Coding RNA HOTAIR in Breast Cancer Therapy.

Breast cancer (BC) is the most common cancer type among women, and morbidity and mortality rates are still very high. Despite new innovative therapeutic approaches for all BC molecular subtypes, the discovery of new molecular biomarkers involved in tumor progression has been fundamental for the implementation of personalized treatment strategies and improvement of patient management. Many experimental studies indicate that long non-coding RNAs (lncRNAs) are strongly involved in BC initiation, metastatic progression, and drug resistance. In particular, aberrant expression of HOX transcript antisense intergenic RNA (HOTAIR) lncRNA plays an important role in BC contributing to its progression and represents a predictor of BC metastasis. For its proven prognostic value, HOTAIR could represent a potential therapeutic target in BC. In the present review, we summarize the role of HOTAIR in cancer progression and drug resistance, in particular in BC, and we illustrate the main approaches for silencing it.

Small Non-Coding RNA Profiling Identifies miR-181a-5p as a Mediator of Estrogen Receptor Beta-Induced Inhibition of Cholesterol Biosynthesis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease, representing the most aggressive breast cancer (BC) subtype with limited treatment options due to a lack of estrogen receptor alpha (ERα), progesterone receptor (PR), and Erb-B2 receptor tyrosine kinase 2 (HER2/neu) expression. Estrogen receptor beta (ERß) is present in a fraction of TNBC patients, where its expression correlates with improved patient outcomes, supported by the fact that it exerts oncosuppressive effects in TNBC cell models in vitro. ERß is involved in microRNA-mediated regulation of gene expression in hormone-responsive BC cells and could mediate its actions through small noncoding RNAs (sncRNAs) in TNBCs also. To verify this possibility, smallRNA sequencing was performed on three ERß-expressing cell lines from different TNBC molecular subtypes. Several sncRNAs resulted modulated by ERß, with a subset being regulated in a tumor subtype-independent manner. Interestingly, sncRNA profiling of 12 ERß+and 32 ERß- primary TNBC biopsies identified 7 microRNAs, 1 PIWI-interacting RNA (piRNA), and 1 transfer RNA (tRNA) differentially expressed in ERß+ compared to ERß- tumors and cell lines. Among them, miR-181a-5p was found to be overexpressed in ERß+ tumors and predicted target key components of the cholesterol biosynthesis pathway previously found to be inhibited by ERß in TNBC cells.

Novel Aptamers Selected on Living Cells for Specific Recognition of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a high heterogeneous group of tumors with a distinctly aggressive nature and high rates of relapse. So far, the lack of any known targetable proteins has not allowed a specific anti-tumor treatment. Therefore, the identification of novel agents for specific TNBC targeting and treatment is desperately needed. Here, by integrating cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) for the specific recognition of TNBC cells with high-throughput sequencing technology, we identified a panel of 2'-fluoropyrimidine-RNA aptamers binding to TNBC cells and their cisplatin- and doxorubicin-resistant derivatives at low nanomolar affinity. These aptamers distinguish TNBC cells from both non-malignant and non-TNBC breast cancer cells and are able to differentiate TNBC histological specimens. Importantly, they inhibit TNBC cell capacity of growing in vitro as mammospheres, indicating they could also act as anti-tumor agents. Therefore, our newly identified aptamers are a valuable tool for selectively dealing with TNBC.