A 15-year experience in pediatric palliative care: a retrospective hospital-based study.

BACKGROUND: The current Italian scenario of pediatric palliative care (PPC) services is characterized by inadequate coverage of the territory. Therefore, it is important to improve the referral of patients to the most appropriate setting (community care, general PPC, or specialized PPC) and to improve the delivery of PPC care. METHODS: Aiming at obtaining information about the referrals to the Padua Pediatric Hospice that could help estimate the investments needed to improve the provision of care, a retrospective analysis has been carried out. The rate of proper referral and discharge, the number of patients followed at the hospice, the mortality rate, and the length of follow-up were analyzed, and, when possible, data were stratified by oncological and non-oncological diseases. RESULTS: The analysis showed that of the 870 patients referred to the Padua Pediatric Hospice between 2008 and 2022, 76% were affected by non-oncological conditions. 82% of patients referred were taken in charge and most of the remaining patients have been inappropriately referred. The analysis showed a growing number of total referrals, which increased by 195% from 2008 to 2022. An increase in proper referrals and referrals of non-oncological patients was observed alongside a decrease in oncological patient referrals and a trend toward a decrease in discharge rates. A decreased mortality was observed in patients with non-oncological conditions, with only 6% of deceased patients in 2022. Moreover, a longer survival with a median follow-up length of 43 months was observed among patients with non-oncological conditions who were followed up at the Padua Pediatric Hospice for more than 12 months. Conversely, the short survival rate observed for oncological patients suggests that those patients should have been referred to PPC earlier to benefit from palliative care for longer periods. CONCLUSIONS: Considering these data, it is expected that the number of patients needing PPC services will steadily increase in the next years. Hence, there is a need to invest resources to provide the best care delivery model encompassing specific pathways for the transition into adulthood, the establishment of networks within all the Italian regions, and an efficient referral to the more suitable setting of care.

Gene expression profile in TNF receptor-associated periodic syndrome reveals constitutively enhanced pathways and new players in the underlying inflammation.

OBJECTIVES: Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is a multisystemic autoinflammatory condition associated with heterozygous TNFRSF1A mutations, presenting with a variety of clinical symptoms, many of which yet unexplained. In this work, we aimed at deepening into TRAPS pathogenic mechanisms sustained by monocytes. METHODS: Microarray experiments were conducted to identify genes whose expression results altered in patients compared to healthy individuals, both under basal condition and following LPS stimulation. RESULTS: An inflammatory state baseline, characterised by constitutive overexpression of IL1ß and IL1R1 receptor, has been shown in TRAPS patients compared to controls, including in non-active disease phases. Following LPS stimulation, IL1RN up-regulation is stronger in controls than in patients and inflammatory pathways and microRNAs undergo differential regulation. Genes involved in post-translational modifications, protein folding and ubiquitination result constitutively up-regulated in TRAPS, while response to interferon types I and II is defective, failing to be up-regulated by LPS. TGFß pathway is down-regulated in untreated TRAPS monocytes, while genes involved in redox regulation result constitutively over-expressed. Finally, additional molecular alterations seem to reflect organ failures sometime complicating the disease. CONCLUSIONS: Gene expression profile in resting TRAPS monocytes has confirmed the patients' chronic inflammatory condition. In addition, pathways not yet associated with the disease have been disclosed, such as interferon types I and II response to LPS stimulation and a downregulation of the TGFß pathway in basal condition. The role of miRNA, suggested by our results, deserves in-depth analyses in light of the possible development of targeted therapies.

Omalizumab management beyond clinical trials: the added value of a network model.

BACKGROUND: Omalizumab is effective and safe in severe allergic asthma. Few data are available about its impact on lung function and on asthma comorbidities, long-term follow-up of treated patients, adherence, non-responders profile, and optimal treatment duration. OBJECTIVE: We aimed at evaluating omalizumab-related clinical outcomes and unmet needs in a real-life setting. METHODS: We created a collaborative network (NEONet - North East Omalizumab Network) involving 9 Allergy and Respiratory referral centres for severe asthma placed in the North-East of Italy. Patients' data were entered into a common study database shared by all the participating physicians. A preliminary retrospective analysis was performed. RESULTS: Patients come from a common well-defined geographical and environmental district providing a homogeneous population sample. A moderate but statistically significant improvement of the FEV1, and an increasing proportion of exacerbations-free patients were observed since the treatment start. These findings were independent of the baseline severity of bronchial obstruction. A positive impact of omalizumab on rhinitis in patients with both asthma and rhinitis was detected. Moreover the efficacy of omalizumab on asthma seemed not to be affected by the baseline severity of rhinitis. CONCLUSION: Our retrospective analysis represents a preliminary report from the NEONet activity. It confirmed omalizumab efficacy and provided some new insights about its impact on lung function and on comorbid rhinitis. The network approach, under a prospective view, allows creating a large uniform database, by means of a standardized shared tool for data collecting, and joining a multidisciplinary expertise.

High-Tech Parameters for the Evaluation of Signs and Symptoms of Dry Eye Disease: Identification of Clinical Cut-Offs and Agreement with Low-Tech Tests.

INTRODUCTION: High-tech devices for the assessment of dry eye disease (DED) are increasingly available. However, the agreement between high- and low-tech parameters has been poorly explored to date. Trying to fill these gaps, we conducted a post hoc analysis on a recently published retrospective study on patients with DED receiving both low- and high-tech (Keratograph®) assessments, and treatment with different lubricating eyedrops. METHODS: Six clinical questions were defined by the authors, considering literature gaps and their clinical experience, namely: (1) are NIKBUT-i and T-BUT interchangeable parameters? (2) What was the correlation between low- and high-tech parameters in untreated and treated patients with DED? (3) What was the correlation between signs and symptoms at baseline and during/after treatment? (4) Which parameters were better associated with symptoms? And with symptoms change over time? (5) What was the performance of NIKBUT-i and T-BUT in detecting clinically relevant changes? (6) What was the clinical advantage of adding other high- and low-tech parameters, respectively, to NIKBUT-i and T-BUT? RESULTS: Low-tech measures were the best descriptors of the Ocular Surface Disease Index (OSDI) at baseline. In contrast, high-tech assessments demonstrate better performance in detecting changes over time. The distribution of NIKBUT-i data was more dispersed than TBUT both at baseline and follow-up. At a fixed specificity of 80%, the sensitivity in detecting clinically relevant ameliorations of symptoms was 42% for NIKBUT-i and 25% for T-BUT. A battery of high-tech tests could detect 90% of clinical amelioration, compared with 45% with low-tech tests (p < 0.001). Correlation between low- and high-tech parameters in both treated and untreated patients is lacking. CONCLUSIONS: Low-tech measures are adequate for diagnostic purposes in DED, whereas high-tech showed better performances at follow-up, particularly when different tests are combined. Overall, poor interchangeability among parameters and agreement with symptoms was reported both with high- and low-tech assessments.

Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells.

BACKGROUND: Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting.We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type). METHODS: We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis. RESULTS: Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion. CONCLUSIONS: This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting.Massimiliano Monticone and Antonio Daga contributed equally to this work.

How do systemic sclerosis manifestations influence patients' lives? Results from a survey on patients and caregivers.

OBJECTIVE: To investigate the patient- and caregiver-reported impact of systemic sclerosis (SSc) manifestations (hand/feet/joint involvement and pulmonary complications) on the diagnostic and therapeutic journey, working productivity, and social life. METHODS: Two questionnaires (one for the patients, n = 260 and one for the caregivers, n = 47) were designed in collaboration with the patients' association Gruppo Italiano per la Lotta alla Sclerodermia (GILS). Validated questionnaires were combined with specific questions relevant to the Italian scenario. RESULTS: Pulmonary fibrosis and hand/feet/joint involvement have a major impact on patient's working status: (85.3% of patients with pulmonary fibrosis and 72.6% with hand/feet/joint involvement report loss of job/job change due to SSc. Productivity was affected as well: 60.6% of the patients (75% of those with fibrosis) reported that working productivity in the previous 4 weeks was restricted by physical limitations. The disease has a significant impact on patients' life, limiting the ability to conduct common activities, especially those related to movement, such as object manipulation (61.1%), doing small manual jobs (44.0%), writing (38.9%), and an increased impact in case of pulmonary fibrosis and hands/feet/joints involvement. Half of the patients also present some difficulties in eating-related activities a Patients also experience poorer social life, personal relationships, and sexual life. Caregivers are also deeply influenced by the manifestations of SSc. Pulmonary fibrosis and hand/feet/joint involvement represent an additional challenge. CONCLUSION: Pulmonary fibrosis and hand/feet/joint involvement are extremely burdensome complications for both SSc patients and caregivers, decreasing work productivity, limiting relationship and social life, and impacting psychological status and everyday activities.

Integrated metabolomic analysis and cytokine profiling define clusters of immuno-metabolic correlation in new-onset psoriasis.

The association between the metabolic profile and inflammatory cytokines in psoriasis is poorly understood. We analyzed the metabolic and cytokine/chemokine profiles in serum and skin from patients with new-onset psoriasis and healthy subjects (n = 7/group) by HR-MAS NMR and Bio-Plex immunoassay. Immuno-metabolic correlation matrix was analyzed in skin and serum to identify a potential immune-metabolic signature. Metabolomics analysis showed a significant increase in ascorbate and a decrease in scyllo-inositol, and a trend towards an increase in eight other metabolites in psoriatic skin. In serum, there was a significant increase of dimethylglycine and isoleucine. In parallel, psoriatic skin exhibited an increase of early inflammatory cytokines (IL-6, IL-8, TNF-α, IL-1ß) and correlation analysis highlighted some major clusters of immune-metabolic correlations. A cluster comprising scyllo-inositol and lysine showed correlations with T-cell cytokines; a cluster comprising serine and taurine showed a negative correlation with early inflammatory cytokines (IL-6, G-CSF, CCL3). A strong positive correlation was enlightened between glutathione and inflammatory cytokines/angiogenesis promoters of psoriasis. The integration of metabolic and immune data indicated a molecular signature constituted by IL-6, IL1-ra, DMG, CCL4, Ile, Gly and IL-8, which could discriminate patients and healthy subjects and could represent a candidate tool in the diagnosis of new-onset psoriasis.

Reference profile correlation reveals estrogen-like trancriptional activity of Curcumin.

BACKGROUND: Several secondary metabolites from herbal nutrient products act as weak estrogens (phytoestrogens), competing with endogenous estrogen for binding to the estrogen receptors and inhibiting steroid converting enzymes. However, it is still unclear whether these compounds elicit estrogen dependent transcription of genes at physiological concentrations. METHODS: We compare the effects of physiological concentrations (100 nM) of the two phytoestrogens Enterolactone and Quercetin and the suspected phytoestrogen Curcumin on gene expression in the breast cancer cell line MCF7 with the effects elicited by 17-beta-estradiol (E2). RESULTS: All three phytocompounds have weak effects on gene transcription; most of the E2 genes respond to the phytoestrogens in the same direction though to a much lesser extent and in the order Curcumin > Quercetin > Enterolactone. Gene regulation induced by these compounds was low for genes strongly induced by E2 and similar to the latter for genes only weakly regulated by the classic estrogen. Of interest with regard to the treatment of menopausal symptoms, the survival factor Birc5/survivin and the oncogene MYBL1 are strongly induced by E2 but only marginally by phytoestrogens. CONCLUSION: This approach demonstrates estrogenic effects of putative phytoestrogens at physiological concentrations and shows, for the first time, estrogenic effects of Curcumin.

Mutually exclusive expression of DLX2 and DLX5/6 is associated with the metastatic potential of the human breast cancer cell line MDA-MB-231.

BACKGROUND: The DLX gene family encodes for homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis. Their expression can be regulated by Endothelin1 (ET1), a peptide associated with breast cancer invasive phenotype. Deregulation of DLX gene expression was found in human solid tumors and hematologic malignancies. In particular, DLX4 overexpression represents a possible prognostic marker in ovarian cancer. We have investigated the role of DLX genes in human breast cancer progression. METHODS: MDA-MB-231 human breast carcinoma cells were grown in vitro or injected in nude mice, either subcutaneously, to mimic primary tumor growth, or intravenously, to mimic metastatic spreading. Expression of DLX2, DLX5 and DLX6 was assessed in cultured cells, either treated or not with ET1, tumors and metastases by RT-PCR. In situ hybridization was used to confirm DLX gene expression in primary tumors and in lung and bone metastases. The expression of DLX2 and DLX5 was evaluated in 408 primary human breast cancers examining the GSE1456 and GSE3494 microarray datasets. Kaplan-Meier estimates for disease-free survival were calculated for the patients grouped on the basis of DLX2/DLX5 expression. RESULTS: Before injection, or after subcutaneous growth, MDA-MB-231 cells expressed DLX2 but neither DLX5 nor DLX6. Instead, in bone and lung metastases resulting from intravenous injection we detected expression of DLX5/6 but not of DLX2, suggesting that DLX5/6 are activated during metastasis formation, and that their expression is alternative to that of DLX2. The in vitro treatment of MDA-MB-231 cells with ET1, resulted in switch from DLX2 to DLX5 expression. By data mining in microarray datasets we found that expression of DLX2 occurred in 21.6% of patients, and was significantly correlated with prolonged disease-free survival and reduced incidence of relapse. Instead, DLX5 was expressed in a small subset of cases, 2.2% of total, displaying reduced disease-free survival and high incidence of relapse which was, however, non-significantly different from the other groups due to the small size of the DLX+ cohort. In all cases, we found mutually exclusive expression of DLX2 and DLX5. CONCLUSIONS: Our studies indicate that DLX genes are involved in human breast cancer progression, and that DLX2 and DLX5 genes might serve as prognostic markers.

The Experience of Oncology Healthcare Providers in the Central Italy during the COVID-19 Lockdown.

While the emotional response of healthcare providers during the COVID-19 pandemic has been extensively investigated in countries in the Far-East, little is known about the psychological impact and the associated emotional distress of healthcare providers in Italy, especially with regard to different regions. The aim of the "VIRARE" survey, which was addressed to all the healthcare providers in the Lazio region (central Italy) and, in particular, to those working in the oncology field, is to analyze their opinion on the impact and management of the pandemic, to better understand the level of their psychological distress. A global good psychological response of healthcare providers to the pandemic has emerged, independently from their different occupations in the oncology field. Healthcare providers show a high degree of resilience, identifying the major causes of distress the difficulty of the management of this situation, the obstacles in their working activity and expressing a high degree of dissatisfaction with how Italian institutions handled this situation. This survey also provides a direct comparison between COVID-19-infected (or directly in contact with COVID-19-infected patients) and uninfected healthcare providers, identifying the sub-category of infected professionals that reported signs of depression as particularly vulnerable.

Survival Online: a web-based service for the analysis of correlations between gene expression and clinical and follow-up data.

BACKGROUND: Complex microarray gene expression datasets can be used for many independent analyses and are particularly interesting for the validation of potential biomarkers and multi-gene classifiers. This article presents a novel method to perform correlations between microarray gene expression data and clinico-pathological data through a combination of available and newly developed processing tools. RESULTS: We developed Survival Online (available at http://ada.dist.unige.it:8080/enginframe/bioinf/bioinf.xml), a Web-based system that allows for the analysis of Affymetrix GeneChip microarrays by using a parallel version of dChip. The user is first enabled to select pre-loaded datasets or single samples thereof, as well as single genes or lists of genes. Expression values of selected genes are then correlated with sample annotation data by uni- or multi-variate Cox regression and survival analyses. The system was tested using publicly available breast cancer datasets and GO (Gene Ontology) derived gene lists or single genes for survival analyses. CONCLUSION: The system can be used by bio-medical researchers without specific computation skills to validate potential biomarkers or multi-gene classifiers. The design of the service, the parallelization of pre-processing tasks and the implementation on an HPC (High Performance Computing) environment make this system a useful tool for validation on several independent datasets.

Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells.

BACKGROUND: Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma. RESULTS: We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFkappaB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFkappaB dependent manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFkappaB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression. CONCLUSION: Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

Curcumin downregulates the inflammatory cytokines CXCL1 and -2 in breast cancer cells via NFkappaB.

The dietary antioxidant Curcumin has been proposed for cancer chemoprevention since it induces apoptosis and inhibits the formation of breast cancer metastases. Curcumin acts through the inhibition of phosphorylation of the inhibitor of kappa B (IkappaB), which in turn reduces the nuclear translocation of nuclear factor kappa B (NFkappaB), an inflammation- and cell survival-related transcription factor. However, it is not clear whether the strong antimetastatic effect can exclusively be explained by inhibition of NFkappaB. Here, we addressed the effects of Curcumin (IC(50) = 17 muM) in MDA-MB-231 breast cancer cells using microarray gene expression analyses. Among the 62 genes whose expression was significantly altered, we found the two inflammatory cytokines CXCL1 and -2 (Groalpha and -beta) that were downregulated. Further validation of the microarray results by quantitative real-time reverse transcription-polymerase chain reaction, western blots and enzyme-linked immunosorbent assay revealed that Curcumin impairs transcription of CXCL1 and -2 >24 h and reduces the corresponding proteins. Using small interfering RNA techniques, we elucidated the underlying molecular mechanism revealing that reduction of CXCL1 and -2 messenger RNA levels is NFkappaB dependent and requires intact IkappaBalpha expression. Moreover, CXCL1 and -2 silencing leads to downregulation of several metastasis-promoting genes among which we found the cytokine receptor CXCR4. We therefore suggest that the decrease of CXCL1 and -2 mediated by Curcumin is involved in the inhibition of metastasis.

Lineage infidelity and expression of melanocytic markers in human breast cancer.

In previous analyses, dual expression of melanocytic and epithelial molecular markers have been described as indicators of lineage infidelity for breast cancer cells that lose their epithelial identity. Here we demonstrated that this is a much more frequent phenomenon in human breast carcinomas, usually affecting only a part of the tumor. Accordingly we detected, in 18 out of 100 breast carcinomas, immunohistochemically focally positive cells for the melanocytic marker Melan A. The presence and extent of Melan A expression was statistically significantly associated with a reduction in tumor cell differentiation, but not tumor type, size, lymph node metastasis, hormone receptor status or Her-2-neu expression. Microarrays of a further 159 breast cancers showed, in several samples, variably low expression levels of Melan A (and other melanocytic markers) that are consistent with focal expression in many tumors. One case strongly overexpressed Melan A. The transition from an epithelial to a melanocytic phenotype (lineage infidelity) appears to occur much more frequently than previously assumed and occurs in restricted areas of breast cancer during tumor progression, a possible association with a reduction in tumor cell differentiation.

A highly invasive subpopulation of MDA-MB-231 breast cancer cells shows accelerated growth, differential chemoresistance, features of apocrine tumors and reduced tumorigenicity in vivo.

The acquisition of an invasive phenotype is a prerequisite for metastasization, yet it is not clear whether or to which extent the invasive phenotype is linked to other features characteristic of metastatic cells. We selected an invasive subpopulation from the triple negative breast cancer cell line MDA-MB-231, performing repeated cycles of preparative assays of invasion through Matrigel covered membranes. The invasive sub-population of MDA-MB-231 cells exhibits stronger migratory capacity as compared to parental cells confirming the highly invasive potential of the selected cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype. ArrayCGH, DNA index quantification and karyotype analyses confirmed a common genetic origin of the parental and invasive subpopulations and revealed discrete structural differences of the invasive subpopulation including increased ploidy and the absence of a characteristic amplification of chromosome 5p14.1-15.33. Gene expression analyses showed a drastically altered expression profile including features of apocrine breast cancers and of invasion related matrix-metalloproteases and cytokines. The invasive cells showed accelerated proliferation, increased apoptosis, and an altered pattern of chemo-sensitivity with lower IC50 values for drugs affecting the mitotic apparatus. However, the invasive cell population is significantly less tumorigenic in orthotopic mouse xenografts suggesting that the acquisition of the invasive capacity and the achievement of metastatic growth potential are distinct events.

Female and male human babies have distinct blood metabolomic patterns.

A sex-gender approach in laboratory medicine is scarce; furthermore, the influence of sex on acylcarnitines and amino acid levels at birth has not been thoroughly investigated, even if sex impacts on newborn screening. We aimed to establish the influence of sex on amino acids and acylcarnitines levels in male and female newborns. Amino acids and acylcarnitines were analysed in dried blood spots using tandem mass spectrometry in male and female newborns. Data were analysed before and after body weight correction also using principal components analysis. This retrospective analytical study showed that females had small but significantly higher levels of amino acids and the correction for body weight amplified these differences. Acylcarnitines were overall higher in males before body weight correction with the exception of isovalerylcarnitine + methylbutyrylcarnitine (C5), which was significantly higher in females. Body weight correction decreased the sex differences in C5. Principal component analysis showed that both amino acids and acylcarnitines were necessary to describe the model for females, whereas only acylcarnitines were required for males. These metabolomics data underline the importance of including sex as a variable in future investigations of circulating metabolites; the existence of sex differences highlights the need for setting distinct reference values for female and male neonates in metabolite concentration.

miR181b is induced by the chemopreventive polyphenol curcumin and inhibits breast cancer metastasis via down-regulation of the inflammatory cytokines CXCL1 and -2.

Chronic inflammation is a major risk factor for the development and metastatic progression of cancer. We have previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast and prostate cancer metastases. In the present study, we have analyzed the effects of Curcumin on miRNA expression and its correlation to the anti-tumorigenic properties of this natural occurring polyphenol. Using microarray miRNA expression analyses, we show here that Curcumin modulates the expression of a series of miRNAs, including miR181b, in metastatic breast cancer cells. Interestingly, we found that miR181b down-modulates CXCL1 and -2 through a direct binding to their 3'-UTR. Overexpression or inhibition of miR181b in metastatic breast cancer cells has a significant impact on CXCL1 and -2 and is required for the effect of Curcumin on these two cytokines. miR181b also mediates the effects of Curcumin on inhibition of proliferation and invasion as well as induction of apoptosis. Importantly, over-expression of miR181b in metastatic breast cancer cells inhibits metastasis formation in vivo in immunodeficient mice. Finally, we demonstrated that Curcumin up-regulates miR181b and down-regulates CXCL1 and -2 in cells isolated from several primary human breast cancers. Taken together, these data show that Curcumin provides a simple bridge to bring metastamir modulation into the clinic, placing it in a primary and tertiary preventive, as well as a therapeutic, setting.

Evidence of epidermal growth factor receptor expression in uveal melanoma: inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity.

Despite advances in surgery and radiotherapy of uveal melanoma (UM), many patients develop distant metastases that poorly respond to therapy. Improved therapies for the metastatic disease are therefore urgently needed. Expression of the epidermal growth factor receptor (EGFR), a target of kinase inhibitors and humanised antibodies in use for several cancers, had been reported. Forty-eight human UMs were analysed by expression profiling. Signalling was tested in three EGFR expressing UM cell lines by Western blotting using phosphorylation specific antibodies for EGFR and the downstream mediators AKT (v-akt murine thymoma viral oncogene homolog) and extracellular signal-regulated kinase (ERK). Evidence for signalling in tumours was obtained through the application of a UM-specific EGF-signature. The EGFR specific kinase inhibitor, Gefitinib and the humanised monoclonal antibody, Cetuximab, were tested for their effect on EGFR signalling. Natural killer cell mediated antibody-dependent cellular cytotoxicity (ADCC) and tumour necrosis factor α (TNF-α) release was analysed for Cetuximab. Fourteen of 48 UMs and three of 14 cell lines (over-)express EGFR, at least in part due to trisomy of the EGFR locus on chromosome 7p12. EGFR and the downstream mediator, AKT, are phosphorylated upon stimulation with EGF in EGFR expressing cell lines. EGFR over-expressing tumours but not EGFR negative tumours show an activated EGF-signature. Gefitinib inhibits EGFR and AKT phosphorylation and Cetuximab induces EGFR phosphorylation but inhibits signalling to AKT induced with EGF. Cetuximab triggers natural killer (NK) cells to lyse EGFR+ cell lines and to release TNF-α. EGFR appears suited as a novel molecular drug target for therapy of uveal melanoma.

Mda-9/syntenin is expressed in uveal melanoma and correlates with metastatic progression.

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound-healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.