First Evidence of Mineralocorticoid Receptor Gene and Protein Expression in Rat and Human Thyroid Tissues and Cell Cultures.

Aldosterone (Aldo) exerts its action through binding with the mineralocorticoid receptor (MR). Clinically, a link between primary aldosteronism (PA) and thyroid diseases has been hypothesised. However, the presence and activity of MR on the thyroid have not yet been demonstrated. We investigated the gene/protein expression and activation of MR in primary thyroid cell cultures (normal rat thyroid [FRTL-5] and human papillary thyroid cancer [PTC] cell lines, BCPAP and K1) through qRT-PCR analysis, immunofluorescence, and confocal microscopy. We also studied the effects of Aldo on thyroid-specific and inflammation genes in vitro. Paired human normal and neoplastic thyroid tissues were also studied. We demonstrated both gene and protein expression and activation of MR in normal rat thyroid and human PTC lines. Incubation with Aldo induced an acute increase in IL-6 expression in both the FRTL-5 and BCPAP lines, which was antagonised by spironolactone, and an acute and late upregulation of thyroid-specific genes in FRTL-5. MR was also expressed at both gene and protein levels in normal human thyroid tissues and in PTC, with a progressive decline during neoplastic tumourigenesis, particularly in more aggressive histotypes. We present the first evidence of MR gene and protein expression in both normal and pathological thyroid cells and tissues. We have shown that MR is present and functionally activated in thyroid tissue. Binding of Aldo to MR induces the expression of inflammatory and thyroid-specific genes, and the thyroid may thus be considered a novel mineralocorticoid target tissue.

Size-Exclusion Chromatography Combined with Ultrafiltration Efficiently Isolates Extracellular Vesicles from Human Blood Samples in Health and Disease.

There is still a need for an efficient method for the isolation of extracellular vesicles (EVs) from human blood that provides a reliable yield with acceptable purity. Blood is a source of circulating EVs, but soluble proteins and lipoproteins hamper their concentration, isolation, and detection. This study aims to investigate the efficiency of EV isolation and characterization methods not defined as "gold standard". EVs were isolated from human platelet-free plasma (PFP) of patients and healthy donors through size-exclusion chromatography (SEC) combined with ultrafiltration (UF). Then, EVs were characterized using transmission electron microscopy (TEM), imaging flow cytometry (IFC), and nanoparticle tracking analysis (NTA). TEM images showed intact and roundish nanoparticles in pure samples. IFC analysis detected a prevalence of CD63+ EVs compared to CD9+, CD81+, and CD11c+ EVs. NTA confirmed the presence of small EVs with a concentration of ~1010 EVs/mL that were comparable when stratifying the subjects by baseline demographics; conversely, concentration differed according to the health status across healthy donors and patients affected with autoimmune diseases (130 subjects in total, with 65 healthy donors and 65 idiopathic inflammatory myopathy (IIM) patients). Altogether, our data show that a combined EV isolation method, i.e., SEC followed by UF, is a reliable approach to isolate intact EVs with a significant yield from complex fluids, which might characterize disease conditions early.

TSH-receptor autoantibodies in patients with chronic thyroiditis and hypothyroidism.

OBJECTIVES: The reported prevalence of TSH-receptor (TSHR) autoantibodies (TRAb) in patients with chronic thyroiditis (CT) range from 0 to 48%. The objective was to study the prevalence of TRAb in patients with CT and hypothyroidism and to correlate it with gender, age, thyroid dimensions, TSH levels, and autoimmune diseases. METHODS: The study comprised 245 patients with CT and hypothyroidism (median age 42 years, 193 females, 52 males) and 123 Italian healthy subjects matched for sex and age as controls. TRAb were tested with ELISA using a >2.5 IU/L cut off for positivity. TSHR blocking (TBAb) and TSHR stimulating autoantibodies (TSAb) were measured in 12 TRAb-positive patients using bioassays with Chinese hamster ovary (CHO) cells expressing wild-type or R255D-mutated TSHR. RESULTS: TRAb positivity was found in 32/245 (13.1%) patients and significantly correlated (p<0.05) with TSH levels. TRAb positivity was significantly higher in males vs. females (p=0.034), in females 16-45 years of age vs. >45 years of age (p<0.05) and in patients with reduced vs. normal/increased thyroid dimensions (p<0.05). Linear regression analysis showed a correlation between TRAb concentrations with age (p<0.05) and TRAb concentrations with TSH (p<0.01). In bioassay with TSHR-R255D all 12 patients tested were TBAb-positive while 33% were also TSAb-positive suggesting the presence of a mixture of TRAbs with different biological activities in some patients. CONCLUSIONS: TRAb have been found in patients with CT and hypothyroidism. A mixture of TBAb and TSAb was found in some patients and this may contribute to the pathogenesis of thyroid dysfunction during the course of the disease.

Overexpression of miR-375 and L-type Amino Acid Transporter 1 in Pheochromocytoma and Their Molecular and Functional Implications.

Pheochromocytoma (Pheo) is a tumor derived from chromaffin cells. It can be studied using 18F-dihydroxyphenylalanine (DOPA)-positron emission tomography (PET) due to its overexpression of L-type amino acid transporters (LAT1 and LAT2). The oncogenic pathways involved are still poorly understood. This study examined the relationship between 18F-DOPA-PET uptake and LAT1 expression, and we explored the role of miR-375 and putative target genes. A consecutive series of 58 Pheo patients were retrospectively analyzed, performing 18F-DOPA-PET in 32/58 patients. Real-time quantitative PCR was used to assess the expression of LAT1, LAT2, phenylethanolamine N-methyltransferase (PNMT), miR-375, and the major components of the Hippo and Wingless/Integrated pathways. Principal germline mutations associated with hereditary Pheo were also studied. Pheo tissues had significantly higher LAT1, LAT2, and PNMT mRNA levels than normal adrenal tissues. MiR-375 was strongly overexpressed. Yes-associated protein 1 and tankyrase 1 were upregulated, while beta-catenin, axin2, monocarboxylate transporter 8, and Frizzled 8 were downregulated. A positive relationship was found between 18F-DOPA-PET SUV mean and LAT1 gene expression and for 24 h-urinary norepinephrine and LAT1. This is the first experimental evidence of 18F-DOPA uptake correlating with LAT1 overexpression. We also demonstrated miR-375 overexpression and downregulated (Wnt) signaling and identified the Hippo pathway as a new potentially oncogenic feature of Pheo.

Biological Effects of EF24, a Curcumin Derivative, Alone or Combined with Mitotane in Adrenocortical Tumor Cell Lines.

BACKGROUND: Curcumin has numerous properties and is used in many preclinical conditions, including cancer. It has low bioavailability, while its derivative EF24 shows enhanced solubility. However, its effects have never been explored in adrenocortical tumor cell models. The efficacy of EF24 alone or combined with mitotane (reference drug for adrenocortical cancer) was evaluated in two adrenocortical tumor cell lines, SW13 and H295R. METHOD AND RESULTS: EF24 reduced cell viability with an IC50 (half maximal inhibitory concentration) of 6.5 ± 2.4 µM and 4.9 ± 2.8 µM for SW13 and H295R cells, respectively. Combination index (EF24 associated with mitotane) suggested an additivity effect in both cell lines. Cell cycle analysis revealed an increase in subG0/G1 phase, while motility assay showed a decrease in migratory cell capacity, and similarly, clonogenic assay indicated that EF24 could reduce colony numbers. Furthermore, Wnt/ß-catenin, NF-κB, MAPK, and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with EF24 alone or combined with mitotane. In addition, intracellular reactive oxygen species levels increased in both cell lines. CONCLUSION: This work analyzed EF24 in adrenocortical tumor cell lines for the first time. These results suggest that EF24 could potentially impact on adrenocortical tumors, laying the foundation for further research in animal models.

The aurora kinase inhibitor VX-680 shows anti-cancer effects in primary metastatic cells and the SW13 cell line.

New therapeutic targets are needed to fight cancer. Aurora kinases (AK) were recently identified as vital key regulators of cell mitosis and have consequently been investigated as therapeutic targets in preclinical and clinical studies. Aurora kinase inhibitors (AKI) have been studied in many cancer types, but their potential capacity to limit or delay metastases has rarely been considered, and never in adrenal tissue. Given the lack of an effective pharmacological therapy for adrenal metastasis and adrenocortical carcinoma, we assessed AKI (VX-680, SNS314, ZM447439) in 2 cell lines (H295R and SW13 cells), 3 cell cultures of primary adrenocortical metastases (from lung cancer), and 4 primary adrenocortical tumor cell cultures. We also tested reversan, which is a P-gp inhibitor (a fundamental efflux pump that can extrude drugs), and we measured AK expression levels in 66 adrenocortical tumor tissue samples. Biomolecular and cellular tests were performed (such as MTT, thymidine assay, Wright's staining, cell cycle and apoptosis analysis, Western blot, qRT-PCR, and mutation analysis). Our results are the first to document AK overexpression in adrenocortical carcinoma as well as in H295R and SW13 cell lines, thus proving the efficacy of AKI against adrenal metastases and in the SW13 cancer cell model. We also demonstrated that reversan and AKI Vx-680 are useless in the H295R cell model, and therefore should not be considered as potential treatments for ACC. Serine/threonine AK inhibition, essentially with VX-680, could be a promising, specific therapeutic tool for eradicating metastases in adrenocortical tissue.

Calcitonin measurement and immunoassay interference: a case report and literature review.

Calcitonin (CT) is currently the most sensitive serological marker of C-cell disease [medullary thyroid carcinoma (MTC) and C-cell hyperplasia]. Starting with a report on a case that occurred at our institution, this review focuses on trying to explain the reasons behind the poor specificity and sensitivity of the various CT immunoassays. A 15-year-old patient was referred to our institution in May 2014 for moderately elevated CT levels. Thyroid ultrasonography (US) documented a colloidal goiter. Secondary causes of the hypercalcitoninemia (hyperCT) were ruled out. The mismatch between the clinical picture and the laboratory results prompted us to search for other reasons for the patient's high CT levels, so we applied the heterophilic blocking tube (HBT) procedure to the patient's sera before the CT assay. Using this pretreatment step, his serum CT concentration dropped to <1 ng/L, as measured at the same laboratory. Measuring plasma CT has an important role in screening for C-cell disease, but moderately elevated serum CT levels need to be placed in their clinical context, bearing in mind all the secondary causes of C-cell hyperplasia and the possibility of laboratory interference, before exposing patients to the risks and costs of further tests.

Iodine status from childhood to adulthood in females living in North-East Italy: Iodine deficiency is still an issue.

PURPOSE: This survey aimed to assess iodine status in a female population at different ages, also investigating their eating habits. METHODS: We measured urinary iodine concentrations (UIC) in: 634 females at puberty and 361 fertile women in 246 of whom were considered also their children (134 daughters and 120 sons). All subjects completed a food frequency questionnaire. RESULTS: Median UIC decreased from childhood to adulthood (median UIC 107, 77 and 55 µg/l in the young girls, females at puberty and fertile women, respectively). Though using iodized salt improved iodine status in all groups, a significantly higher UIC was only noted in females at puberty. Milk consumption significantly increased UIC at all ages. In mother-child (both daughters and sons) pairs, the children's median UIC was nearly twice as high as their mothers' (UIC 115 vs. 57 µg/l). Milk consumption varied significantly: 56% of the mothers and 76% of their children drank milk regularly. The children (both daughters and sons) and mothers who drank milk had UIC ≥100 µg/l in 59 and 34% of cases, respectively, among the pairs who did not drink milk, 44% of the children and 19% of the mothers had UIC ≥100 µg/l. On statistical regression, 3.6% of the variability in the children's UIC depended on that of their mothers. CONCLUSIONS: Dietary iodine status declines from childhood to adulthood in females due to different eating habits. A mild iodine deficiency emerged in women of child-bearing age that could have consequences during pregnancy and lactation.

Synergistic antitumour activity of RAF265 and ZSTK474 on human TT medullary thyroid cancer cells.

Medullary thyroid cancer (MTC) is an aggressive malignancy responsible for up to 14% of all thyroid cancer-related deaths. It is characterized by point mutations in the rearranged during transfection (RET) proto-oncogene. The activated RET kinase is known to signal via extracellular signal regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine-protein kinase B-Raf (BRAF) inhibitors (RAF265 and SB590885), and a PI3K inhibitor (ZSTK474), on RET-mediated signalling and proliferation in a MTC cell line (TT cells) harbouring the RETC634W activating mutation. The effects of the inhibitors on VEGFR2, PI3K/Akt and mitogen-activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the RAF265+ ZSTK474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated VEGFR2 for RAF265+ ZSTK474 and a signal reduction in activated Akt for ZSTK474. The activated ERK signal also decreased after RAF265 and RAF265+ ZSTK474 treatments. Alone and in combination with ZSTK474, RAF265 induced a sustained increase in necrosis. Only RAF265, alone and combined with ZSTK474, prompted a significant drop in calcitonin production. Combination therapy using RAF265 and ZSTK47 proved effective in MTC, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive MTC.

Mitogen-Activated Protein Kinase Pathway: Genetic Analysis of 95 Adrenocortical Tumors.

Mitogen-activated protein kinase (MAPK) pathway is often deregulated in adrenocortical tumors (ACT) but with no concrete data confirming alteration rate. The objective of this study was to evaluate genetic alterations in key components of MAPK pathway. We found one BRAF mutation (p.V600E) and four HRAS silent mutations. No alteration was found in NRAS, KRAS, EGFR genes. The patient carrying BRAF mutation was further characterized by investigating his biomolecular and clinico-pathological findings. Therefore, even if MAPK signaling is activated in ACT, our results suggest that genetic alterations do not seem to represent a frequent mechanism of ACT tumorigenesis.

The combination of RAF265, SB590885, ZSTK474 on thyroid cancer cell lines deeply impact on proliferation and MAPK and PI3K/Akt signaling pathways.

Papillary thyroid cancer (PTC) is the most frequent thyroid cancer entity, accounting for 88 % of cases. It may metastasize and loose iodine uptake capability, preventing any radioiodine or surgical treatment. The main gene altered in PTC is BRAF, which is found altered in over 50 % of cases. Moreover MAPK and PI3K/Akt pathways are greatly implicated in PTC development. Many target therapies for PTC are currently under investigation, unfortunately without the expected results. Aim of this study was to characterized the preclinical effectiveness of novel promising drugs, RAF265, SB590885 and ZSTK474 in 3 thyroid cancer cell lines (BCPAP, K1, 8505C). RAF265 and SB590885 target differentially BRAF, while ZSTK474 acts on PI3K. IC50 demonstrated high drug activities ranging from 0.1 to 6.2 µM, depending on drugs and cell type, while combination index revealed an interesting synergistic effect of combination regimen (RAF265 + ZSTK474 and SB590885 + ZSTK474) in almost all cell lines. Moreover this synergistic effect was particularly evident by Western blot, whereas dual MAPK and PI3K/Akt inhibition was detected. In addition, treating cells with SB590885 induced marked morphological changes, leading to massive vacuolization. This suggests an activation of apoptotic process, as underlined by Annexin V flow cytometry analysis. Also cell cycle was altered in treated cells, without evidence of a common pattern, but rather with a more specific effect relying on single drug or combination regimen used. Since beneficial effects of in vitro combination regimen (RAF265 + ZSTK474 and SB590885 + ZSTK474), it is recommended additional investigation. These data suggest the potential use of combination regimen in in vivo experiment or afterwards in human PTC.

A Novel TSH Receptor Gene Variant Associated with Non-Autoimmune Hyperthyrotropinemia: A Case Report.

BACKGROUND: Resistance to TSH is defined as reduced sensitivity to normal, biologicallyactive TSH, and abnormally high levels of TSH are needed to achieve normal levels of thyroid hormones. CASE PRESENTATION: A 15-year-old female patient, having been treated since childhood with levothyroxine for hyperthyrotropinemia was referred to our institution complaining of tachycardia after the levothyroxine therapy had been increased. Thyroid ultrasound features were normal, and thyroid antibodies were negative. The therapy was gradually tapered in light of the symptoms, although subclinical hypothyroidism was evident at thyroid function tests. First-degree relatives were tested for thyroid function, and the father was also found to have a previously-unknown subclinical hypothyroidism. The patient underwent genetic testing for TSH receptor (TSHR) gene mutations, which revealed a gene variant hitherto not described: p.C598R (c.1792T>C). The father was also tested and was found to carry the same mutation, while other first-degree relatives were wild-type for the TSHR gene. An in-silico analysis was performed, which revealed a loss-of-function phenotype corresponding to the described variant, suggesting a novel loss-of-function TSH receptor gene mutation. CONCLUSION: In this case report, we present a novel loss-of-function gene mutation in the TSH receptor gene associated with a TSH resistance phenotype.

Tumor Grade and Molecular Characteristics Associated with Survival in Sporadic Medullary Thyroid Carcinoma.

Background: The International Medullary Thyroid Carcinoma Grading System (IMTCGS) divides medullary thyroid carcinoma (MTC) into two categories, high- and low-grade tumors, which has a profound impact on patient outcomes. The aim of this study was to explore the association between IMTCGS grading, clinical data, and molecular status in sporadic MTC. Methods: A retrospective cohort study was performed on consecutive sporadic MTCs from patients undergoing initial surgery between January 2000 and January 2022 at the Padua Endocrine Surgery Unit. Clinical, pathological, and follow-up data were collected, tumors were graded, and somatic mutations of RET and RAS genes were analyzed. Patient outcomes were based on Ct levels and MTC-related deaths. Survival analyses were carried out employing the Kaplan-Meier method and the log-rank test. A Cox proportional hazard regression model was employed for multivariable survival analysis with the following covariates: somatic RET mutation, MTC stage at diagnosis, sex, age at diagnosis, and IMTCGS grade. Results: We included 141 consecutive sporadic MTCs. The median follow-up was 80.0 months (interquartile ranges: 41.5-122.5 months). Seventeen patients (12.1%) died from disease-related causes. 107/141 (76.9%) were classified as low-grade tumors, 32/141 (23.1%) as high-grade. Patients carrying a RET mutation had more aggressive features and shorter disease-specific survival (DSS) (p = 0.001) and were more frequently classified high-grade than low-grade MTC (p < 0.001). At multivariable survival analysis, only IMTCGS grading was independently associated with DSS (hazard ratio 8.8 [confidence interval: 2.7-28.3], p = 0.005). RET mutations, in particular RET-M918T, were more frequent in high-grade than in low-grade MTC (68.8% vs. 29.4% mutated in RET, 46.9% vs. 12.7% mutated in RET-M918T; p < 0.001). None of the high-grade tumors was mutated in the RAS gene, but the mutation was present in 11.8% of low-grade tumors. Conclusions: IMTCGS grading was associated with DSS independently of other clinical, pathological, and molecular factors. Moreover, MTC grading was associated with RET and RAS patterns, which explains, at least in part, the molecular basis of the aggressive behavior of high-grade MTC.

Sentinel lymph node mapping: current applications and future perspectives in thyroid carcinoma.

Sentinel lymph node (SLN) mapping is a standard, minimally-invasive diagnostic method in the surgical treatment of many solid tumors, as for example melanoma and breast cancer, for detecting the presence of regional nodal metastases. A negative SLN accurately indicates the absence of metastases in the other regional lymph nodes (LN), thus avoiding unnecessary lymph nodal dissection. Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma (TC) with cervical LN metastases at diagnosis in 20-90%, and nodal involvement correlates with local persistence/recurrence. The SLN in PTC is an intraoperative method for staging preoperative N0 patients and for detecting metastatic LNs "in and outside" the cervical LN central compartment; it represents an alternative method to prophylactic central neck node dissection. In this review we summarize different methods and results of the use of SLN in TC. The SLN identification techniques currently used include the selective vital-dye (VD) method, 99mTc-nanocolloid planar lymphoscintigraphy with intraoperative use of a hand-held gamma probe (LS), the combination LS + VD, and the combination LS and preoperative SPECT-CT (LS + SPECT/CT). The application of the SLN procedure in TC has been described in many studies, however, the techniques are heterogeneous, and the role of SLN in TC, with indications, results, advantages and limits, is still debated.

Biological activities of the aerial and undergound parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) from the Italian Alps.

This study investigated the bioactivity of both aerial (GNAR) and underground (GNUG) parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) (Orchidaceae). The obtained data proved interesting when the samples were tested in two adrenocortical cancer cell lines (SW13 and H295R). In particular, the GNAR 80% methanol extract distinctly inhibited their viability after 24 h at a concentration of 1 µg/µL by MTT assay and trypan blue dye exclusion method. Cell morphology evaluation by means Wright's staining also showed significant results, particularly in SW13 cells under the effect of both extracts. GNAR extract was able to scavenge the DPPH radical better than GNUG extract. It also was more active in albumin denaturation (a maximum % denaturation equal to 463.0 ± 8.3 vs 77.3 ± 13.3) and protease inhibition (a maximum % inhibition equal to 138.5 ± 7.0 vs 2.1 ± 2.0) tests. The results highlighted an important antitumor activity of G. nigra in vitro that deserves to be further studied.

Effects of Handroanthus impetiginosus (Mart. ex DC.) Mattos extract on inflammatory, immune, atherogenic profile and differentiation in THP-1 cell line.

Handroanthus impetiginosus (Mart. ex DC.) Mattos is a plant from Central-South America that possesses different pharmacological activities. Plant extract was tested in THP-1 cell model, a human cell line used to study monocyte/macrophage functions. First, the plant effects on cell viability were evaluated, demonstrating no harmful consequences even at the higher concentrations (200 µg/ml). Thus, anti-inflammatory activity was investigated in gene and protein expression by RT-qPCR and ELISA methods, resulting in a reduction of pro-inflammatory cytokines after Handroanthus impetiginosus treatment. Similarly, NF-kB nuclear translocation was decreased according to confocal images and ImageStream X -analysis. Subsequently, in macrophage differentiated THP-1, CD36 mRNA and protein expression was inhibited in a concentration-dependent manner together with cell morphology changes during treatment. In addition, modified LDL-derived cholesterol uptake by THP-1 cells was reduced after plant extract incubation. Handroanthus impetiginosus showed anti-inflammatory and immunomodulating properties that may pave the way for future characterization in higher models.

Circulating miR-146a predicts glucocorticoid response in thyroid eye disease.

Objective: Thyroid eye disease (TED) is an immune-mediated disorder of the eye. Intravenous glucocorticoid (GC) is the first-line treatment for patients with active moderate-to-severe TED. However, the response rate is between 50% and 80%. There are still no simple and reliable markers of responsiveness to GC therapy. We aimed to explore the possible role of miR-146a and miR-21 as predictors of responsiveness to GC treatment in TED. Methods: We carried out a prospective longitudinal study on 30 consecutive adult patients with active moderate-to-severe TED and eligible for GC therapy. All patients received the standard GC treatment with methylprednisolone i.v. In cases of progressive worsening of Gorman Score for diplopia or with duction restriction <30° in at least two consecutive controls, patients also underwent orbital radiotherapy. Response to GC treatment was defined as a decrease of two or more points in the clinical activity score (CAS) or CAS <4/10 at 24 weeks. Circulating miRNAs were extracted from patients' serum and quantified by real-time PCR. Results: Twenty-three (77%) patients responded to GC. Thyroid surgery, higher CAS, greater proptosis and higher pre-treatment circulating levels of miR-146a emerged as predictive factors of responsiveness to GC. A ROC analysis revealed that miR-146a could predict responsiveness to GC with a positive predictive value of 100%. Conclusion: This is the first study investigating the role of pre-treatment circulating miR-21 and miR-146a to predict responsiveness to GC in TED. miR-146a emerged as a simple, objective, new marker of GC sensitivity that could be used to avoid ineffective administration of GC therapy to TED patients.

SerpinB3 Upregulates Low-Density Lipoprotein Receptor-Related Protein (LRP) Family Members, Leading to Wnt Signaling Activation and Increased Cell Survival and Invasiveness.

Abnormal activation of the Wnt-ß-catenin signaling cascade is involved in tumor growth and dissemination. SerpinB3 has been shown to induce ß-catenin, and both molecules are overexpressed in tumors, particularly in those with poor prognoses. The aim of this study was to evaluate the ability of SerpinB3 to modulate the Wnt pathway in liver cancer and in monocytic cells, the main type of inflammatory cells in the tumor microenvironment. The Wnt cascade, Wnt co-receptors, and low-density lipoprotein receptor-related protein (LRP) members were analyzed in different cell lines and human monocytes in the presence or absence of SerpinB3. The Wnt-ß-catenin axis was also evaluated in liver tumors induced in mice with different extents of SeprinB3 expression. In monocytic cells, SerpinB3 induced a significant upregulation of Wnt-1/7, nuclear ß-catenin, and c-Myc, which are associated with increased cell lifespan and proliferation. In liver tumors in mice, the expression of ß-catenin was significantly correlated with the presence of SerpinB3. In hepatoma cells, Wnt co-receptors LRP-5/6 and LRP-1, implicated in cell survival and invasiveness, were upregulated by SerpinB3. The LRP pan-inhibitor RAP not only induced a decrease in LRP expression, but also a dose-dependent reduction in SerpinB3-induced invasiveness. In conclusion, SerpinB3 determines the activation of the Wnt canonical pathway and cell invasiveness through the upregulation of LRP family members.

Validation of miRNAs as diagnostic and prognostic biomarkers, and possible therapeutic targets in medullary thyroid cancers.

Introduction: Medullary thyroid cancer (MTC) is a rare type of neuroendocrine tumor that produces a hormone called calcitonin (CT). Thyroidectomy is the preferred treatment for MTC, as chemotherapy has been shown to have limited effectiveness. Targeted therapy approaches are currently being used for patients with advanced, metastatic MTC. Several studies have identified microRNAs, including miR-21, as playing a role in the development of MTC. Programmed cell death 4 (PDCD4) is a tumor suppressor gene that is an important target of miR-21. Our previous research has shown that high levels of miR-21 are associated with low PDCD4 nuclear scores and high CT levels. The aim of this study was to investigate the potential of this pathway as a novel therapeutic target for MTC. Methods: We used a specific process to silence miR-21 in two human MTC cell lines. We studied the effect of this anti-miRNA process alone and in combination with cabozantinib and vandetanib, two drugs used in targeted therapy for MTC. We analyzed the effect of miR-21 silencing on cell viability, PDCD4 and CT expression, phosphorylation pathways, cell migration, cell cycle, and apoptosis. Results: Silencing miR-21 alone resulted in a reduction of cell viability and an increase in PDCD4 levels at both mRNA and protein levels. It also led to a reduction in CT expression at both mRNA and secretion levels. When combined with cabozantinib and vandetanib, miR-21 silencing did not affect cell cycle or migration but was able to enhance apoptosis. Conclusion: Silencing miR-21, although not showing synergistic activity with TKIs (tyrosine kinase inhibitors), represents a potential alternative worth exploring as a therapeutic target for MTC.

Prognostic value of putative circulating cancer stem cells in patients undergoing hepatic resection for colorectal liver metastasis.

BACKGROUND: Although surgery is the gold standard treatment of hepatic metastasis from colorectal cancer (CRC), many patients ultimately die of their disease. We tested the hypothesis that the detection of circulating tumor cells (CTC) might identify patients at high risk of dying of disease recurrence after apparently radical liver surgery. METHODS: We considered 50 patients undergoing radical surgery for liver-confined hepatic metastasis from CRC. The expression of a panel of cancer-related genes, as assessed by quantitative real-time PCR, was used to detect CTC in the peripheral blood of these patients immediately before surgery. Survival analysis was performed by the Cox regression model. RESULTS: Univariate analysis of the expression levels of CD133 (a marker of colon cancer stem cells) and survivin (an antiapoptotic factor) resulted in statistically significant association with patient survival [hazard ratio (HR) 2.7, 95% confidence interval (CI) 1.9-3.7, P < 0.0001; and hazard ratio 2.1, 95% CI 1.4-3.2, P < 0.0001, respectively]. Remarkably, multivariate analysis found that only the transcriptional amount of CD133 resulted in statistical significance (HR 2.6, 95% CI 1.9-3.6, P < 0.0001), indicating that this biomarker can independently predict the survival of these patients. CONCLUSIONS: CD133-positive CTC may represent a suitable prognostic marker to stratify the risk of patients who undergo liver resection for CRC metastasis, which opens the avenue to identifying and potentially monitoring the patients who are most likely to benefit from adjuvant treatments.