Luca Lavra

The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas

Background Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC. Methodology/Principal Findings We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation. Conclusions/Significance Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers.

Repression of the Antiapoptotic Molecule Galectin-3 by Homeodomain-Interacting Protein Kinase 2-Activated p53 Is Required for p53-Induced Apoptosis

ABSTRACT Galectin 3 (Gal-3), a member of the β-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3.

Large needle aspiration biopsy and galectin-3 determination in selected thyroid nodules with indeterminate FNA-cytology.

Thyroid fine-needle aspiration biopsy (FNA)-cytology is widely used for the preoperative characterisation of thyroid nodules but this task is difficult for follicular lesions, which often remain undefined. We propose a strategy for improving the preoperative characterisation of selected follicular thyroid proliferations, which is based on large needle aspiration biopsy (LNAB) and galectin-3 expression analysis. Eighty-five thyroid specimens were obtained by LNAB (20-gauge needles) from thyroid nodules with indeterminate follicular FNA-cytology. Aspirated material was processed as a tissue microbiopsy to obtain cell blocks for both cyto/histo-morphological evaluation and galectin-3 expression analysis, by using a purified monoclonal antibody to galectin-3 and a biotin-free immunoperoxidase staining method. Preoperative diagnosis was compared to the final histology. LNAB and cell-block technique allow a preliminary distinction between nodules with a homogeneous microfollicular/trabecular structure, as frequently observed in tumours, and lesions with mixed normo–micro–macrofollicular architecture, as observed in goitre. Furthermore, LNAB provides optimal substrates for galectin-3 expression analysis. Among 85 cases tested, 14 galectin-3-positive cases were discovered preoperatively (11 thyroid cancers and three adenomas confirmed at the final histology), whereas galectin-3-negative cases were 71 (one carcinoma and 70 benign proliferations at the final histology). Sensitivity, specificity and diagnostic accuracy of this integrated morphologic and phenotypic diagnostic approach were 91.6, 97.2 and 95.3%, respectively. In conclusion, LNAB plus galectin-3 expression analysis when applied preoperatively to selected thyroid nodules candidate to surgery can potentially reduce unnecessary thyroid resections.

MicroRNA miR-24 promotes cell proliferation by targeting the CDKs inhibitors p27Kip1 and p16INK4a

Cell cycle progression is controlled by numerous mechanisms ensuring correct cell division. The transition from one cell cycle phase to another occurs in an orderly fashion and is regulated by different cellular proteins. Therefore an alteration of the regulatory mechanisms of the cell cycle results in uncontrolled cell proliferation, which is a distinctive feature of human cancers. Recent evidences suggest that microRNAs (miRs) may also control the levels of multiple cell cycle regulators and therefore control cell proliferation. In fact miRs are a class of small non‐coding RNAs, which modulate gene expression. They are involved in numerous physiological cellular processes and most importantly accumulating evidence indicates that many miRs are aberrantly expressed in human cancers. In this report we describe that miR‐24 directly targets p27Kip1 and p16Ink4a in primary keratinocyte and in different cancer derived cell lines promoting their proliferation, suggesting that miR‐24 is involved in cyclin‐dependent kinase inhibitors post‐transcriptional regulation and that upregulation of miR‐24 may play a role in carcinogenesis. J. Cell. Physiol. 228: 2015–2023, 2013.

More than apples and oranges - Detecting cancer with a fruit fly's antenna

Cancer cells and non-cancer cells differ in their metabolism and they emit distinct volatile compound profiles, allowing to recognise cancer cells by their scent. Insect odorant receptors are excellent chemosensors with high sensitivity and a broad receptive range unmatched by current gas sensors. We thus investigated the potential of utilising the fruit flys olfactory system to detect cancer cells. Using in vivo calcium imaging, we recorded an array of olfactory receptor neurons on the fruit flys antenna. We performed multidimensional analysis of antenna responses, finding that cell volatiles from different cell types lead to characteristic response vectors. The distances between these response vectors are conserved across flies and can be used to discriminate healthy mammary epithelial cells from different types of breast cancer cells. This may expand the repertoire of clinical diagnostics, and it is the first step towards electronic noses equipped with biological sensors, integrating artificial and biological olfaction.

Gal-3 is stimulated by gain-of-function p53 mutations and modulates chemoresistance in anaplastic thyroid carcinomas.

Galectin‐3 (Gal‐3) is an anti‐apoptotic molecule of the β‐galactoside‐binding lectin family. Gal‐3 is down‐regulated by wt‐p53 and this repression is required for p53‐induced apoptosis. Since poorly differentiated thyroid carcinomas (PDTCs) and anaplastic thyroid carcinomas (ATCs) frequently harbour p53 mutations, we asked whether Gal‐3 expression and activity could be influenced by such mutations in these tumours. We found a positive correlation between Gal‐3 expression and p53 mutation in human thyroids and in thyroid carcinoma cell lines (TCCLs) harbouring different p53 mutations. Gal‐3 was over‐expressed in most ATCs and TCCLs, especially those with the most frequently detected p53 mutation (p53R273H). Over‐expression of p53R273H in two p53‐null cells (SAOS‐2 and SW‐1736) as well as in two wt‐p53‐carrying TCCLs (TPC‐1 and K1), stimulated Gal‐3 expression, while interference with p53R273H endogenous expression in ARO cells down‐regulated Gal‐3 expression. Conversely, over‐expression of wt‐p53 in ARO cells restored the inhibitory effect on Gal‐3 expression. ARO cells are highly resistant to apoptosis and express both p53 and Gal‐3, which are increased upon cisplatin treatment. Interference with Gal‐3 expression in these cells stimulated their chemosensitivity. In conclusion, gain‐of‐function p53 mutant acquires the de novo ability to stimulate Gal‐3 expression and to increase chemoresistance in ATCs. Copyright

Investigation of VOCs associated with different characteristics of breast cancer cells.

The efficacy of breath volatile organic compounds (VOCs) analysis for the screening of patients bearing breast cancer lesions has been demonstrated by using gas chromatography and artificial olfactory systems. On the other hand, in-vitro studies suggest that VOCs detection could also give important indications regarding molecular and tumorigenic characteristics of tumor cells. Aim of this study was to analyze VOCs in the headspace of breast cancer cell lines in order to ascertain the potentiality of VOCs signatures in giving information about these cells and set-up a new sensor system able to detect breast tumor-associated VOCs. We identified by Gas Chromatography-Mass Spectrometry analysis a VOCs signature that discriminates breast cancer cells for: i) transformed condition; ii) cell doubling time (CDT); iii) Estrogen and Progesterone Receptors (ER, PgR) expression, and HER2 overexpression. Moreover, the signals obtained from a temperature modulated metal oxide semiconductor gas sensor can be classified in order to recognize VOCs signatures associated with breast cancer cells, CDT and ER expression. Our results demonstrate that VOCs analysis could give clinically relevant information about proliferative and molecular features of breast cancer cells and pose the basis for the optimization of a low-cost diagnostic device to be used for tumors characterization.

Thyroid cancer imaging in vivo by targeting the anti-apoptotic molecule galectin-3.

Background The prevalence of thyroid nodules increases with age, average 4–7% for the U.S.A. adult population, but it is much higher (19–67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. Methods The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific 99mTc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 µCi of 99mTc-labeled mAb to galectin-3 (30 µg protein/in 100 µl saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. Findings Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. Conclusions These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed.

Homeodomain-interacting protein kinase2 in human idiopathic pulmonary fibrosis.

Homeodomain‐interacting protein kinase 2 (Hipk2) is an emerging player in cell response to genotoxic agents that contributes to the cells decision between cell cycle arrest or apoptosis. HIPK2 acts as co‐regulator of an increasing number of transcription factors and modulates many different basic cellular processes such as apoptosis, proliferation, DNA damage response, differentiation. Idiopathic pulmonary fibrosis (IPF) is characterized by an anatomical disarrangement of the lung due to fibroblast proliferation, extracellular matrix deposition and lung function impairment. Although the role of inflammation is still debated, attention has been focused on lung cell functions as fibroblast phenotype and activity. Aim of the present study was to analyze the loss of heterozygosity (LOH) at HIPK2 locus 7q32.34 in human lung fibroblasts and the HIPK2 expression in 15 IPF samples and in four primary fibroblast cell cultures isolated from IPF biopsies using semi‐quantitative RT‐PCR, Western blots and immunohistochemistry. We demonstrated a frequency of LOH in IPF fibroblasts of 46% for the internal D7S6440 microsatellite and 26.6% for the external D7S2468 microsatellite. Furthermore, we demonstrated low HIPK2 protein expression in those fibroblasts from IPF patients that present the HIPK2 LOH. The restoration of HIPK2 expression in IPF derived cells induced a significant reduction of chemoresistance after treatment with cisplatin. The results obtained allow us to hypothesize that HIPK2 dysfunction may play a role in fibroblasts behavior and in IPF pathogenesis. HIPK2 may be considered as a novel potential target for anti‐fibrosis therapy. J. Cell. Physiol. 228: 235–241, 2013.

Frizzled-1 is down-regulated in follicular thyroid tumours and modulates growth and invasiveness†

The mechanisms of follicular thyroid carcinoma (FTC) transformation and progression are not well understood. Previously, we detected LOH at 7q21 in all FTCs examined, indicating that loss of genetic material in that region is a common trait in these lesions. To analyse the effects of LOH on gene expression, we performed an analysis of the mRNA expression levels of six different genes, located at 7q21.1–7q21.3. A total of 23 lesions, including eight follicular hyperplasias (FHs), eight follicular adenomas (FAs), two FTCs and five papillary thyroid carcinomas (PTCs) were analysed. The Frizzled‐1 (FZD‐1) gene, located at 7q21.13, showed the lowest levels of mRNA expression. Down‐regulation of FZD‐1 expression was also confirmed in an independent series of 69 follicular neoplastic lesions compared to 25 PTCs, analysed by quantitative RT–PCR. In vitro studies showed that FZD‐1 expression was also markedly reduced at both protein and mRNA levels in three FTC‐derived cell lines (FRO, WRO and FTC‐133), while it was normal in the three PTC‐derived cell lines (Ca300, Ca301 and K1) examined. We demonstrated that over‐expression of FZD‐1 in 3 FTC‐derived cells decreased invasiveness and proliferation rate, indicating a possible pathogenetic role. In addition, FZD‐1 RNA interference in the PTC‐derived cell line K1 increased invasiveness. Our data indicated that FZD‐1 is involved in growth of follicular tumours and may be considered as a novel marker of this type of tumour. Copyright