Luciana Bueno Ferreira

Both osteopontin-c and osteopontin-b splicing isoforms exert pro-tumorigenic roles in prostate cancer cells.

Alternative splicing of the osteopontin (opn, spp1) gene generates three protein splicing isoforms (OPN‐SI), designated as OPNa, OPNb, and OPNc, which have demonstrated specific roles in different tumor models. This work aims to investigate the roles of each OPN‐SI in prostate cancer (PCa) progression by using in vivo and in vitro functional assays.

Influence of Lycopene on Cell Viability, Cell Cycle, and Apoptosis of Human Prostate Cancer and Benign Hyperplastic Cells

Prostate cancer is the most common malignancy in men and the second leading cause of cancer-related mortality in men of the Western world. Lycopene has received attention because of its expcted potential to prevent cancer. In the present study, we evaluated the influence of lycopene on cell viability, cell cycle, and apoptosis of human prostate cancer cells and benign prostate hyperplastic cells. Using MTT assay, we observed a decrease of cell viability in all cancer cell lines after treatment with lycopene, which decreased the percentage of cells in G0/G1 phase and increased in S and G2/M phases after 96 h of treatment in metastatic prostate cancer cell lineages. Flow citometry analysis of cell cycle revealed lycopene promoted cell cycle arrest in G0/G1 phase after 48 and 96 h of treatment in a primary cancer cell line. Using real time PCR assay, lycopene also induced apoptosis in prostate cancer cells with altered gene expression of Bax and Bcl-2. No effect was observed in benign prostate hyperplasia cells. These results suggest an effect of lycopene on activity of human prostate cancer cells.

Extracellular matrix secreted by reactive stroma is a main inducer of pro-tumorigenic features on LNCaP prostate cancer cells

Tumor microenvironment modifications are related to the generation of reactive stroma and to critical events in cancer progression, such as proliferation, migration and apoptosis. In order to clarify these cellular interactions mediated by reactive stroma, we investigated the effects of cell-cell contacts, and the influence of soluble factors and extracellular matrix (ECM) secreted by Benign Prostate Hyperplasia (BPH) reactive stroma over LNCaP prostate tumor cells. Using in vitro functional assays, we demonstrated that ECM strongly stimulated LNCaP cell proliferation and migration, while inhibiting apoptosis, and inducing a deregulated expression pattern of several genes related to prostate cancer (PCa) progression. Conversely, reactive stromal cells per se and their secreted conditioned medium partially modulated these pro-tumorigenic events. These data indicate that secreted ECM in reactive stroma microenvironment contains key molecules that positively modulate important cancer hallmarks.

Osteopontin expression is correlated with differentiation and good prognosis in medullary thyroid carcinoma

BACKGROUND Osteopontin (OPN) or secreted phosphoprotein 1 (SPP1) is a matricellular glycoprotein whose expression is elevated in various types of cancer and has been shown to be involved in tumourigenesis and metastasis in many malignancies, including follicular cell-derived thyroid carcinomas. Its role in C-cell-derived thyroid lesions and tumours remains to be established. OBJECTIVE The objective of this study is to clarify the role of OPN expression in the development of medullary thyroid carcinoma (MTC). METHODS OPN expression was analysed in a series of 116 MTCs by immunohistochemistry and by qPCR mRNA quantification of the 3 OPN isoforms (OPNa, OPNb and OPNc) in six cases from which fresh frozen tissue was available. Statistical tests were used to evaluate the relationship of OPN expression and the clinicopathological and molecular characteristics of patients and tumours. RESULTS OPN expression was detected in 91 of 116 (78.4%) of the MTC. We also observed high OPN expression in C-cell hyperplasia as well as in C-cells scattered in the thyroid parenchyma adjacent to the tumours. OPN expression was significantly associated with smaller tumour size, PTEN nuclear expression and RAS status, and suggestively associated with non-invasive tumours. OPNa isoform was expressed significantly at higher levels in tumours than in non-tumour samples. OPNb and OPNc presented similar levels of expression in all samples. Furthermore, OPNa isoform overexpression was significantly associated with reduced growth and viability in the MTC-derived cell line (TT). CONCLUSION The expression of OPN in normal C-cells and C-cell hyperplasia suggests that OPN is a differentiation marker of C-cells, rather than a marker of biological aggressiveness in this setting. At variance with other cancers, OPN expression is associated with good prognostic features in MTC.

Osteopontin-a splice variant is overexpressed in papillary thyroid carcinoma and modulates invasive behavior

Osteopontin (OPN) is a matricellular protein overexpressed in cancer cells and modulates tumorigenesis and metastasis, including in thyroid cancer (TC). The contribution of each OPN splice variant (OPN-SV), named OPNa, OPNb and OPNc, in TC is currently unknown. This study evaluates the expression of total OPN (tOPN) and OPN-SV in TC tissues and cell lines, their correlation with clinicopathological, molecular features and their functional roles. We showed that tOPN and OPNa are overexpressed in classic papillary thyroid carcinoma (cPTC) in relation to adjacent thyroid, adenoma and follicular variant of papillary thyroid carcinoma (fvPTC) tissues. In cPTC, OPNa overexpression is associated with larger tumor size, vascular invasion, extrathyroid extension and BRAFV600E mutation. We found that TC cell lines overexpressing OPNa exhibited increased proliferation, migration, motility and in vivo invasion. Conditioned medium secreted from cells overexpressing OPNa induce MMP2 and MMP9 metalloproteinases activity. In summary, we described the expression pattern of OPN-SV in cPTC samples and the key role of OPNa expression on activating TC tumor progression features. Our findings highlight OPNa variant as TC biomarker, besides being a putative target for cPTC therapeutic approaches.

Poorly Differentiated and Undifferentiated Thyroid Carcinomas.

Thyroid cancer is the most common endocrine malignancy and its incidence goes on increasing worldwide. The majority of thyroid tumours comprise well-differentiated (papillary and follicular) thyroid carcinomas that usually carry an excellent prognosis, while a minority progress to poorly differentiated carcinoma (PDTC) and, ultimately, to the highly aggressive and lethal undifferentiated carcinoma (UTC). Recently, some major advances have been made on the histologic and imunohistochemical identification, as well as on the molecular characterization of PDTC and UTC. In this review we summarize the most recent immunohistochemical and molecular findings in PDTC and UTC, giving a particular emphasis to the diagnostic and prognostic meaning of the genetic alterations.

Calcitonin receptor expression in medullary thyroid carcinoma

Background Calcitonin expression is a well-established marker for medullary thyroid carcinoma (MTC); yet the role of calcitonin receptor (CTR), its seven-transmembrane G-protein coupled receptor, remains to be established in C-cells derived thyroid tumors. The aim of this work was to investigate CTR expression in MTC and to correlate such expression with clinicopathological features in order to evaluate its possible role as a prognostic indicator of disease aggressiveness and outcome. Methods Calcitonin receptor expression was analyzed in a series of 75 MTCs by immunohistochemistry, and by qPCR mRNA quantification in specimens from four patients. Statistical tests were used to evaluate the correlation between CTR expression and the clinicopathological and molecular characteristics of patients and tumors. Results Calcitonin receptor expression was detected in 62 out of 75 samples (82.7%), whereas 13 of the 75 samples (17.3%) were completely negative. CTR expression was significantly associated with expression of cytoplasmatic phosphatase and tensin homologue deleted on chromosome 10 and osteopontin, as well as with wild type RET/RAS genes and absence of tumor stroma, suggesting that CTR expression do not associate with clinicopathological signs of worse prognosis. Discussion Calcitonin receptor expression appears to be associated in MTC with more differentiated status of the neoplastic cells.

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression.

OPNa Overexpression Is Associated with Matrix Calcification in Thyroid Cancer Cell Lines

Osteopontin (OPN) spliced variants (OPN-SV: OPNa, OPNb, and OPNc) are aberrantly expressed in tumors and frequently associated with cancer progression. This holds true for papillary thyroid carcinoma (PTC), which is the most common type of thyroid cancer (TC). PTC often presents with desmoplasia and dystrophic calcification, including psammoma bodies (PB). This work aimed to investigate total OPN (tOPN) and OPN-SV expression and their association with the presence of PB in the PTC classical variants (cPTC), as well as the involvement of OPN-SV in matrix calcification of TC cell lines. We found that cPTC samples presenting PB showed higher OPN expression levels. In TC cell lines, OPNa overexpression promotes higher matrix calcification and collagen synthesis when compared to that of clones overexpressing OPNb or OPNc. In response to OPN knockdown, calcification was inhibited, paralleled with the downregulation of calcification markers. In conclusion, our data evidenced that OPN expression is associated with the presence of PB in cPTC samples. Among the OPN-SV, OPNa is the main contributor to matrix calcification in tested TC cells, providing clues to a better understanding on the biology and ethiopathogenesis of the calcification process in TC cells.

Abstract 163: Investigation of molecular mechanisms by which PCA3 modulates prostate cancer cell survival

PCA3 is a prostate-specific non coding RNA (ncRNA), involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. We aimed to investigate whether several cancer related genes, including those involved in epithelial-mesenchymal transition (EMT) and AR co-regulators could modulate LNCaP cell survival in response to PCA3 downregulation. We also aimed to promote PCA3 stable silencing through a lentiviral vector containing a PCA3 short hairpin RNA (shRNA) specific sequence. Small interfering RNA (siRNA) or lentivirus vector-based shRNA were used to downregulate PCA3 expression. Upon PCA3 downregulation, cells were analyzed by qRT-PCR to investigate mRNA expression. Flow cytometry was performed to analyze the percentage of LNCaP GFP + cells and trypan blue staining to analyze the number of viable cells after PCA3 stable silencing. Among 14 AR co-regulators genes investigated, 12 were upregulated in siPCA3-transfected cells in relation to controls. We also found that among 84 cancer-related genes tested, 16 were differentially expressed in LNCaP siPCA3-transfected cells when compared to transfected cells with a scramble sequence. Of these, 30% are genes coding for signal transduction molecules and transcription factors. Gene expression profile of EMT-related genes revealed that E-cadherin, Claudin-3, Cytokeratin-18, Snail, Twist and Slug are upregulated in LNCaP siPCA3-transfected cells compared to control, while Claudin-4, Cytokeratin-8 and Vimentin are downregulated. LNCaP cells transduced with lentiviral vectors carrying the GFP gene and shPCA3 stably dowregulated PCA3 expression, producing a reduction of 60% of LNCaP GFP+ cells compared to shScr transduced or non-transduced LNCaP cells. Our results suggest that the decrease in the number of LNCaP viable cells upon siPCA3 transfection is not modulated by the classical EMT program. Observed upregulation of most tested AR co-regulators upon PCA3 silencing indicates that this non-coding RNA could negatively modulate the transcription of these genes. Our data further suggest that that this ncRNA perform a regulatory role in gene expression, being able to modulate gene expression of several signaling pathways related to cancer. Finally, stable silencing of PCA3 expression using lentivirus vector shows potential as a PCa therapeutic approach by negatively modulating cell survival. Citation Format: Ana Emilia Goulart, Luciana B. Ferreira, Paula Priscilla de Freitas, Nadia Batoreu, MARTIN H. BONAMINO, Etel Rodrigues Pereira Gimba. Investigation of molecular mechanisms by which PCA3 modulates prostate cancer cell survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 163. doi:10.1158/1538-7445.AM2015-163