Resolving the role of podoplanin in the motility of papillary thyroid carcinoma-derived cells using RNA sequencing.

The intracellular level of podoplanin (PDPN), a transmembrane protein of still unclear function, is frequently altered in metastatic tumors. High expression of PDPN is frequently observed in papillary thyroid cancer (PTC) specimens. Similarly, PTC-derived cell lines (BCPAP and TPC1, harboring the BRAF V600E mutation and RET/PTC1 fusion, respectively), also present enhanced PDPN yield. We previously reported that depletion of PDPN impairs migration of TPC1 cells, but augments metastasis of BCPAP cells. Interestingly, this phenomenon stays in contrast to the migratory pattern observed for wild-type cells, where TPC1 exhibited higher motility than BCPAP cells. Here, we aimed to elucidate the potential role of PDPN in regulation of molecular mechanisms leading to the diverse metastatic features of the studied PTC-derived cells. We consider that this phenomenon may be caused by alternative regulation of signaling pathways due to the presence of the mutated BRAF allele or RET/PTC1 fusion. The high-throughput RNA sequencing (RNA-seq) technique was used to uncover the genes and signaling pathways affected in wild-type and PDPN-depleted TPC1 and BCPAP cells. We found that changes in the expression of various factors of signaling pathways, like RHOA and RAC1 GTPases and their regulators, are linked with both high PDPN levels and presence of the BRAF V600E mutation. We imply that the suppressed motility of wild-type BCPAP cells results from overactivation of RHOA through natively high PDPN expression. This process is accompanied by inhibition of the PI3K kinase and consequently RAC1, due to overactivation of RAS-mediated signaling and the PTEN regulator.

Simvastatin Coadministration Modulates the Electrostatically Driven Incorporation of Doxorubicin into Model Lipid and Cell Membranes.

Understanding the interactions between drugs and lipid membranes is a prerequisite for finding the optimal way to deliver drugs into cells. Coadministration of statins and anticancer agents has been reported to have a positive effect on anticancer therapy. In this study, we elucidate the mechanism by which simvastatin (SIM) improves the efficiency of biological membrane penetration by the chemotherapeutic agent doxorubicin (DOX) in neutral and slightly acidic solutions. The incorporation of DOX, SIM, or a combination of them (DOX:SIM) into selected single-component lipid membranes, zwitterionic unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), neutral cholesterol, and negatively charged 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) was assessed using the Langmuir method. The penetration of neutral lipid monolayers by the codelivery of SIM and DOX was clearly facilitated at pH 5.5, which resembles the pH conditions of the environment of cancer cells. This effect was ascribed to partial neutralization of the DOX positive charge as the result of intermolecular interactions between DOX and SIM. On the other hand, the penetration of the negatively charged DMPS monolayer was most efficient in the case of the positively charged DOX. The efficiency of the drug delivery to the cell membranes was evaluated under in vitro conditions using a panel of cancer-derived cell lines (A172, T98G, and HeLa). MTS and trypan blue exclusion assays were performed, followed by confocal microscopy and spheroid culture tests. Cells were exposed to either free drugs or drugs encapsulated in lipid carriers termed cubosomes. We demonstrated that the viability of cancer cells exposed to DOX was significantly impaired in the presence of SIM, and this phenomenon was greatly magnified when DOX and SIM were coencapsulated in cubosomes. Overall, our results confirmed the utility of the DOX:SIM combination delivery, which enhances the interactions between neutral components of cell membranes and positively charged chemotherapeutic agents.

Expression of pendrin and NIS iodide transporters in human breast tumor and peri-tumoral tissue.

Introduction: Thyroid iodide transporters, Na+/I- symporter (NIS) and pendrin (PDS), are responsible for supplying this vital micronutrient for thyroid hormone synthesis by thyroid peroxidase (TPO). Both proteins were shown to be expressed, apart from the thyroid, also in other human tissues, including lactating mammary gland. NIS expression in human breast cancers has been widely studied. On the other hand, until now PDS mRNA levels in breast tumor tissue have been estimated only in high throughput analyses. Previously, we have observed that TPO is expressed in normal and cancerous human breast tissues and shows enzymatic activity. However, biochemical activity of TPO in human breast cancer cells requires iodide transport by NIS and PDS. Therefore, to extend our previous study on TPO expression and function in human breast tumors we performed analysis of NIS and PDS levels in the same group of patients. Material and methods: The study involved detection of NIS and PDS protein levels by immunohistochemistry and Western blotting, as well as mRNA levels by real-time quantitative polymerase chain reaction. Results: Here we provide direct evidence that NIS and PDS are expressed in human breast cancer tissue, with NIS levels being increased and PDS levels decreased in tumor tissue. Interestingly, PDS mRNA levels in breast cancer tissue seem to be influenced by the estrogen receptor status and age of the patients, while NIS mRNA levels were dependent on histological type of the tumor. Conclusions: This study provides valuable information important for consideration in diagnostic or therapeutic application of radioiodine in breast cancer management.

Cellular and molecular basis of thyroid autoimmunity.

Autoimmune thyroid disease (AITD) is the most common human autoimmune disease. The two major clinical manifestations of AITD are Graves' disease and Hashimoto's thyroiditis (HT). AITD is characterized by lymphocytic infiltration of the thyroid gland, leading either to follicular cell damage, thyroid gland destruction, and development of hypothyroidism (in HT) or thyroid hyperplasia, induced by thyroid antibodies which activate thyrotropin receptor (TSHR) on thyrocytes, leading to hyperthyroidism. The aim of this review is to present up-to-date picture of the molecular and cellular mechanisms that underlie the pathology of AITD. Based on studies involving patients, animal AITD models, and thyroid cell lines, we discuss the key events leading to the loss of immune tolerance to thyroid autoantigens as well as the signaling cascades leading to the destruction of thyroid gland. Special focus is given on the interplay between the environmental and genetic factors, as well as ncRNAs and microbiome contributing to AITD development. In particular, we describe mechanistic models by which SNPs in genes involved in immune regulation and thyroid function, such as CD40, TSHR, FLT3, and PTPN22, underlie AITD predisposition. The clinical significance of novel diagnostic and prognostic biomarkers based on ncRNAs and microbiome composition is also underscored. Finally, we discuss the possible significance of probiotic supplementation on thyroid function in AITD.

The Anti-Tumoral Potential of Phosphonate Analog of Sulforaphane in Zebrafish Xenograft Model.

Isothiocyanates (ITCs) show strong activity against numerous human tumors. Five structurally diverse ITCs were tested in vivo using the zebrafish embryos 6 and 48 h post-fertilization (hpf). The survival rate, hatching time, and gross morphological changes were assessed 24, 48, and 72 h after treatment with all compounds in various doses (1-10 µM). As a result, we selected a phosphonate analog of sulforaphane (P-ITC; 1-3 µM) as a non-toxic treatment for zebrafish embryos, both 6 and 48 hpf. Furthermore, the in vivo anti-cancerogenic studies with selected 3 µM P-ITC were performed using a set of cell lines derived from the brain (U87), cervical (HeLa), and breast (MDA-MB-231) tumors. For the experiment, cells were labeled using red fluorescence dye Dil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine, 10 µg/mL) and injected into the hindbrain ventricle, yolk sac region and Cuvier duct of zebrafish embryos. The tumor size measurement after 48 h of treatment demonstrated the significant inhibition of cancer cell growth in all tested cases by P-ITC compared to the non-treated controls. Our studies provided evidence for P-ITC anti-cancerogenic properties with versatile activity against different cancer types. Additionally, P-ITC demonstrated the safety of use in the living organism at various stages of embryogenesis.

Novel Inhibitor-Based Therapies for Thyroid Cancer-An Update.

Thyroid cancers (TCs) are the most common tumors of the endocrine system and a constant rise in the number of TC cases has been observed for the past few decades. TCs are one of the most frequent tumors in younger adults, especially in women, therefore early diagnosis and effective therapy are especially important. Ultrasonography examination followed by fine needle biopsy have become the gold standard for diagnosis of TCs, as these strategies allow for early-stage detection and aid accurate qualification for further procedures, including surgical treatment. Despite all the advancements in detection and treatment of TCs, constant mortality levels are still observed. Therefore, a novel generation line of targeted treatment strategies is being developed, including personalized therapies with kinase inhibitors. Recent molecular studies on TCs demonstrate that kinase inhibitor-based therapies might be considered as the most promising. In the past decade, new kinase inhibitors with different mechanisms of action have been reported and approved for clinical trials. This review presents an up-to-date picture of new approaches and challenges of inhibitor-based therapies in treatment of TCs, focusing on the latest findings reported over the past two years.

Analysis of the Role of FRMD5 in the Biology of Papillary Thyroid Carcinoma.

BACKGROUND: Thyroid carcinoma (TC) is the most common endocrine system malignancy, and papillary thyroid carcinoma (PTC) accounts for >80% of all TC cases. Nevertheless, PTC pathogenesis is still not fully understood. The aim of the study was to elucidate the role of the FRMD5 protein in the regulation of biological pathways associated with the development of PTC. We imply that the presence of certain genetic aberrations (e.g., BRAF V600E mutation) is associated with the activity of FRMD5. METHODS: The studies were conducted on TPC1 and BCPAP (BRAF V600E) model PTC-derived cells. Transfection with siRNA was used to deplete the expression of FRMD5. The mRNA expression and protein yield were evaluated using RT-qPCR and Western blot techniques. Proliferation, migration, invasiveness, adhesion, spheroid formation, and survival tests were performed. RNA sequencing and phospho-kinase proteome profiling were used to assess signaling pathways associated with the FRMD5 expressional status. RESULTS: The obtained data indicate that the expression of FRMD5 is significantly enhanced in BRAF V600E tumor specimens and cells. It was observed that a drop in intracellular yield of FRMD5 results in significant alternations in the migration, invasiveness, adhesion, and spheroid formation potential of PTC-derived cells. Importantly, significant divergences in the effect of FRMD5 depletion in both BRAF-wt and BRAF-mutated PTC cells were observed. It was also found that knockdown of FRMD5 significantly alters the expression of multidrug resistant genes. CONCLUSIONS: This is the first report highlighting the importance of the FRMD5 protein in the biology of PTCs. The results suggest that the FRMD5 protein can play an important role in controlling the metastatic potential and multidrug resistance of thyroid tumor cells.

The Role of miRNA-7 in the Biology of Cancer and Modulation of Drug Resistance.

MicroRNAs (miRNAs, miRs) are small non-coding RNA (ncRNA) molecules capable of regulating post-transcriptional gene expression. Imbalances in the miRNA network have been associated with the development of many pathological conditions and diseases, including cancer. Recently, miRNAs have also been linked to the phenomenon of multidrug resistance (MDR). MiR-7 is one of the extensively studied miRNAs and its role in cancer progression and MDR modulation has been highlighted. MiR-7 is engaged in multiple cellular pathways and acts as a tumor suppressor in the majority of human neoplasia. Its depletion limits the effectiveness of anti-cancer therapies, while its restoration sensitizes cells to the administered drugs. Therefore, miR-7 might be considered as a potential adjuvant agent, which can increase the efficiency of standard chemotherapeutics.

Lipid membranes exposed to dispersions of phytantriol and monoolein cubosomes: Langmuir monolayer and HeLa cell membrane studies.

The interactions of liquid-crystalline nanoparticles based on lipid-like surfactants, glyceryl monooleate, monoolein (GMO) and 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane, phytantriol (PT) with selected model lipid membranes prepared by Langmuir technique were compared. Monolayers of DPPC, DMPS and their mixture DPPC:DMPS 87:13 mol% were used as simple models of one leaflet of a cell membrane. The incorporation of cubosomes into the lipid layers spread at the air-water interface was followed by surface-pressure measurements and Brewster angle microscopy. The cubosome - membrane interactions lead to the fluidization of the model membranes but this effect depended on the composition of the model membrane and on the type of cubosomes. The interactions of PT cubosomes with lipid layers, especially DMPS-based monolayer were stronger compared with those of GMO-based nanoparticles. The kinetics of incorporation of cubosomal material into the lipid layer was influenced by the extent of hydration of the polar headgroups of the lipid: faster in the case of smaller, less hydrated polar groups of DMPS than for strongly hydrated uncharged choline of DPPC. The membrane disrupting effect of cubosomes increased at longer times of the lipid membrane exposure to the cubosome solution and at larger carrier concentrations. Langmuir monolayer observations correspond well to results of studies of HeLa cells exposed to cubosomes. The larger toxicity of PT cubosomes was confirmed by MTS. Their ability to disrupt lipid membranes was imaged by confocal microscopy. On the other hand, PT cubosomes easily penetrated cellular membranes and released cargo into various cellular compartments more effectively than GMO-based nanocarriers. Therefore, at low concentrations, they may be further investigated as a promising drug delivery tool.

Combinatory Treatment with miR-7-5p and Drug-Loaded Cubosomes Effectively Impairs Cancer Cells.

BACKGROUND: Multidrug resistance (MDR) is an emerging problem in the treatment of cancer. Therefore, there is a necessity for novel strategies that would sensitize tumor cells to the administered chemotherapeutics. One of the innovative approaches in fighting drug-resistant tumors is the treatment of cancer with microRNA (miRNA), or the use of cubosomes (lipid nanoparticles) loaded with drugs. Here, we present a study on a novel approach, which combines both tools. METHODS: Cubosomes loaded with miR-7-5p and chemotherapeutics were developed. The effects of drug- and miRNA-loaded vehicles on glioma- (A172, T98G), papillary thyroid- (TPC-1) and cervical carcinoma-derived (HeLa) cells were analyzed using molecular biology techniques, including quantitative real-time PCR, MTS-based cell proliferation test, flow cytometry and spheroids formation assay. RESULTS: The obtained data indicate that miR-7-5p increases the sensitivity of the tested cells to the drug, and that nanoparticles loaded with both miRNA and the drug produce a greater anti-tumor effect in comparison to the free drug treatment. It was found that an increased level of apoptosis in the drug/miRNA co-treated cells is accompanied by an alternation in the expression of the genes encoding for key MDR proteins of the ABC family. CONCLUSIONS: Overall, co-administration of miR-7-5p with a chemotherapeutic can be considered a promising strategy, leading to reduced MDR and the induction of apoptosis in cancer cells.

Structural Studies of Thyroid Peroxidase Show the Monomer Interacting With Autoantibodies in Thyroid Autoimmune Disease.

Thyroid peroxidase (TPO) is a critical membrane-bound enzyme involved in the biosynthesis of multiple thyroid hormones, and is a major autoantigen in autoimmune thyroid diseases such as destructive (Hashimoto) thyroiditis. Here we report the biophysical and structural characterization of a novel TPO construct containing only the ectodomain of TPO and lacking the propeptide. The construct was enzymatically active and able to bind the patient-derived TR1.9 autoantibody. Analytical ultracentrifugation data suggest that TPO can exist as both a monomer and a dimer. Combined with negative stain electron microscopy and molecular dynamics simulations, these data show that the TR1.9 autoantibody preferentially binds the TPO monomer, revealing conformational changes that bring together previously disparate residues into a continuous epitope. In addition to providing plausible structural models of a TPO-autoantibody complex, this study provides validated TPO constructs that will facilitate further characterization, and advances our understanding of the structural, functional, and antigenic characteristics of TPO, an autoantigen implicated in some of the most common autoimmune diseases.

A rapid assay of human thyroid peroxidase activity.

Impaired synthesis or action of thyroid hormones (THs) during critically sensitive periods of development can have long term adverse effects on health. Development of rapid assays to identify chemicals that impair THs physiology is an important goal for reducing risks from chemical use. Thyroid peroxidase (TPO) is a key enzyme regulating THs synthesis in thyroid gland and a vulnerable target for chemicals that disrupt THs synthesis. To develop a human-relevant, rapid assay for TPO inhibition, we have engineered two cell lines (CHO and LentiX- 293) to express active human TPO (hTPO) enzyme and applied them in a recently-described assay using a stable fluorescent product (Amplex UltraRed). Assay performance was assessed by comparing activity of 19 reference chemicals with known strong, weak or no TPO inhibitory activity. The assay using hTPO from either cell line consistently identified the relative potency of strong to moderate inhibitors and chemicals known to be inactive. Results were less consistent for chemicals reported to be weak inhibitors of rodent TPO, possibly suggesting some species specificity. Our studies support the use of hTPO from stably transfected cell lines to substitute for animal-derived thyroid microsomes for rapid high throughput screening assays to identify and characterize TPO inhibitors.

Thyroid Peroxidase Revisited - What's New?

Thyroid peroxidase (TPO) is an enzyme that participates in thyroid hormone biosynthesis. TPO is also a major autoantigen in autoimmune thyroid diseases (AITD). In this review, we summarize the latest developments in the field of TPO research. We present the current understanding of immunodominant serologic determinants, frequency of TPO-specific autoantibodies in the population, as well as genetic and environmental factors contributing to their development. Moreover, we report recent progress in the clinical utilities of TPO autoantibody testing, including thyroid dysfunctions and extra-thyroidal disorders.

Trastuzumab-Modified Gold Nanoparticles Labeled with 211At as a Prospective Tool for Local Treatment of HER2-Positive Breast Cancer.

Highly localized radiotherapy with radionuclides is a commonly used treatment modality for patients with unresectable solid tumors. Herein, we propose a novel α-nanobrachytherapy approach for selective therapy of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This uses local intratumoral injection of 5-nm-diameter gold nanoparticles (AuNPs) labeled with an α-emitter (211At), modified with polyethylene glycol (PEG) chains and attached to HER2-specific monoclonal antibody (trastuzumab). The size, shape, morphology, and zeta potential of the 5 nm synthesized AuNPs were characterized by TEM (Transmission Electron Microscopy) and DLS (Dynamic Light Scattering) techniques. The gold nanoparticle surface was modified by PEG and subsequently used for antibody immobilization. Utilizing the high affinity of gold for heavy halogens, the bioconjugate was labelled with 211At obtained by α irradiation of the bismuth target. The labeling yield of 211At was greater than 99%. 211At bioconjugates were stable in human serum. Additionally, in vitro biological studies indicated that 211At-AuNP-PEG-trastuzumab exhibited higher affinity and cytotoxicity towards the HER2-overexpressing human ovarian SKOV-3 cell line than unmodified nanoparticles. Confocal and dark field microscopy studies revealed that 211At-AuNP-PEG-trastuzumab was effectively internalized and deposited near the nucleus. These findings show promising potential for the 211At-AuNP-PEG-trastuzumab radiobioconjugate as a perspective therapeutic agent in the treatment of unresectable solid cancers expressing HER2 receptors.

Mobilization of Stem and Progenitor Cells in Septic Shock Patients.

Septic shock is associated with multiple injuries to organs and tissues. These events may induce the regenerative response of adult stem cells. However, little is known about how endogenous stem cells are modulated by sepsis. This study analyzed the circulation of hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs) and very small embryonic-like stem cells (VSELs) in the peripheral blood of patients with septic shock. Thirty-three patients with septic shock and twenty-two healthy control subjects were enrolled in this prospective observational study. Blood samples were collected on the first, third and seventh days of septic shock. Populations of stem cells were analyzed by flow cytometry. Chemotactic mediators were analyzed by HPLC and ELISA. Populations of early HSCs (Lin-CD133+CD45+ and CD34+CD38-) were mobilized to the peripheral blood after an initial decrease. Mobilized HSCs showed significantly increased expression of Ki-67, a marker of cell proliferation. Circulating EPCs and VSELs were mobilized to the blood circulation upon the first day of sepsis. Patients with a greater number of Lin-CD133+CD45+ HSCs and Lin-CD34+CD45- VSELs had a significantly lower probability of 60-day survival. The concentration of CXCL12 was elevated in the blood of septic patients, while the concentration of sphingosine-1-phosphate was significantly decreased. As an emergency early response to sepsis, VSELs and EPCs were mobilized to the peripheral blood, while the HSCs showed delayed mobilization. Differential mobilization of stem cell subsets reflected changes in the concentration of chemoattractants in the blood. The relationship between the probability of death and a large number of HSCs and VSELs in septic shock patients can be used as a novel prognostic marker and may provide new therapeutic approaches.

Thyroid peroxidase as a dual active site enzyme: Focus on biosynthesis, hormonogenesis and thyroid disorders of autoimmunity and cancer.

Thyroid peroxidase (TPO) is the key enzyme involved in thyroid hormone synthesis. Autoantibodies to TPO (TPOAbs) are a hallmark of autoimmune thyroid disease (AITD). Here, we highlight recent progress over several years in understanding TPO biochemistry and function in various pathologies. TPO undergoes complex post-translational modifications as a dimer in endoplasmic reticulum during secretory pathway to apical membrane of thyrocytes. In silico modelling of TPO dimer has provided new information into the two enzyme active site regions and autoantigenic determinants. TPO and hydrogen peroxide generating DUOX and caveolin-1 form a complex known as thyroxisome to bring together in close proximity the components of hormone synthesis in apical membrane. Autoimmunity to TPO is characterised by autoantibodies and T cell reactivity in Hashimoto's disease and Graves' disease. TPOAbs are directed predominantly to two immunodominant determinants (IDR) termed IDR-A and IDR-B regions, with the latter antibodies more predominant in autoimmune disease. Strong genetic risk has been shown to be associated with TPOAbs for AITD development. A different antibody with unusual features of bispecificity for both TPO and thyroglobulin may play protective role in Hashimoto's disease. In the context of TPO biology in human cancer, thyroid tumor tissue and breast cancer differ in TPO expression and isoform composition. In thyroid cancer, TPO expression is decreased partly by the BRAF(V600E) mutation, with direct impact on significant hormone production. TPOAbs may play a protective role in breast cancer development. An understanding of TPO and its unique two enzymatic active sites and autoantigenic determinants continues to add new knowledge on the biochemistry and immunology of this enzyme.

MIX2: A Novel Natural Multi-Component Modulator of Multidrug-Resistance and Hallmarks of Cancer Cells.

Multidrug resistance is one of the key barriers suppressing the effectiveness of drug therapies of malignant tumors. Here, we report a study on the effect of a mix of natural extracts (MIX2) prepared from fresh fruits of Prunus spinosa, Crataegus monogyna, Sorbus aucuparia, and Euonymus europaeus on the classic hallmarks of cancer cells and the expression of multidrug resistance proteins. In the studies, HeLa and T98G cell lines, and classic methods of molecular biology, including RT-qPCR, Western blot, flow cytometry, and confocal imaging, were used. Additionally, migration, adhesion, and proliferation assays were performed. The obtained results indicate that the MIX2 cocktail presents strong anti-cancer properties. MIX2 is not toxic, but at the same time significantly alters the migration, proliferation, and adhesion of tumor cells. Furthermore, it was found that cells exposed to the mixture presented a significantly reduced expression level of genes associated with MDR, including ABCB1, which encodes for glycoprotein P. In vitro data showed that MIX2 effectively sensitizes tumor cells to doxorubicin. We postulate that modulation of the multidrug resistance phenotype of tumors with the use of MIX2 may be considered as a safe and applicable tool in sustaining drug delivery therapies of malignancies.

Thyroid Peroxidase as an Autoantigen in Hashimoto's Disease: Structure, Function, and Antigenicity.

Human thyroid peroxidase (TPO), is an important enzyme responsible for the biosynthesis of thyroid hormones and is a major autoantigen in autoimmune thyroid diseases (AITDs) such as the destructive Hashimoto's thyroiditis. Although the structure of TPO has yet to be determined, its extracellular domain consists of three regions that exhibit a high degree of sequence similarity to domains of known three-dimensional structure: the myeloperoxidase (MPO)-like domain, complement control protein (CCP)-like domain, and epidermal growth factor (EGF)-like domain. Homology models of TPO can therefore be constructed, providing some structural context to its known function, as well as facilitating the mapping of regions that are responsible for its autoantigenicity. In this review, we highlight recent progress in this area, in particular how a molecular modelling approach has advanced the visualisation and interpretation of epitope mapping studies for TPO, facilitating the dissection of the interplay between TPO protein structure, function, and autoantigenticity.

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines.

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

Thyroid peroxidase (TPO) expressed in thyroid and breast tissues shows similar antigenic properties.

BACKGROUND: Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. METHODS: In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. RESULTS: We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. CONCLUSIONS: Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.