The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

The selenoenzyme type I iodothyronine deiodinase (DIO1) catalyzes removal of iodine atoms from thyroid hormones. Although DIO1 action is reported to be disturbed in several malignancies, no work has been conducted in high-grade serous ovarian carcinoma (HGSOC), the most lethal gynecologic cancer. We studied DIO1 expression in HGSOC patients [The Cancer Genome Atlas (TCGA) data and tumor tissues], human cell lines (ES-2 and Kuramochi), normal Chinese hamster ovarian cells (CHO-K1), and normal human fallopian tube cells (FT282 and FT109). To study its functional role, DIO1 was overexpressed, inhibited [by propylthiouracil (PTU)], or knocked down (KD), and cell count, proliferation, apoptosis, cell viability, and proteomics analysis were performed. Lower DIO1 levels were observed in HGSOC compared to normal cells and tissues. TCGA analyses confirmed that low DIO1 mRNA expression correlated with worse survival and therapy resistance in patients. Silencing or inhibiting the enzyme led to enhanced ovarian cancer proliferation, while an opposite effect was shown following DIO1 ectopic expression. Proteomics analysis in DIO1-KD cells revealed global changes in proteins that facilitate tumor metabolism and progression. In conclusion, DIO1 expression and ovarian cancer progression are inversely correlated, highlighting a tumor suppressive role for this enzyme and its potential use as a biomarker in this disease.

Three-Dimensional Modeling of Thyroid Hormone Metabolites Binding to the Cancer-Relevant αvß3 Integrin: In-Silico Based Study.

Background: Thyroid hormones (TH), T4 and T3, mediate pro-mitogenic effects in cancer cells through binding the membrane receptor αvß3 integrin. The deaminated analogue tetrac effectively blocks TH binding to this receptor and prevents their action. While computational data on TH binding to the αvß3 integrin was published, a comprehensive analysis of additional TH metabolites is lacking. Methods: In-silico docking of 26 TH metabolites, including the biologically active thyroid hormones (T3 and T4) and an array of sulfated, deiodinated, deaminated or decarboxylated metabolites, to the αvß3 receptor binding pocket was performed using DOCK6, based on the three-dimensional representation of the crystallographic structure of the integrin. As the TH binding site upon the integrin is at close proximity to the well-defined RGD binding site, linear and cyclic RGD were included as a reference. Binding energy was calculated for each receptor-ligand complex using Grid score and Amber score with distance movable region protocol. Results: All TH molecules demonstrated negative free energy, suggesting affinity to the αvß3 integrin. Notably, based on both Grid and Amber scores sulfated forms of 3,3' T2 (3,3' T2S) and T4 (T4S) demonstrated the highest binding affinity to the integrin, compared to both cyclic RGD and an array of examined TH metabolites. The major thyroid hormones, T3 and T4, showed high affinity to the integrin, which was superior to that of linear RGD. For all hormone metabolites, decarboxylation led to decreased affinity. This corresponds with the observation that the carboxylic group mediates binding to the integrin pocket via divalent cations at the metal-ion-dependent adhesion (MIDAS) motif site. A similar reduced affinity was documented for deaminated forms of T3 (triac) and T4 (tetrac). Lastly, the reverse forms of T3, T3S, and T3AM showed higher Amber scores relative to their native form, indicating that iodination at position 5 is associated with increased binding affinity compared to position 5'. Summary: Three-dimensional docking of various TH metabolites uncovered a structural basis for a differential computational free energy to the αvß3 integrin. These findings may suggest that naturally occurring endogenous TH metabolites may impact integrin-mediate intracellular pathways in physiology and cancer.

Nuclear αvß3 integrin expression, post translational modifications and regulation in hematological malignancies.

αvß3 integrin, a plasma membrane protein, is amply expressed on an array of tumors. We identified nuclear αvß3 pool in ovarian cancer cells and were interested to explore this phenomenon in two rare and aggressive types of leukemia, T-cell acute lymphoblastic leukemia (T-ALL) and Mast cell leukemia (MCL) using Jurkat and HMC-1 cell lines, respectively. Moreover, we collected primary cells from patients with chronic lymphocytic leukemia (CLL, n = 11), the most common chronic adult leukemia and used human lymphoblastoid cell lines (LCL) generated from normal B cells. Nuclear αvß3 integrin was assessed by Western blots, confocal microscopy, and the ImageStream technology which combines flow-cytometry with microscopy. We further examined post translational modifications (phosphorylation/glycosylation), nuclear trafficking regulation using inhibitors for MAPK (U0126) and PI3K (LY294002), as well as nuclear interactions by performing Co-immunoprecipitation (Co-IP). αvß3 integrin was identified in all cell models within the nucleus and is N-glycosylated. In primary CLL cells the ß3 integrin monomer is tyrosine Y759 phosphorylated, suggesting an active receptor conformation. MAPK and PI3K inhibition in Jurkat and CLL cells led to αvß3 enhancement in the nucleus and a reduction in the membrane. The nuclear αvß3 integrin interacts with ERK, Histone H3 and Lamin B1 in Jurkat, Histone H3 in CLL cells, but not in control LCL cells. To conclude, this observational study provides the identification of nuclear αvß3 in hematological malignancies and lays the basis for novel cancer-relevant actions, which may be independent from the membrane functions.

Targeting the DIO3 enzyme using first-in-class inhibitors effectively suppresses tumor growth: a new paradigm in ovarian cancer treatment.

The enzyme iodothyronine deiodinase type 3 (DIO3) contributes to cancer proliferation by inactivating the tumor-suppressive actions of thyroid hormone (T3). We recently established DIO3 involvement in the progression of high-grade serous ovarian cancer (HGSOC). Here we provide a link between high DIO3 expression and lower survival in patients, similar to common disease markers such as Ki67, PAX8, CA-125, and CCNE1. These observations suggest that DIO3 is a logical target for inhibition. Using a DIO3 mimic, we developed original DIO3 inhibitors that contain a core of dibromomaleic anhydride (DBRMD) as scaffold. Two compounds, PBENZ-DBRMD and ITYR-DBRMD, demonstrated attenuated cell counts, induction in apoptosis, and a reduction in cell proliferation in DIO3-positive HGSOC cells (OVCAR3 and KURAMOCHI), but not in DIO3-negative normal ovary cells (CHOK1) and OVCAR3 depleted for DIO3 or its substrate, T3. Potent tumor inhibition with a high safety profile was further established in HGSOC xenograft model, with no effect in DIO3-depleted tumors. The antitumor effects are mediated by downregulation in an array of pro-cancerous proteins, the majority of which known to be repressed by T3. To conclude, using small molecules that specifically target the DIO3 enzyme we present a new treatment paradigm for ovarian cancer and potentially other DIO3-dependent malignancies.

Pre-diagnosis thyroid hormone dysfunction is associated with cancer mortality.

Research on the association between thyroid hormone levels and cancer mortality remains limited and inconclusive. We determined the relation of thyroid stimulating hormone (TSH), free T4 (FT4), and free T3 (FT3) levels with mortality in overall cancer and specific tumor types. Thyroid hormone levels 1-5 years prior to cancer diagnosis, as well as multiple clinical and demographic parameters, were retrospectively collected for 10,325 Israeli cancer patients, diagnosed between 2000 and 2016. Patients treated with thyroid altering medications were excluded. Cancer diagnosis was determined via the Israel National Cancer Registry. Multivariate-adjusted Cox proportional hazards model was used to assess the hazard ratios (HRs) based on thyroid hormone function for cancer mortality. A total of 5265 patients died during the follow-up period (median of 4.4 years). TSH, FT4, and FT3 levels in the hypothyroid range were associated with increase in overall mortality (adjusted HR 1.20, 1.74, 1.87, respectively). We further analyzed the association between TSH and mortality in 14 cancer subgroups. Specifically, TSH in both the hyperthyroid and hypothyroid range was associated with melanoma mortality (adjusted HR 2.20, 4.47, respectively). In conclusion, pre-diagnosis of thyroid dysfunction is associated with increased cancer mortality, a relation likely driven by specific cancer types. These findings suggest that thyroid hormones may potentially serve as prognostic markers in cancer.

αvß3 Integrin Expression and Mitogenic Effects by Thyroid Hormones in Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. The thyroid hormones, T3 and T4, bind the αvß3 integrin and activate phosphorylates ERK (pERK). These tumor-promoting actions were reported in a number of malignancies, but not in CLL. METHODS: Primary cells from 22 CLL patients were verified for disease markers (CD5/CD19/CD23) and analyzed for αvß3 by flow cytometry (FC), ImageStream, Western blots (WB), and immunohistochemistry (IHC) in archival bone marrow (BM, n = 6) and lymph node (LN, n = 5) tissues. Selected samples (n = 8) were incubated with T3 (1-100 nM) or T4 (0.1-10 µM) for 30 min, and the expression levels of αvß3, pERK and PCNA (cell proliferation marker) were determined (WB). RESULTS: αvß3 was detected on the membrane of circulating CLL cells and in the BM but not in the LN. T3 and T4 enhanced αvß3 protein levels in primary CLL cells. Similarly, pERK and PCNA were rapidly induced in response to T3 and T4 exposure. CONCLUSIONS: αvß3 integrin is expressed on primary CLL cells and is induced by thyroid hormones. We further suggest that the hormones are mitogenic in these cells, presumably via αvß3-mediated signaling.

Opposing effects of thyroid hormones on cancer risk: a population-based study.

OBJECTIVE: The association between dysregulated thyroid hormone function and cancer risk is inconclusive, especially among different age groups and uncommon malignancies. We sought to determine the relation of TSH and free T4 levels with overall cancer risk as well as risk of specific cancer types. DESIGN AND METHODS: Data on thyroid hormone profile was collected from 375 635 Israeli patients with no prior history of cancer. Cancer cases were identified via the Israel National Cancer Registry. Cox proportional hazards model was used to assess hazard ratios for overall cancer as well as 20 cancer subgroups. RESULTS: In this study, 23 808 cases of cancer were detected over median follow up of 10.9 years. Among patients younger than 50 at inclusion, TSH in the hyperthyroid range, elevated free T4 and subclinical hyperthyroidism were associated with increased cancer risk (HR: 1.3, 1.28 and 1.31, respectively). In contrast, patients 50 or older with clinical hyperthyroidism were at lower cancer risk (HR: 0.64). Elevated TSH was associated with decreased risk of prostate cancer (HR: 0.67). Log-TSH elevation was associated with decreased risk of thyroid cancer (HR: 0.82) and increased risk of melanoma (HR: 1.11) and uterine cancer (HR: 1.27). Elevated free T4 was associated with increased lung cancer risk (HR: 1.54), while free T4 levels above the normal range and clinical hyperthyroidism were related to lower colorectal cancer risk (HR: 0.59 and 0.08, respectively). CONCLUSIONS: Thyroid hormones display opposing effects on cancer risk, based on patient age and cancer type.

Enhanced expression of αVß3 integrin in villus and extravillous trophoblasts of placenta accreta.

BACKGROUND: Placenta accreta is one of the most serious complications in obstetrics and gynecology. Villous trophoblasts (VT) and extravillous trophoblasts (EVT) play a central role in normal placentation. Placenta accreta is characterized by abnormal invasion of EVT cells through the uterine layers, due to changes in several parameters, including adhesion proteins. Although αvß3 integrin is a central adhesion molecule, participating in multiple invasive pathological conditions including cancer, data on placenta accreta are lacking. OBJECTIVE: To study the expression pattern of αvß3 integrin in placenta accreta in comparison with normal placentas. STUDY DESIGN: We collected tissue samples from placentas defined as percreta, the most severe presentation of placenta accreta and from normal control placentas (n = 10 each). The samples underwent protein extractions for analyses of αvß3 expression by Western blots (WB) and a parallel tissue assessment by immunohistochemistry (IHC). RESULTS: WB results indicated significantly elevated αvß3 integrin expression in the percreta samples compared to normal placentas. These elevated levels were mainly contributed by EVT cells, as demonstrated by IHC. αvß3 integrin demonstrated a classical membranal expression in the VT cells, whereas a uniformly distributed expression was documented in the EVT cells. These patterns of the αvß3 integrin localization were similar in both accreta and normal placental samples. CONCLUSIONS: Enhanced αvß3 integrin expression, mainly in extra villous trophoblasts of placenta percreta, implies for a role of this adhesion molecule in pathological placentation.

DIO3, the thyroid hormone inactivating enzyme, promotes tumorigenesis and metabolic reprogramming in high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy with a need for better understanding the disease pathogenesis. The biologically active thyroid hormone, T3, is considered a tumor suppressor by promoting cell differentiation and mitochondrial respiration. Tumors evolved a strategy to avoid these anticancer actions by expressing the T3 catabolizing enzyme, Deiodinase type 3 (DIO3). This stimulates cancer proliferation and aerobic glycolysis (Warburg effect). We identified DIO3 expression in HGSOC cell lines, tumor tissues from mice and human patients, fallopian tube (FT) premalignant lesion and secretory cells of normal FT, considered the disease site-of-origin. Stable DIO3 knockdown (DIO3-KD) in HGSOC cells led to increased T3 bioavailability and demonstrated induced apoptosis and attenuated proliferation, migration, colony formation, oncogenic signaling, Warburg effect and tumor growth in mice. Proteomics analysis further indicated alterations in an array of cancer-relevant proteins, the majority of which are involved in tumor suppression and metabolism. Collectively this study establishes the functional role of DIO3 in facilitating tumorigenesis and metabolic reprogramming, and proposes this enzyme as a promising target for inhibition in HGSOC.

Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent.

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO2 as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin-mediated drug-delivery of RGD-modified DLDH (DLDHRGD ), leading to apoptotic cell death. In these experiments, DLDHRGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA-binding capability of this enzyme. To explore this interaction under cell-free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel-shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA-binding. A Docking algorithm for prediction of protein-DNA complexes, Paradoc, identified a putative DNA binding site at the C-terminus of DLDH. Our finding that TiO2 -bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.

The identification of nuclear αvß3 integrin in ovarian cancer: non-paradigmal localization with cancer promoting actions.

Nuclear translocation of transmembrane proteins was reported in high-grade serous ovarian cancer (HGSOC), a highly aggressive gynecological malignancy. Although the membrane receptor αvß3 integrin is amply expressed in HGSOC and involved in disease progression, its nuclear localization was never demonstrated. Nuclear αvß3 was explored in HGSOC cells (OVCAR3, KURAMOCHI, and JHOS4), nuclear localization signal (NLS) modified ß3 OVCAR3, Chinese hamster ovaries (CHO-K1) and human embryonic kidney (HEK293) before/after transfections with ß3/ß1 integrins. We used the ImageStream technology, Western blots (WB), co immunoprecipitations (Co-IP), confocal immunofluorescence (IF) microscopy, flow cytometry for cell counts and cell cycle, wound healing assays and proteomics analyses. Fresh/archived tumor tissues were collected from nine HGSOC patients and normal ovarian and fallopian tube (FT) tissues from eight nononcological patients and assessed for nuclear αvß3 by WB, confocal IF microscopy and immunohistochemistry (IHC). We identified nuclear αvß3 in HGSOC cells and tissues, but not in normal ovaries and FTs. The nuclear integrin was Tyr 759 phosphorylated and functionally active. Nuclear αvß3 enriched OVCAR3 cells demonstrated induced proliferation and oncogenic signaling, intact colony formation ability and inhibited migration. Proteomics analyses revealed a network of nuclear αvß3-bound proteins, many of which with key cancer-relevant activities. Identification of atypical nuclear localization of the αvß3 integrin in HGSOC challenges the prevalent conception that the setting in which this receptor exerts its pleiotropic actions is exclusively at the cell membrane. This discovery proposes αvß3 moonlighting functions and may improve our understanding of the molecular basis of ovarian cancer pathogenesis.

Targeting the Achilles' heel of cancer cells via integrin-mediated delivery of ROS-generating dihydrolipoamide dehydrogenase.

Cancer cells frequently exhibit higher levels of reactive oxygen species (ROS) than normal cells and when ROS levels increase beyond a cellular tolerability threshold, cancer cell death is enhanced. The mitochondrial dihydrolipoamide dehydrogenase (DLDH) is an enzyme which produces ROS in association with its oxidoreductive activity and may be thus utilized as an exogenous anticancer agent. As cancer cells often overexpress integrins that recognize RGD-containing proteins, we have bioengineered the human DLDH with RGD motifs (DLDHRGD) for integrin-mediated drug delivery. The modified protein fully retained its enzyme activity and ROS-production capability. DLDHRGD uptake by cells was shown to depend on the presence of cell-associated integrin αvß3, as comparatively demonstrated with normal kidney cells (HEK293) transfected with either ß1 (αvß1 positive) or ß3 integrins (αvß3 positive). The interaction with ß3 integrins was shown to be competitively inhibited by an RGD peptide. In mice melanoma cells (B16F10), which highly express an endogenous αvß3 integrin, fast cellular uptake of DLDHRGD which resulted in cell number reduction, apoptosis induction, and a parallel intracellular ROS production was shown. Similar results were obtained with additional human melanoma cell models (A375, WM3314, and WM3682). In contrast, HEK293ß3 cells remained intact following DLDHRGD uptake. The high pharmacological safety profile of DLDHRGD has been observed by several modes of administrations in BALB/C or C57Bl/6 mouse strains. Treatments with DLDHRGD in a subcutaneous melanoma mice model resulted in significant tumor inhibition. Our study demonstrated, in vitro and in vivo, the development of a unique platform, which targets cancer cells via integrin-mediated drug delivery of an exogenous ROS-generating drug.

Thyroid Hormones and Cancer: A Comprehensive Review of Preclinical and Clinical Studies.

Thyroid hormones take major part in normal growth, development and metabolism. Over a century of research has supported a relationship between thyroid hormones and the pathophysiology of various cancer types. In vitro studies as well as research in animal models demonstrated an effect of the thyroid hormones T3 and T4 on cancer proliferation, apoptosis, invasiveness and angiogenesis. Thyroid hormones mediate their effects on the cancer cell through several non-genomic pathways including activation of the plasma membrane receptor integrin αvß3. Furthermore, cancer development and progression are affected by dysregulation of local bioavailability of thyroid hormones. Case-control and population-based studies provide conflicting results regarding the association between thyroid hormones and cancer. However, a large body of evidence suggests that subclinical and clinical hyperthyroidism increase the risk of several solid malignancies while hypothyroidism may reduce aggressiveness or delay the onset of cancer. Additional support is provided from studies in which dysregulation of the thyroid hormone axis secondary to cancer treatment or thyroid hormone supplementation was shown to affect cancer outcomes. Recent preclinical and clinical studies in various cancer types have further shown promising outcomes following chemical reduction of thyroid hormones or inhibition or their binding to the integrin receptor. This review provides a comprehensive overview of the preclinical and clinical research conducted so far.

Contributions of Thyroid Hormone to Cancer Metastasis.

Acting at a cell surface receptor on the extracellular domain of integrin αvß3, thyroid hormone analogues regulate downstream the expression of a large panel of genes relevant to cancer cell proliferation, to cancer cell survival pathways, and to tumor-linked angiogenesis. Because αvß3 is involved in the cancer cell metastatic process, we examine here the possibility that thyroid hormone as l-thyroxine (T4) and the thyroid hormone antagonist, tetraiodothyroacetic acid (tetrac), may respectively promote and inhibit metastasis. Actions of T4 and tetrac that are relevant to cancer metastasis include the multitude of synergistic effects on molecular levels such as expression of matrix metalloproteinase genes, angiogenesis support genes, receptor tyrosine kinase (EGFR/ERBB2) genes, specific microRNAs, the epithelial⁻mesenchymal transition (EMT) process; and on the cellular level are exemplified by effects on macrophages. We conclude that the thyroid hormone-αvß3 interaction is mechanistically linked to cancer metastasis and that modified tetrac molecules have antimetastatic activity with feasible therapeutic potential.

Chemical and thyroid hormone profile of the bone marrow interstitial fluid in hematologic disorders and patients without primary hematologic disorders.

Bone marrow interstitial fluid (BMIF) has not been well characterized. BMIF was isolated from 60 patients including plasma cell dyscrasias (PCD, n = 33), other primary hematologic disorders (OHD, n = 15), and patients with secondary or nonhemtologic disorders (NHD, n = 12) and analyzed for an array of chemical constituents. These included total cholesterol, glucose, phosphate, creatinine, urea, total protein, albumin, globulins, total bilirubin, aspartate aminotransferase, lactate dehydrogenase, sodium, osmolarity, free triiodothyronine (free T3), total triiodothyronine (total T3), and free tetraiodothyronine (free T4). Levels of BMIF components were compared between patient groups and to plasma levels. Compared with plasma, total cholesterol, total protein, total bilirubin, sodium, and calculated osmolarity were lower in BMIF in all groups (P < 0.05). Calculated globulins and aspartate aminotransferase were lower in BMIF of PCD patients and patients with NHD. Albumin was lower in BMIF of patients with PCD and patients with OHD. Lastly, free T4 was significantly higher in BMIF of patients with PCD and patients with OHD. Similar results were demonstrated in a separate analysis performed in patients with multiple myeloma. To conclude, the chemical and thyroid hormone composition of BMIF differs significantly from plasma in several key constituents.

RGD-modified dihydrolipoamide dehydrogenase conjugated to titanium dioxide nanoparticles - switchable integrin-targeted photodynamic treatment of melanoma cells.

The photocytotoxic effect of UVA-excited titanium dioxide (TiO2), which is caused by the generation of reactive oxygen species (ROS), is often used in medical applications, such as cancer treatment. Photodynamic-therapy (PDT) is applied in several cancer models including cutaneous melanoma (CM), however the lack of selectivity causing damage to surrounding healthy tissues limits its applicability and novel targeted-delivery approaches are required. As cancer cells often overexpress integrin receptors (e.g. αvß3) on their cell surface, targeted delivery of TiO2 nanoparticles (NPs) via an Arg-Gly-Asp (RGD) motif would make PDT more selective. We have recently reported that the mitochondrial enzyme dihydrolipoamide dehydrogenase (DLDH) strongly and specifically conjugates TiO2 via coordinative bonds. In this work we have modified DLDH with RGD moieties (DLDHRGD), creating a molecular bridge between the integrin-expressing cancer cells and the photo-excitable TiO2 nanoparticles. Physicochemical assays have indicated that the hybrid-conjugated nanobiocomplex, TiO2-DLDHRGD, is producing controlled-release ROS under UVA illumination, with anatase NPs being the most photoreactive TiO2 form. This drug delivery system exhibited a cytotoxic effect in αvß3 integrin-expressing mice melanoma cells (B16F10), but not in normal cells lacking this integrin (HEK293). No cytotoxic effect was observed in the absence of UV illumination. Our results demonstrate the feasibility of combining the high efficiency of TiO2-based PDT, with an integrin-mediated tumor-targeted drug delivery for nanomedicine.