Effects of dabrafenib and erlotinib combination treatment on anaplastic thyroid carcinoma.

Dabrafenib is a BRAF kinase inhibitor approved for treatment of BRAF-mutated anaplastic thyroid carcinoma (ATC) in combination with trametinib. Erlotinib is a tyrosine kinase inhibitor of EGF receptor (EGFR). We evaluated effects of dabrafenib and erlotinib combination treatment on ATC cells in vitro and in vivo. Cell proliferation, colony formation, apoptosis, and migration of ATC cells harboring a BRAF mutation (BHT101, 8505C, and SW1736) were evaluated after treatment with dabrafenib in combination with erlotinib or trametinib. The changes in activation of mitogen extracellular kinase (MEK) and extracellular signal-related kinase (ERK) signaling were also evaluated by Western blot analysis. Effects of these combinations were also evaluated using an in vivo xenograft model. First, we detected EGFR activation in dabrafenib-resistant SW1736 cells using a phospho-receptor tyrosine kinase array. A dabrafenib and erlotinib combination synergistically inhibited cell proliferation, colony formation, and migration, with an induction of apoptotic cell death in all three ATC cells, compared with dabrafenib or erlotinib alone. This synergistic effect was comparable with a dabrafenib and trametinib combination. The dabrafenib and erlotinib combination effectively inhibited phosphorylated (p)-MEK, p-ERK, and p-EGFR expressions compared with dabrafenib or erlotinib alone, while the dabrafenib and trametinib combination only inhibited p-MEK and p-ERK expressions. The dabrafenib with erlotinib or trametinib combinations also significantly suppressed tumor growth and induced apoptosis in a BHT101 xenograft model. The dabrafenib and erlotinib combination could be a potential novel treatment regimen to overcome drug resistance to dabrafenib alone in patients with BRAF-mutated ATC.

Death-Associated Protein Kinase 1 Inhibits Progression of Thyroid Cancer by Regulating Stem Cell Markers.

The activation of metastatic reprogramming is vital for cancer metastasis, but little is known about its mechanism. This study investigated the potential role of death-associated protein kinase 1 (DAPK1) in thyroid cancer progression. We generated knockdown (KD) DAPK1 using siRNA or shRNA in 8505C and KTC-1 cell lines, which we transiently or stably overexpressed in MDA-T32 and BCPAP cell lines. DAPK1 KD in 8505C and KTC-1 cells significantly increased cell proliferation and colony formation compared with controls. We observed significant inhibition of cancer cell invasion in cells overexpressing DAPK1, but the opposite effect in KD cells. Tumorsphere formation significantly increased after inhibition of DAPK1 expression in 8505C cells and was significantly suppressed in DAPK1-overexpressing MDA-T32 and BCPAP cells. DAPK1 overexpression inhibited mRNA and protein levels of stem markers (OCT4, Sox2, KLF4, and Nanog). Furthermore, the expression of these markers increased after KD of DAPK1 in 8505C cells. Mechanistic studies suggest that DAPK1 may modulate the expression of stem cell markers through the inhibition of ß-catenin pathways. These findings were consistent with the public data and our thyroid tissue analysis, which showed higher DAPK1 expression was associated with advanced-stage papillary thyroid cancer with a higher stemness index and lower disease-free survival.

SHMT2 expression as a diagnostic and prognostic marker for thyroid cancer.

BACKGROUND: Catabolism of serine via serine hydroxymethyltransferase2 (SHMT2) through the mitochondrial one-carbon unit pathway is important in tumorigenesis. Therefore, SHMT2 may play a role in thyroid cancer. METHODS: Thyroid tissue samples and The Cancer Genome Atlas (TCGA) database were used to evaluate SHMT2 expression in thyroid tissues and the association with clinical outcomes. RESULTS: SHMT2 protein expression was evaluated in thyroid tissues consisting of 52 benign nodules, 129 papillary thyroid carcinomas (PTC) and matched normal samples, and 20 anaplastic thyroid carcinomas (ATC). ATCs presented the highest (95.0%) positivity of SMHT2 protein expression. PTCs showed the second highest (73.6%) positivity of SHMT2 expression, which was significantly higher than that of benign nodules (19.2%, P = 0.016) and normal thyroid tissues (0%, P < 0.001). Analysis of TCGA data showed that SHMT2 messenger RNA (mRNA) expression was significantly higher in tumors than in normal tissues (P < 0.001). When we classified thyroid cancer into high and low groups according to SHMT2 mRNA expression levels, the thyroid differentiation score for the high SHMT2 group was significantly lower than that of the low SHMT2 group (P < 0.001). There was also a significant correlation between SHMT2 mRNA expression and the stemness index (r = 0.41, P < 0.001). The high SHMT2 group had more advanced TNM stages and shorter progression-free survival rates than the low SHMT2 group (P < 0.01 and P = 0.007, respectively). CONCLUSION: SHMT2 expression is higher in thyroid cancers than normal or benign tissues and is associated with de-differentiation and poor clinical outcomes. Thus, SHMT2 might be useful as a diagnostic and prognostic marker for thyroid cancer.

Mitofusin-2 modulates the epithelial to mesenchymal transition in thyroid cancer progression.

Here, we investigated the potential roles of Mitofusin-2 (MFN2) in thyroid cancer progression. MFN2 regulates mitochondrial fusion/division in cells and plays an important role in various aspects of cell metabolism. MFN2 might involve in cell cycle regulation, apoptosis, and differentiation, and it might play a role as a tumor suppressor in carcinogenesis. We evaluated the prognostic impacts of MFN2 expression in thyroid cancer by analyzing TCGA data. In vitro and in vivo, MFN2 was knocked out using CRISPR/Cas9 or siRNA, and MFN2 was stably overexpressed in two thyroid cancer cell lines (Cal62 and HTH83). TCGA analysis revealed that MFN2 expression was lower in thyroid cancer than in normal tissues and significantly associated with a degree of differentiation, RAS mutations, and less lymph node metastasis. MFN2 expression was significantly correlated with cell adhesion molecules and epithelial to mesenchymal transition (EMT) in a gene-set enrichment assay. MFN2 knock-out (KO) in Cal62 and HTH83 cells using CRISPR/Cas9 or siRNA significantly promoted cell migration and invasion in vitro. The same trends were observed in MFN2 KO mouse embryonic fibroblasts (MEFs) compared to those in the controls (MFN2 WT MEFs). Conversely, MFN2 overexpression in cancer cell lines greatly inhibited cell migration and invasion. However, there was no difference in colony formation and proliferation in Cal62 and HTH83 cells after modulating MFN2, although there were significant differences between MFN KO and WT MEFs. EMT-associated protein expression was induced after MFN2 KO in both cancer cell lines. The mechanistic results suggest that MFN2 might modulate EMT through inducing the AKT signaling pathway. EMT-associated changes in protein expression were also confirmed by modulating MFN2 in xenograft tumors. Thus, MFN2 acts as a tumor suppressor in thyroid cancer progression and metastasis by modulating EMT.

High Phosphoglycerate Dehydrogenase Expression Induces Stemness and Aggressiveness in Thyroid Cancer.

Background: We examined the changes in glucose metabolites of papillary thyroid cancer (PTC) and identified phosphoglycerate dehydrogenase (PHGDH) as a potential target. The role of PHGDH in the proliferation and tumorigenesis of thyroid cancer cells and its clinical significance were analyzed. Methods: Glucose metabolites of various thyroid tissues were analyzed via targeted metabolomics analysis. In vitro experiments using shPHGDHs, inhibitor (NCT503), or PHGDH overexpression in thyroid cell lines (BCPAP, 8505C, and Nthy-Ori) were performed. In vivo experiments were performed by using shPHGDH. Human tissue samples and The Cancer Genome Atlas (TCGA) data were used to validate the experimental findings. Results:PHGDH knockdown in BCPAP and 8505c cell lines significantly inhibited cell viability, colony formation, and tumor spheroid formation compared with the control. In addition, treatment with NCT503 showed similar results. PHGDH inhibition by both knockdown and treatment with NCT503 significantly inhibited the expression of embryonic cancer stemness markers (Oct4, Sox2, KLF4, and Nanog). PHGDH overexpression in Nthy-Ori cells significantly increased cell viability and colony formation. The stemness markers were significantly increased after PHGDH overexpression. PHGDH knockdown significantly inhibited tumor growth in an in vivo mouse xenograft study using 8505c cells. The protein expression of Oct4 in tumors was significantly reduced after PHGDH knockdown. The associations between PHGDH expression and stemness markers were confirmed in the TCGA data and human thyroid tissue samples. Positive PHGDH protein expression was associated with metastases of PTC. Conclusions:PHGDH expression is induced in thyroid cancer and is associated with stemness and aggressiveness of PTC.

Expression of NF2 Modulates the Progression of BRAFV600E Mutated Thyroid Cancer Cells.

BACKGROUND: We previously reported the frequent neurofibromatosis 2 (NF2) gene mutations in anaplastic thyroid cancers in association with the BRAFV600E mutation. We aimed to investigate the role of NF2 in thyroid cancer with BRAF mutation. METHODS: To identify the function of NF2 in thyroid cancers, we investigated the changes in cell proliferation, colon formation, migration and invasion of thyroid cancer cells (8505C, BHT101, and KTC-1) with BRAFV600E mutation after overexpression and knock-down of NF2. We also examined how cell proliferation changed when NF2 was mutagenized. Human NF2 expression in papillary thyroid carcinoma (PTC) was analyzed using the The Cancer Genome Atlas (TCGA) data. RESULTS: First, NF2 was overexpressed in 8505C and KTC-1 cells. Compared to control, NF2 overexpressed group of both thyroid cancer cells showed significant inhibition in cell proliferation and colony formation. These results were also confirmed by cell migration and invasion assay. After knock-down of NF2 in 8505C cells, there were no significant changes in cell proliferation and colony formation, compared with the control group. However, after mutagenized S288* and Q470* sites of NF2 gene, the cell proliferation increased compared to NF2 overexpression group. In the analysis of TCGA data, the mRNA expression of NF2 was significantly decreased in PTCs with lateral cervical lymph node (LN) metastasis compared with PTCs without LN metastasis. CONCLUSION: Our study suggests that NF2 might play a role as a tumor suppressor in thyroid cancer with BRAF mutation. More studies are needed to elucidate the mechanism how NF2 acts in thyroid cancer with BRAF mutation.

The role of Slit2 as a tumor suppressor in thyroid cancer.

Slits, representative axon guidance molecules, and their Roundabout (Robo) transmembrane receptors play roles in the progression of many cancers. We investigated the effects of Slit2 on the proliferation, migration, and invasion of thyroid cancer cells, and on the prognosis of papillary thyroid cancer (PTC). Slit2 overexpression inhibited the proliferation, migration and invasion of thyroid cancer cells by inhibiting transcriptional activity of beta-catenin and regulating Rho GTPase activity. Slit2 knockdown activated the migration and invasion of thyroid cancer cells and transcriptional activity of beta-catenin. Fragment Slit2 treatment inhibited thyroid cancer cell proliferation in a dose dependent manner, and also inhibited migration and invasion. When we evaluated the protein expression of Slit2 in PTCs, 24 of 160 PTCs (15%) were negative for Slit2 protein expression and these patients had significantly increased risk of cervical lymph node metastasis (P < 0.001), distant metastasis (P < 0.001) and recurrence of PTC (P < 0.001). Our findings suggest a role for Slit2 as a tumor suppressor, and also as a novel prognostic and potential therapeutic target for thyroid cancer.

Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer.

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML-RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.

Pin1 cysteine-113 oxidation inhibits its catalytic activity and cellular function in Alzheimer's disease.

The unique proline isomerase Pin1 is pivotal for protecting against age-dependent neurodegeneration in Alzheimer's disease (AD), with its inhibition providing a molecular link between tangle and plaque pathologies. Pin1 is oxidatively modified in human AD brains, but little is known about its regulatory mechanisms and pathological significance of such Pin1 modification. In this paper, our determination of crystal structures of oxidized Pin1 reveals a series of Pin1 oxidative modifications on Cys113 in a sequential fashion. Cys113 oxidization is further confirmed by generating antibodies specifically recognizing oxidized Cys113 of Pin1. Furthermore, Pin1 oxidation on Cys113 inactivates its catalytic activity in vitro, and Ala point substitution of Cys113 inactivates the ability of Pin1 to isomerize tau as well as to promote protein turnover of tau and APP. Moreover, redox regulation affects Pin1 subcellular localization and Pin1-mediated neuronal survival in response to hypoxia treatment. Importantly, Cys113-oxidized Pin1 is significantly increased in human AD brain comparing to age-matched controls. These results not only identify a novel Pin1 oxidation site to be the critical catalytic residue Cys113, but also provide a novel oxidative regulation mechanism for inhibiting Pin1 activity in AD. These results suggest that preventing Pin1 oxidization might help to reduce the risk of AD.

Voltage-gated K+ channels in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells.

AIM: To determine the presence of voltage-gated K(+) (Kv) channels in bone marrow-derived human mesenchymal stem cells (hMSCs) and their impact on differentiation of hMSCs into adipocytes. METHODS: For adipogenic differentiation, hMSCs were cultured in adipogenic medium for 22 d. The degrees of adipogenic differentiation were examined using Western blot, Oil Red O staining and Alamar assay. The expression levels of Kv channel subunits Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv2.1, Kv3.1, Kv3.3, Kv4.2, Kv4.3, and Kv9.3 in the cells were detected using RT-PCR and Western blot analysis. RESULTS: The expression levels of Kv2.1 and Kv3.3 subunits were markedly increased on d 16 and 22. In contrast, the expression levels of other Kv channel subunits, including Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv4.2, Kv4.3, and Kv9.3, were decreased as undifferentiated hMSCs differentiated into adipocytes. Addition of the Kv channel blocker tetraethylammonium (TEA, 10 mmol/L) into the adipogenic medium for 6 or 12 d caused a significant decrease, although not complete, in lipid droplet formation and adipocyte fatty acid-binding protein 2 (aP(2)) expressions. Addition of the selective Kv2.1 channel blocker guangxitoxin (GxTX-1, 40 nmol/L) into the adipogenic medium for 21 d also suppressed adipogenic differentiation of the cells. CONCLUSION: The results demonstrate that subsets of Kv channels including Kv2.1 and Kv3.3 may play an important role in the differentiation of hMSCs into adipocytes.

Nasopharyngeal oncocytoma in a cat.

A 5-year-old male neutered Siamese cat was referred because of nasal swelling, nasal discharge, and oral respiration. Computed tomography and necropsy following euthanasia revealed a firm tan mass in the nasopharynx, occluding the nasal passage. Histologically, the nasopharyngeal mass was composed of solid nests, anastomosing cords, and closely packed glands separated by a delicate fibrovascular stroma. Individual neoplastic cells were cylindrical to polyhedral in shape, had distinct cell borders, and contained moderate amounts of finely granular eosinophilic cytoplasm and round to oval nuclei. The cytoplasmic granules were positive on phosphotungstic acid-hematoxylin staining. Immunohistochemically, the neoplastic cells were positive for cytokeratin and negative for chromogranin A, neuron-specific enolase, and vimentin. Ultrastructurally, the neoplastic cells contained numerous mitochondria.

Synergistically enhanced osteogenic differentiation of human mesenchymal stem cells by culture on nanostructured surfaces with induction media.

We have examined the effects of surface nanotopography on in vitro osteogenesis of human mesenchymal stem cells (hMSCs). UV-assisted capillary force lithography was employed to fabricate a scalable (4x5 cm), well-defined nanostructured substrate of a UV curable polyurethane polymer with dots (150, 400, 600 nm diameter) and lines (150, 400, 600 nm width). The influence of osteogenic differentiation of hMSCs was characterized at day 8 by alkaline phosphatase (ALP) assay, RT-PCR, and real-time PCR analysis. We found that hMSCs cultured on the nanostructured surfaces in osteogenic induction media showed significantly higher ALP activity compared to unpatterned PUA surface (control group). In particular, the hMSCs on the 400 nm dot pattern showed the highest level of ALP activity. Further investigation with real-time quantitative RT-PCR analysis demonstrated significantly higher expression of core binding factor 1 (Cbfa1), osteopontin (OP), and osteocalcin (OC) levels in hMSCs cultured on the 400 nm dot pattern in osteogenic induction media. These findings suggest that surface nanotopography can enhance osteogenic differentiation synergistically with biochemical induction substance.

Cytosine deaminase-producing human mesenchymal stem cells mediate an antitumor effect in a mouse xenograft model.

BACKGROUND AND AIM: Stem cell transplantation offers potential gene therapy for brain tumors. However, this approach has received little attention as a treatment for gastrointestinal tumors. In the present study, we explored the possibility of human bone marrow-derived mesenchymal stem cells (hMSC) producing cytosine deaminase (CD), followed by systemic 5-fluorocytosine (5-FC) administration, showing an antitumor effect on a mouse gastric cancer xenograft. METHODS: We first explored the ability of hMSC, coated with fluorescent dye, to migrate to human gastric cancer MKN45 cells in vitro and in vivo. We then used hMSC in which a gene expressed the prodrug-activating enzyme CD, which can convert the prodrug 5-FC into the cytotoxic agent 5-fluorouracil (5-FU), and further investigated the potential of these cells to deliver the CD gene and to reduce tumor growth in nude mice. The migratory capacity of hMSC was confirmed by an in vitro migration assay, as well as in an in vivo model of nude mice bearing subcutaneous tumors of MKN45 cells when hMSC were injected. RESULTS: The migration ability of hMSC towards MKN45 cells was confirmed by migration assay. Effective conversion of 5-FC to 5-FU by hMSC transfected with the CD gene (CD-hMSC) showed therapeutic anticancer potential in a MKN45 cell co-culture system, as confirmed by thin layer chromatography. Nude mice bearing MKN45 tumors were intravenously injected with CD-hMSC, followed by systemic 5-FC treatment (500 mg/kg/day) for 7 days. Tumor volumes and weights of mice injected with CD-hMSC decreased significantly after treatment with 5-FC. However, the 5-FC-treated group without CD-hMSC injection showed neither a decrease in tumor volume nor bodyweight loss. CONCLUSION: The CD-hMSC system showed anticancer therapeutic potential, and minimized the side-effects of 5-FU.

Hemangiosarcoma in a South American sea lion (Otaria byronia).

A 10-yr-old male South American sea lion (Otaria byronia) died after several weeks of depression, anorexia, weight loss, and progressive respiratory distress. At necropsy, three confluent, lobulated, dark-red masses were noted in the mesentery. Similar masses were also observed in the lung and both kidneys. Hemangiosarcoma was diagnosed based on gross findings, histopathology, and immunohistochemistry. This is the first case of hemangiosarcoma reported in pinnipeds.

Retrospective study of canine cutaneous tumors in Korea.

Over the 42 month period from January 2003 to June 2006, a total of 2,952 canine biopsy specimens were received from the Veterinary Medical Teaching Hospital of Seoul National University and from veterinary practitioners across the nation. Out of these, 748 (25.34%) cases were diagnosed as canine cutaneous tumors in the Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Korea. Thirty-eight different types of cutaneous tumors were identified and categorized into epithelial and melanocytic tumors (56.95%), mesenchymal tumors (38.90%), and hematopoietic tumors (4.14%) located in the skin. Among these, 69.25% were benign and 30.74% were malignant. The top ten most frequently diagnosed cutaneous tumors were epidermal and follicular cysts (12.70%), lipoma (11.36%), mast cell tumors (8.82%), cutaneous histiocytoma (7.49%), basal cell tumors (6.82%), sebaceous gland adenoma (6.68%), sebaceous gland hyperplasia (5.08%), hepatoid gland adenoma (3.61%), apocrine adenocarcinoma (3.07%), and fibroma (2.81%), in order of prevalence. They comprised 68.45% of all cutaneous tumors. These top ten cutaneous tumors were distributed on the trunk (30.08%), head and neck (20.9%), extremities (19.14%), anal and perianal area (8.59%), and tail (3.91%). The age of the dogs with the ten most frequent tumors had a mean age of 8.3 years, with a range of 2 months to 19 years. When all types of tumors were considered together in the entire population, there was no difference in incidence according to sex.