Thyroid Cancer Cell Proliferation Research Articles

Circular RNA circZFR contributes to papillary thyroid cancer cell proliferation and invasion by sponging miR-1261 and facilitating C8orf4 expression

In recent years, more and more circular RNAs (circRNAs) have been identified in multiple tissues and cells. Increasing evidences show circRNAs play important roles in human cancers. However, the role of circRNAs in papillary thyroid carcinoma (PTC) remains largely unknown. In this study, we identified a new circRNA circZFR that was significantly upregulated in PTC tissues compared to adjacent normal tissues. Furthermore, circZFR expression level was negatively correlated with clinical severity. We found that circZFR knockdown dramatically inhibited the proliferation, migration and invasion of PTC cells in vitro. Mechanistically, we found circZFR could promote C8orf4 expression via serving as a competing endogenous RNA (ceRNA) of miR-1261 in PTC cells. Rescue assays indicated that restoration of C8orf4 significantly attenuated the inhibitory effects of circZFR knockdown on PTC cell proliferation, migration and invasion. In summary, our findings demonstrated that circRNA circZFR exerted oncogenic roles via regulating miR-1261/C8orf4 axis in PTC, which suggested circZFR might be a potential therapeutic target.

Telomerase reverse transcriptase induced thyroid carcinoma cell proliferation through PTEN/AKT signaling pathway

Thyroid carcinoma is the most common endocrine malignant tumor in the world, and so, there is a requirement to develop novel molecular targets for thyroid cancer diagnosis and treatment. Telomerase reverse transcriptase (TERT) was revealed to promote cell proliferation in a number of types of cell. To evaluate whether and how TERT functioned on papillary thyroid cancer (PTC) cell proliferation, the present study constructed TERT over-expression [recombined (r)TERT plasmid group] and interference [small interfering RNA (si)-TERT group] models by liposome transfection respectively to study the molecular mechanisms. The transfection efficiency was first detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting to analyze TERT levels compared with the negative control (NC) and control groups. Then MTT and carboxyfluorescein diacetate succinimidyl ester assays were performed to determine living cell proliferation and total cell proliferation respectively. Propidium iodide assay was used to detect alterations in cell cycle progression. RT-qPCR and western blotting were performed to detect associated factor variation. The results demonstrated that, following the generation of TERT overexpression or silencing PTC cells, the living cells and also total cell proliferation increased significantly in the rTERT group, and decreased significantly in siTERT group, when compared with the NC and control groups. The cell cycle was accelerated in the rTERT group, and blocked in the G1/S transition in the siTERT group. The mRNA and protein levels of P27, P53 and phosphatase and tensin homolog (PTEN) decreased significantly in the rTERP group and increased in the siTERP group, while cyclin dependent kinase 2 and Cyclin D1 increased significantly in the rTERP group and decreased in the siTERP group. The expression of cell division cycle 25A did not alter significantly. The protein levels of β-catenin and retinoblastoma were also unaltered. Protein kinase B (AKT) was detected once activated by TERT, and there were increased phosphorylated (p)-AKT protein levels in the rTERT group, and decreased p-AKT protein levels in the siTERT group. In conclusion, TERT could induce thyroid carcinoma cell proliferation mainly through the PTEN/AKT signaling pathway.

MiR-524 inhibits cell proliferation and induces cell apoptosis in thyroid cancer via targeting SPAG9.

To investigate the effect of miR-524 on the proliferation of thyroid cancer and its underlying mechanism. MiR-524 expression levels in thyroid cancer samples and para-cancer tissues were tested by quantitative Real-time polymerase chain reaction (qRT-PCR). Cells proliferative ability and apoptosis were evaluated through methyl thiazolyl tetrazolium (MTT) and apoptosis assays, respectively. Luciferase reporter assay was used to confirm the regulatory mechanism. MiR-524 expression was reduced in thyroid cancer specimen (p<0.05). Up-regulated miR-524 expression inhibited the proliferative ability and enhanced cell apoptosis of thyroid cancer cells. SPAG9 was a target gene of miR-524, and was reversely regulated by miR-524. MiR-524 represses thyroid cancer cell proliferation and induces cell apoptosis via targeting SPAG9.

Lenvatinib in the Therapy of Aggressive Thyroid Cancer: State of the Art and New Perspectives with Patents Recently Applied.

Lenvatinib is an oral, multitargeted Tyrosine Kinase Inhibitor (TKI) of Vascular Endothelial Growth Factor Receptors (VEGFR1-VEGFR3), fibroblast growth factor receptors (FGFR1-FGFR4), Platelet-Derived Growth Factor Receptor (PDGFR)α, rearranged during transfection (RET), and v-kit (KIT) signaling networks implicated in tumor angiogenesis. Here, we review the scientific literature about lenvatinib in the treatment of thyroid cancer. In vitro studies have shown antineoplastic activity of lenvatinib in Differentiated Thyroid Cancer (DTC), mainly because of its antiangiogenetic effects, but a slight effect on thyroid cancer cell proliferation has been shown. In vivo Phase II, and Phase III studies in patients with aggressive DTC not responsive to radioiodine, have shown that lenvatinib administration was associated with an amelioration in Progression-Free Survival (PFS) with respect to placebo (median PFS 18.2 vs. 3.6 months). However, overall survival was not significantly changed. Lenvatinib is also effective in patients resistant to sorafenib as salvage therapy. Adverse effects of any grade occur in more than 40% of lenvatinib-treated patients, mainly hypertension, diarrhea, asthenia or fatigue, nausea, decreased appetite, and decreased weight. Discontinuations of the therapy because of adverse effects occur in about 14% of patients. Moreover, deaths considered to be drug-related can occur. On the basis of the above-mentioned considerations, it is necessary to prove the effectiveness of lenvatinib in the context of associated moderate to severe toxicities requiring frequent dose reduction and delays, and for this reason, many interesting patents have been recently applied.

Loss-of-function of IFT88 determines metabolic phenotypes in thyroid cancer.

Primary cilia are microtubule-based, dynamic organelles characterized by continuous assembly and disassembly. The intraflagellar transport (IFT) machinery, including IFT88 in cilia, is involved in the maintenance of bidirectional motility along the axonemes, which is required for ciliogenesis and functional competence. Cancer cells are frequently associated with loss of primary cilia and IFT functions. However, there is little information on the role of IFT88 or primary cilia in the metabolic remodeling of cancer cells. Therefore, we investigated the cellular and metabolic effects of the loss-of-function (LOF) mutations of IFT88/primary cilia in thyroid cancer cells. IFT88-deficient 8505C thyroid cancer cells were generated using the CRISPR/Cas9 system, and RNA-sequencing analysis was performed. LOF of the IFT88 gene resulted in a marked defect in ciliogenesis and mitochondrial oxidative function. Gene expression patterns in IFT88-deficient thyroid cancer cells favored glycolysis and lipid biosynthesis. However, LOF of IFT88/primary cilia did not promote thyroid cancer cell proliferation, migration, and invasion. The results suggest that IFT88/primary cilia play a role in metabolic reprogramming in thyroid cancer cells.

Inverse agonist of ERRγ inhibits the cell growth through the regulation of cell cycle and apoptosis in anaplastic thyroid cancer cells

In the previous study, we investigated that inverse agonist of ERRγ (estrogen‐related receptor gamma), GSK5182, enhances the responsiveness of radioiodine therapy by the modulation of sodium iodide symporter, NIS function in anaplastic thyroid cancer cells via the regulation of ERRγ and the MAPK signaling pathway. However, there is no reports showing the direct molecular mechanisms supporting the anti‐ cancer effect of inverse agonist of ERRγ. By the screening of inverse agonist of ERRγ, NDDC‐1228 which has more potent activity compared to GSK5182 was identified. Next, the functional activity of NDDC‐1228 was evaluated in the anaplastic thyroid cancer cells. At first, we evaluated the role of NDDC‐1228 in the regulation of thyroid cancer cell proliferation. NDDC‐1228 specifically inhibited the proliferation of CAL62, anaplastic thyroid carcinoma cell line under the cancer environment mimic condition but did not affect the proliferation of other organ derived cancer cell lines. When we examine the effect on the cell cycle, NDDC‐1228 treatment effectively induced the GI‐arrest by enhancing the expression of p21 and reducing the expression of Cyclin D and CDK proteins. In addition, NDDC‐1228 suppressed the expression of survivin, XIAP1 which are the inhibitors of apoptosis as well as activated the pro‐apoptotic protein, Caspase‐3. These results suggest that inverse agonist of ERRγ has anti‐cancer effects on anaplastic thyroid cancer cells via the regulation of cell cycle and apoptosis pathways and NDDC‐1228 could be used as a lead compound for the thyroid cancer therapy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-625-3p promotes the proliferation, migration and invasion of thyroid cancer cells by up-regulating astrocyte elevated gene 1

BackgroundThyroid cancer is the most common malignancy in human endocrine system. This study aimed to investigate the effects of microRNA-625-3p (miR-625-3p) on thyroid cancer cell proliferation, migration, invasion and apoptosis, as well as underlying potential mechanism. MethodsThe relative expressions of miR-625-3p in tumor tissues and adjacent normal tissues of 20 patients with papillary thyroid cancer (PTC) were assessed using qRT-PCR. Cell transfection was used to up-regulate or down-regulate the expressions of miR-625-3p in thyroid cancer SW579 and TPC-1 cells. Effects of miR-625-3p overexpression or suppression on SW579 and TPC-1 cell viability, migration, invasion and apoptosis were detected respectively. The regulatory effect of miR-625-3p on astrocyte elevated gene 1 (AEG-1) expression was also analyzed. Then, the roles of AEG-1 in SW579 and TPC-1 cell proliferation, migration, invasion and apoptosis, as well as Wnt/β-catenin and c-Jun N-terminal kinase (JNK) pathways activation, were evaluated. ResultsmiR-625-3p had high expressions in tumor tissues, compared to adjacent normal tissues. Overexpression of miR-625-3p significantly promoted SW579 and TPC-1 cell proliferation, migration and invasion but had no influence on cell apoptosis. Knockdown of miR-625-3p had opposite effects, but induced cell apoptosis. AEG-1 was up-regulated by miR-625-3p overexpression and participated in the effects of miR-625-3p on SW-579 and TPC-1 cells. In addition, overexpression of AEG-1 induced the activation of Wnt/β-catenin and JNK pathways in SW579 and TPC-1 cells. ConclusionmiR-625-3p promoted proliferation, migration and invasion of thyroid cancer cells by enhancing the expression of AEG-1 and activating downstream Wnt/β-catenin and JNK pathways.

Long glucocorticoid-induced leucine zipper regulates human thyroid cancer cell proliferation

Long glucocorticoid-induced leucine zipper (L-GILZ) has recently been implicated in cancer cell proliferation. Here, we investigated its role in human thyroid cancer cells. L-GILZ protein was highly expressed in well-differentiated cancer cells from thyroid cancer patients and differentiated thyroid cancer cell lines, but poorly expressed in anaplastic tumors. A fusion protein containing L-GILZ, when overexpressed in an L-GILZ-deficient 8505C cell line derived from undifferentiated human thyroid cancer tissue, inhibited cellular proliferation in vitro. In addition, when this protein was injected into nude mice, in which cells from line 8505C had been transplanted, xenograft growth was reduced. Since the mitogen-activated protein kinase (MAPK) pathway is frequently hyperactivated in thyroid cancer cells as a result of the BRAFV600E or Ras mutation, we sought to further investigate the role of L-GILZ in the MAPK pathway. To this end, we analyzed L-GILZ expression and function in cells treated with MAPK inhibitors. We used 8505C cells, which have the BRAFV600E mutation, or the CAL-62 cell line, which harbors a Ras mutation. The cells were treated with the BRAF-specific drug vemurafenib (PLX4032) or the MEK1/2 inhibitor, U0126, respectively. Treatment with these agents inhibited MAPK activation, reduced cell proliferation, and upregulated L-GILZ expression. L-GILZ silencing reversed the antiproliferative activity of the MAPK inhibitors, consistent with an antiproliferative role. Treatment with MAPK inhibitors led to the phosphorylation of the cAMP/response element-binding protein (CREB), and active CREB bound to the L-GILZ promoter, contributing to its transcription. We suggest that the CREB signaling pathway, frequently deregulated in thyroid tumors, is involved in L-GILZ upregulation and that L-GILZ regulates thyroid cancer cell proliferation, which may have potential in cancer treatment.

Up-regulation of miR-340-5p promotes progression of thyroid cancer by inhibiting BMP4.

The incidence of thyroid cancer is increasing and the proliferation of thyroid cancer cells is incompletely understood. microRNAs may play key roles in thyroid cancer progression. We analyzed miR-340-5p in thyroid cancer tissue and normal tissue, and using informatics to predict its target. Cell lines and a mouse model were used to study the role of miR-340-5p in cancer proliferation. Overexpression of miR-340-5p was found in thyroid cancer specimens. Tumors with higher pathological grade had higher levels of miR-340-5p. Overexpression of miR-340-5p significantly enhanced cell viability and colony formation. Treatment of anti-miR-340-5p, however, showed opposite alterations. We predicted that bone morphogenetic protein 4 (BMP4) is a possible target, and found a negative correlation between miR-340-5p and BMP4 levels in thyroid cancer tissue. miR-340-5p reduced BMP4 expression. BMP4 overexpression attenuated the effects of miR-340-5p in cell viability and colony formation. In addition, using a xenograft mouse model we proved that anti-miR-340-5p was able to inhibit tumor growth. miR-340-5p promotes thyroid cancer proliferation by inhibiting BMP4. Anti-miR-340-5p can be a promising strategy to control thyroid cancer.

Shikonin Inhibites Migration and Invasion of Thyroid Cancer Cells by Downregulating DNMT1.

BackgroundShikonin is a component of Chinese herbal medicine. The aim of this study was to investigate the effects of shikonin on cell migration of papillary thyroid cancer cells of the TPC-1 cell line in vitro and expression levels of the phosphate and tensin homolog deleted on chromosome 10 (PTEN) and DNA methyltransferase 1 (DNMT1) genes.Material/MethodsThe Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the proliferation of TPC-1 papillary thyroid cancer cells, and the normal thyroid cells, HTori-3, in vitro. A transwell motility assay was used to analyze the migration of TPC-1 cells. Western blot was performed to determine the expression levels of PTEN and DNMT1 genes. A methylation-specific polymerase chain reaction (PCR) (MSP) assay was used to evaluate the methylation of PTEN.ResultsFollowing treatment with shikonin, the cell survival rate of TPC-1 cells decreased in a dose-dependent manner; the inhibitory effects on HTori-3 cells were less marked. Shikonin inhibited TPC-1 cell migration and invasion in a dose-dependent manner. The methylation of PTEN was suppressed by shikonin, which also reduced the expression of DNMT1 in a dose-dependent manner, and increased the expression of PTEN. Overexpression of DNMT1 promoted the migration of TPC-1 cells and the methylation of PTEN. Levels of protein expression of PTEN in TPC-1 cells treated with shikonin decreased, and were increased by DNMT1 knockdown.ConclusionsShikonin suppressed the expression of DNMT1, reduced PTEN gene methylation, and increased PTEN protein expression, leading to the inhibition of TPC-1 cell migration.

Ginsenoside improves papillary thyroid cancer cell malignancies partially through upregulating connexin 31

Connexin 31 (Cx31) is considered a suppressor for many tumors. Ginsenoside (Rg1) is a traditional Chinese herb that is widely acknowledged due to its anti-tumor characteristics. However, limited studies have focused on the role of Rg1 in papillary thyroid cancer (PTC) cells. In the current study, we found that the expression of Cx31 in thyroid cancer tissues and thyroid cancer cell lines was significantly lower than that in normal thyroid epithelial tissues and cell lines. Overexpression of Cx31 reduced thyroid cancer cell proliferation, migration and invasion. Furthermore, we found that Rg1 significantly enhanced the expression of Cx31. Moreover, the proliferation and migration of IHH-4 and BCPAP cells were significantly reduced by Rg1 treatment. In contrast, the silencing of Cx31 enhanced the expression of Ki67 and proliferating cell nuclear antigen (PCNA). Meanwhile, treatment with Rg1 significantly decreased the protein levels of Ki67 and PCNA, but these effects could be abolished by transfection with si-Cx31. In summary, we provide novel evidence that the expression of Cx31 was decreased in thyroid cancer cells, but Rg1 treatment could significantly enhance the expression of Cx31 thereby suppressing thyroid cancer cell proliferation and migration.

Smad Ubiquitination Regulatory Factor 1 (Smurf1) Promotes Thyroid Cancer Cell Proliferation and Migration via Ubiquitin-Dependent Degradation of Kisspeptin-1

Background/Aims: Thyroid cancer is the most common malignancy in human endocrine system. Smad ubiquitination regulatory factor 1 (Smurf1) is an E3 ubiquitin-protein ligase in ubiquitin-proteasome pathway (UPP) system. This study aimed to investigate the effects of Smurf1 on thyroid cancer proliferation and metastasis, as well as underlying potential mechanism. Methods: The expression levels of Smurf1 in thyroid tumor tissues and thyroid cancer cells were detected by western blotting and qRT-PCR. Then, the effects of up-regulation or down-regulation of Smurf1 on thyroid cancer cell viability, migration, invasion, proliferation and apoptosis were measured using trypan blue exclusion assay, two-chamber migration (invasion) assay, cell colony formation assay and Guava Nexin assay, respectively. The ubiquitination of kisspeptin-1 (KISS-1) was assessed by protein ubiquitination assay. Finally, the effects of KISS-1 overexpression on activity of nuclear factor-kappa B (NF-κB) signaling pathway, as well as thyroid cancer cell viability, migration, invasion, proliferation and apoptosis were also detected, respectively. Results: Smurf1 was highly expressed in thyroid tumor tissues and thyroid cancer cells. Up-regulation of Smurf1 promoted the viability, migration, invasion and proliferation of thyroid cancer cells. Knockdown of Smurf1 had opposite effects. Moreover, smurf1 promoted the ubiquitination of KISS-1. Overexpression of KISS-1 inactivated NF-κB pathway, suppressed thyroid cancer cell viability, migration, invasion and proliferation, and induced cell apoptosis. Conclusion: Up-regulation of Smurf1 exerted important roles in thyroid cancer formation and development by promoting thyroid cancer proliferation and metastasis. The ubiquitin-dependent degradation of KISS-1 induced by Smurf1 and the activation of NF-κB signaling pathway might be involved in this process. Smurf1 could be an effective therapy target and biomarker for thyroid cancer treatment.

Synergistic Antitumor Effect of BKM120 with Prima-1Met Via Inhibiting PI3K/AKT/mTOR and CPSF4/hTERT Signaling and Reactivating Mutant P53

Background/Aims: PI3KCA and mutant p53 are associated with tumorigenesis and the development of cancers. NVP-BKM120, a selective pan-PI3K inhibitor, exerts the antitumor activity by suppressing the PI3K signaling pathway. Prima-1^Met, a low molecular weight compound, can rescue the gain-of-function of mutant p53 by restoring its transcriptional function. In this study, we investigated whether PI3K inhibition combined with mutant p53 reactivation could enhance the antitumor effect in thyroid cancer cells. Methods: The effects of BKM120 and Prima-1^Met on the proliferation, apoptosis, migration and invasion of thyroid cancer cells were measured by MTT, colony formation, flow cytometry, wound-healing and transwell assays, respectively. Thyroid differentiation was assessed by detecting the expression levels of specific markers using RT-PCR and Western blot. The in vivo antitumor efficacy was analyzed in a mouse xenograft model. Results: The combinational treatment of BKM120 and Prima-1^Met significantly enhanced the inhibitions of cell viability, colony formation, migration and invasion, and the induction of apoptosis in thyroid cell lines, and synergistically suppressed tumor xenograft growth by inhibiting the PI3K/Akt/mTOR and EMT signaling pathways, up-regulating p53 targeted genes, and triggering the release of cytochrome c. Moreover, the combination of BKM120 and Prima-1^Met suppressed the stemlike traits of thyroid cancer cells and promoted their differentiation by upregulating the expression of thyroid-specific differentiation markers and repressing the expression of cancer stem cell markers. Furthermore, the mechanism study demonstrated that the combinational treatment synergistically abrogated the binding of CPSF4 at the promoter of hTERT and thus suppressed hTERT expression. Consistently, overexpression of hTERT rescued the inhibitions of cell viability, invasion and stem-like traits mediated by the combination of BKM120 and Prima-1^Met. Conclusion: Our results showed that the combination of BKM120 with Prima-1^Met synergistically suppressed the growth of thyroid cancer cells and tumor xenografts via inhibiting PI3K/Akt/mTOR and CPSF4/hTERT signaling and reactivating mutant p53.

Overexpression of ING5 inhibits HGF-induced proliferation, invasion and EMT in thyroid cancer cells via regulation of the c-Met/PI3K/Akt signaling pathway

The inhibitor of growth 5 (ING5), a novel member of the ING family, is involved in diverse biological processes such as cell growth, apoptosis and DNA repair. Recently, ING5 has been reported to be associated with cancer development. However, its specific role in thyroid cancer has yet to be elucidated. In this study, we found that the expression of ING5 was significantly down-regulated in human thyroid cancer tissues and cell lines. In addition, overexpression of ING5 markedly inhibited hepatocyte growth factor (HGF)-induced proliferation, invasion and epithelial-mesenchymal transition (EMT) of thyroid cancer cells as well as suppressed the tumor growth and metastasis in vivo. Furthermore, our data showed that the c-Met/PI3K/Akt signaling pathway was responsible for the inhibitory effect of ING5 on the thyroid cancer. Taken together, these findings provided an essential basis for the tumor-suppression role of ING5 in thyroid cancer.

SGK1 Is a Critical Component of an AKT-Independent Pathway Essential for PI3K-Mediated Tumor Development and Maintenance.

Activation of the PI3K-AKT signaling cascade is a common critical event during malignant transformation. In this study, we used thyroid gland epithelial cells and a series of genetically engineered mouse strains as model systems to demonstrate that, although necessary, AKT activation is not sufficient for PI3K-driven transformation. Instead, transformation requires the activity of the PDK1-regulated AGC family of protein kinases. In particular, SGK1 was found to be essential for proliferation and survival of thyroid cancer cells harboring PI3K-activating mutations. Notably, cotargeting SGK1 and AKT resulted in significantly higher growth suppression than inhibiting either PI3K or AKT alone. Overall, these findings underscore the clinical relevance of AKT-independent pathways in tumors driven by genetic lesions targeting the PI3K cascade. Cancer Res; 77(24); 6914-26. ©2017 AACR.

Simvastatin Inhibits Cell Proliferation and Migration in Human Anaplastic Thyroid Cancer.

Malignant human anaplastic thyroid cancer (ATC) is pertinacious to conventional therapies. The present study investigated the anti-cancer activity of simvastatin and its underlying regulatory mechanism in cultured ATC cells. Simvastatin (0–20 μM) concentration-dependently reduced cell viability and relative colony formation. Depletions of mevalonate (MEV) and geranylgeranyl pyrophosphate (GGpp) by simvastatin induced G1 arrest and increased apoptotic cell populations at the sub-G1 phase. Adding MEV and GGpp prevented the simvastatin-inhibited cell proliferation. Immunoblotting analysis illustrated that simvastatin diminished the activation of RhoA and Rac1 protein, and this effect was prevented by pre-treatment with MEV and GGpp. Simvastatin increased the levels of p21cip and p27kip proteins and reduced the levels of hyperphosphorylated-Rb, E2F1 and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression.

Upregulation of RSPO2-GPR48/LGR4 signaling in papillary thyroid carcinoma contributes to tumor progression.

The signaling pathway involving the R-spondins and its cognate receptor, GPR48/LGR4, is crucial in development and carcinogenesis. However, the functional implications of the R-spondin-GPR48/LGR4 pathway in thyroid remain to be identified. The aim of this study was to investigate the role of R-spondin-GPR48/LGR4 signaling in papillary thyroid carcinomas. We retrospectively reviewed a total of 214 patients who underwent total thyroidectomy and cervical lymph node dissection for papillary thyroid carcinoma. The role of GPR48/LGR4 in proliferation and migration was examined in thyroid cancer cell lines. R-spondin 2, and GPR48/LGR4 were expressed at significantly higher levels in thyroid cancer than in normal controls. Elevated GPR48/LGR4 expression was significantly associated with tumor size (P=0.049), lymph node metastasis (P=0.004), recurrence (P=0.037), and the BRAFV600E mutation (P=0.003). Moreover, high GPR48/LGR4 expression was an independent risk factor for lymph node metastasis (P=0.027) and the BRAFV600E mutation (P=0.009). in vitro assays demonstrated that elevated expression of GPR48/LGR4 promoted proliferation and migration of thyroid cancer cells, whereas downregulation of GPR48/LGR4 decreased proliferation and migration by inhibition of the β-catenin pathway. Moreover, treatment of thyroid cancer cells with exogenous R-spondin 2 induced activation of the β-catenin pathway through GPR48/LGR4. The R-spondin 2-GPR48/LGR4 signaling axis also induced the phosphorylation of ERK, as well as phosphorylation of LRP6 and serine 9 of GSK3β. Our findings demonstrate that upregulation of the R-spondin 2-GPR48/LGR4 pathway contributes to tumor aggressiveness in papillary thyroid carcinoma by promoting ERK phosphorylation, suggesting that this pathway represents a novel therapeutic target for treatment of differentiated thyroid cancer.

RasGRP3 controls cell proliferation and migration in papillary thyroid cancer by regulating the Akt-MDM2 pathway

Accumulating evidence has shown that Ras guanylnucleotide releasing peptide 3 (RasGRP3) is up-regulated in several distinct cancer types; however, its role in papillary thyroid cancer (PTC) remains unclear. In this study, we demonstrate that RasGRP3 was overexpressed in PTC tissues and cell lines. Downregulation of RasGRP3 using small interfering (si) RNA significantly inhibited PTC cell proliferation and migration in vitro, and tumor growth in vivo, reflecting an oncogenic role of RasGRP3 in PTC. We subsequently identified that the expression of mouse double minute 2 homolog (MDM2) and phosphorylated Akt (p-Akt) was significantly decreased in RasGRP3-downregulated PTC cells. Overexpression of MDM2 attenuated the function of si-RasGRP3. Taken together, our data show that RasGRP3 exerts its oncogenic effect in PTC through Akt-mediated MDM2 activation. RasGRP3 may serve as a potential new therapeutic target for PTC.

Silencing of TCTN1 inhibits proliferation, induces cell cycle arrest and apoptosis in human thyroid cancer.

Tectonic family member 1 (TCTN1) is one of the tectonic family members, and a regulator of the hedgehog signaling pathway, which has been studied in various cancer types, including prostate and pancreatic cancer. However, its function in thyroid cancer has not been well documented. Therefore, the present study investigated the function of TCTN1 in thyroid cancer using a loss-of-function assay. Lentivirus-mediated RNA interference was applied to downregulate TCTN1 in the thyroid cancer cell lines, CAL62 and 8305C. A series of functional properties, including cell viability, colony formation, cell cycle and apoptosis were determined using MTT, colony formation assay and flow cytometry analyses, respectively. The results demonstrated that lentivirus-medicated RNAi could specifically suppress the expression of TCTN1 at the mRNA and protein levels in CAL62, and 8305C cells. Knockdown of TCTN1 inhibited cell growth and proliferation via inducing S phase arrest, and apoptosis. Mechanistically, the S phase arrest was accompanied by the upregulation of cyclin dependent kinase 2, cyclin A2 and downregulation of cyclin B1. Knockdown of TCTN1 induced apoptosis through increasing the expression of Bcl2-associated agonist of cell death, cleaved caspase-3 and poly(ADP-ribose)polymerase, and decreasing apoptosis regulator Bcl-2 expression. The current study highlights the essential role of TCTN1 in promoting thyroid cancer cell proliferation, and its knockdown may serve as a potential therapeutic treatment for thyroid cancer.

PATZ1 knockdown enhances malignant phenotype in thyroid epithelial follicular cells and thyroid cancer cells.

This study was designed to examine the involvement of PATZ1 in carcinogenesis and dedifferentiation of thyroid cancer. Immunohistochemistry on clinical specimens indicated nuclear PATZ1 expression in all normal thyroid glands and adenomatous goiter, while nuclear PATZ1 expression decreased along with the dedifferentiation of thyroid cancer. Knockdown of nuclear PATZ1 by siRNA in an immortalized normal follicular epithelial cell line (Nthy-ori 3-1) altered cellular morphology and significantly increased cell proliferation, migration, and invasion. In addition, the expression of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP) 2, MMP9, and MMP11 was increased by PATZ1 knockdown in Nthy-ori 3-1 cells. When PATZ1 was silenced in differentiated thyroid cancer (DTC) cell lines (TPC-1 and FTC-133), proliferation, cellular motility, and expression of uPA and MMPs were significantly increased. Forced expression of exogenous PATZ1 decreased proliferation, cellular motility, and the expression of uPA and MMPs in ATC cell lines (ACT-1 and FRO). In thyroid cancer cell lines, PATZ1 functioned as a tumor suppressor regardless of p53 status. Moreover, the ratio of nuclear PATZ1 positive tumors was significantly decreased in ATC irrespective of p53 status. Our study demonstrates that PATZ1 knockdown enhances malignant phenotype both in thyroid follicular epithelial cells and thyroid cancer cells, suggesting that PATZ1 functions as a tumor suppressor in thyroid follicular epithelial cells and is involved in the dedifferentiation of thyroid cancer.