SIRT4 inhibits the proliferation, migration, and invasion abilities of thyroid cancer cells by inhibiting glutamine metabolism.

BACKGROUND: SIRT4, a protein localized in the mitochondria, is one of the least characteristic members of the sirtuin family. It is known that SIRT4 has deacetylase activity and plays a role in energy metabolism, but little is known about its possible role in carcinogenesis. Recently, several studies have suggested that SIRT4 may function as either a tumor oncogene or a tumor suppressor gene. However, its relationship with thyroid cancer remains unclear. METHODS: We stably overexpressed SIRT4 or silenced its expression in the human thyroid cancer cell line BCPAP by means of lentiviral vectors. We conducted a variety of tests, such as CCK-8, wound healing, migration, and invasion assays, to investigate the role of SIRT4 in the proliferation, migration, and invasion abilities of thyroid cancer cells. We also investigated the effects of SIRT4 overexpression on cell cycle progression and apoptosis of BCPAP cells and studied the role of glutamine metabolism in the effects of SIRT4 on BCPAP cell migration and invasion. Finally, we analyzed SIRT4 expression levels in thyroid cancer specimens by immunohistochemistry and investigated their association with clinicopathological features. RESULTS: Overexpression of SIRT4 inhibited the proliferation, migration, and invasion abilities of BCPAP thyroid cancer cells, blocked the cell cycle in the G0/G1 phase, and induced apoptosis. Mechanistically, SIRT4 inhibited BCPAP migration and invasion by inhibiting glutamine metabolism. Moreover, we found that SIRT4 protein levels in thyroid cancer tissues were markedly lower than in their non-neoplastic tissue counterparts (P<0.001). CONCLUSION: SIRT4 plays a pivotal role in the growth and metastasis of thyroid cancer cells and could be a potential therapeutic target in thyroid cancer.

Short hairpin RNA targeting autotaxin reduces human gastric carcinoma AGS cell proliferative, migratory and invasive capabilities in vitro and causes tumor regression in vivo.

The aim of this study was to investigate the effect of short hairpin RNA (shRNA) targeting autotaxin (ATX) on the migratory and invasive capability of AGS human gastric cancer cells and the growth of xenografts in nude mice. pSUPER-ATX and pSUPER-mock (non-specific), which were constructed corresponding to the ATX-shRNA and negative control mock-shRNA synthesized based on gene sequence, were transfected with blank plasmid pSUPER-control into AGS human gastric cancer cells using Lipofectamine. At 24, 48 and 72 h post-transfection, the cells were harvested and analyzed. The endogenous ATX mRNA and protein of the different groups of AGS cells were detected by RT-PCR and western blot assays. Cell proliferation was measured by MTT assay. In vitro Transwell and Matrigel assays were used to detect the cell migratory and invasive capabilities. A tumor xenograft model was generated by subcutaneous injection of AGS cells into the dorsum of nude mice. The growth of xenograft tumors was monitored and measured; changes in tumor morphology and the organs of mice were observed by H&E staining. The expression of ATX, MMP-2 and MMP-9 protein in xenograft tumor tissues was detected by immunohistochemistry and western blotting. In vitro, both the mRNA and protein levels of ATX in the pSUPER-ATX group were significantly downregulated (P<0.01), and the cell proliferative, migratory and invasive capabilities were also significantly decreased. In vivo, no obvious damage to the organs was found. pSUPER-ATX significantly suppressed the tumor volume and weight from the 7th week after cell transplantation, compared to the pSUPER-mock, pSUPER-control and WT groups; the inhibition rates were approximately 50% (P<0.05). However, no significant differences in these parameters were found among the WT, pSUPER-mock and pSUPER-control groups. Furthermore, ATX, MMP-2 and MMP-9 were expressed at significantly lower levels in the pSUPER-ATX group compared to the levels in the other three groups (P<0.05), and a significant correlation between ATX and MMP-2 expression was found (r=0.869, P<0.01). The specific shRNA targeting ATX downregulated the endogenous ATX of AGS human gastric cancer cells, and inhibited AGS cell proliferative, migratory and invasive potentials. Moreover, shRNA targeting ATX inhibited the growth of human gastric cancer xenografts in nude mice.

Targeting of the anti-apoptotic gene survivin in human thyroid carcinoma.

Survivin is a novel apoptosis inhibitor. Its gene is related to the baculovirus gene, which is believed to play a crucial role in fetal development and in cancer. We attempted to determine the expression of survivin in both thyroid goiter and carcinoma tissues, and to evaluate its prognostic value in human thyroid disease. In the present study, we applied small interfering RNA (siRNA) directed against survivin to determine the effects of decreasing the high constitutive levels of this protein in the FTC-133 thyroid follicular cancer cell line. Using reverse transcription PCR and immunohistochemistry, we compared the expression of survivin with relevant clinical and pathological data of 90 postsurgical specimens from patients with primary thyroid carcinoma and patients with benign goiter (33 with papillary thyroid cancer, 24 with follicular thyroid cancer, 18 with undifferentiated thyroid cancer and 15 cases with goiter). For the siRNA treatment in a human follicular thyroid carcinoma cell line, fluorescein-labeled double-stranded ultrapure siRNAs were used. RT-PCR identified the survivin transcript in 67/75 (89.3%) tumor samples and in 4/15 benign goiter samples. Immunohistochemical analysis showed positive immunoreactivity in 65/75 (86.7%) carcinomas while no expression was noted in all of the 15 benign goiter tissues. Survivin mRNA and protein levels were significantly higher in cancer tissues compared to benign goiter tissues (P<0.001). Higher survivin expression was found in the tumor tissues of pT3/pT4 and in the tumors with lymph node metastasis (P<0.05). Tumors with distant metastasis demonstrated higher survivin expression compared to the tumors without distant metastasis. Additionally, the expression of survivin in undifferentiated carcinomas was higher than that in differentiated ones. There was no significant correlation between survivin expression and age, gender, histological subtype and pathological stage. Our additional studies demonstrated that siRNA directed against survivin markedly decreased the protein expression of survivin. In conclusion, we conclude that survivin expression indicates more aggressive behavior and metastatic ability in thyroid cancer cells in vivo. Survivin can be used as a diagnostic and therapeutic marker for thyroid carcinoma and an important target in the strategy of thyroid cancer therapy. Our results of siRNA silencing indicate that siRNA may have potential as a therapeutic modality in the treatment of human thyroid cancer.

Down-regulation of TM4SF is associated with the metastatic potential of gastric carcinoma TM4SF members in gastric carcinoma.

BACKGROUND: The aim of this study was to clarify the clinical significance of TM4SF members CD9, CD63 and CD82 in human gastric carcinoma. METHODS: By employing RT-PCR and immunohistochemistry, we studied the expression of CD9, CD63 and CD82 in 49 paired tissue specimens of normal gastric mucosa and carcinoma. All tissues were obtained from patients who underwent curative surgery. RESULTS: All normal gastric epithelium and gastric ulcer tissues strongly expressed transcripts and proteins of CD9, CD63 and CD82 as compared with corresponding controls. We found a significant correlation between CD63 mRNA level and different pM statuses (P = 0.036). Carcinomas in M0 stage revealed a stronger expression of CD63 than carcinomas in M1 stage. Expression of CD9 protein was found significantly stronger in pN0, pM0 than in advanced pN stages (P = 0.03), pM1 (P = 0.013), respectively. We found the relationship between CD63 expression, gender (p = 0.09) and nodal status (p = 0.028), respectively. Additionally, advanced and metastasized tumor tissues revealed significantly down-regulated CD82 protein expression (p = 0.033 and p = 0, respectively), which correlated with the tumor pTNM stage (p = 0.001). CONCLUSION: The reduction of CD9, CD63 and CD82 expression are indicators for the metastatic potential of gastric carcinoma cells. Unlike their expression in other tumor types, the constitutive expression of CD63 may indicate that this factor does play a direct role in human gastric carcinogenesis.

Retinoic acid-mediated down-regulation of ENO1/MBP-1 gene products caused decreased invasiveness of the follicular thyroid carcinoma cell lines.

Retinoic acid (RA) acts as an anti-proliferative and redifferentiation agent in the therapy of thyroid carcinoma. Our previous studies demonstrated that pretreatment of follicular thyroid carcinoma cell lines FTC-133 and FTC-238 resulted in decreased in vitro proliferation rates and reduced tumor cell growth of xenotransplants. In addition to the previous results, we found that RA led to decreased vitality and invasiveness of FTC-133 and FTC-238 cells as they reacted with reduction of intracellular ATP levels and number of migrated cells respectively. However, the molecular mechanisms by which RA mediates these effects are not well understood. Two-dimensional (2D) screening of the proteins related to ATP metabolism and western blot analysis revealed alpha-enolase (ENO1) to be down-regulated in FTC-133 and FTC-238 cells after RA treatment. 2D gel detection and mass spectrometric analysis revealed that ENO1 existed as three separate protein spots of distinct pIs (ENO1-A1-A3). Comparative 2D difference gel electrophoresis analysis of fluorescently labeled protein samples of RA-treated and untreated FTC-133 demonstrated a selective down-regulation of ENO1-A1 which we identified as a phosphoprotein. RA caused the dephosphorylation of ENO1-A1. Both, RA-mediated and specific knock-down of ENO1/MBP-1 resulted in the reduction of MYC oncoprotein, and simultaneously decreased proliferation rates of FTC-133 and FTC-238 cell lines. In summary, the RA-mediated down-regulation of the ENO1 gene products and MYC oncoprotein provides a novel molecular mechanism facilitating the anti-proliferative effect of RA in human thyroid carcinoma cells and suggests new pathways for supportive RA therapies.

The expression of CD97EGF and its ligand CD55 on marginal epithelium is related to higher stage and depth of tumor invasion of gastric carcinomas.

CD97EGF is a member of the EGF-TM7 family of class II seven-transmembrane (7TM), and its cellular ligand CD55 (also known as decay accelerating factor; DAF) protects host cells from complement attack. To determine whether the expression levels of these two molecules are correlated with the clinicopathological features of gastric carcinomas, a total of 35 gastric carcinomas and their corresponding margins and normal specimens were investigated by RT-PCR, Western blot analysis and immunohistochemistry. Transcript levels of CD97EGFand CD55 were higher in tumors than those in the margin and normal epithelial mucous tissues (P<0.05). However, the expression levels of CD97EGF and CD55 mRNA had no correlation with the clinicopathological features of gastric carcinoma patients. All three groups of specimens were immunoreactive for CD97EGF and the CD55 protein. Strong and specific immunoreactivities of CD97EGF were located in the mucosal epithelia of the marginal basal membrane. Expression of CD97(EGF) in the margins showed a marked difference between the depth of tumor invasion T1 and T2, 3 and 4, and stages I and II/III/IV of gastric carcinomas (P<0.05). The expression of CD55 protein was highly correlated with CD97EGF (R=0.6483, P<0.001). Western blot analysis confirmed the expression and distribution patterns of CD97EGF and CD55. Our findings suggest that CD97EGF may play a role in the development and invasion of gastric carcinomas by binding its cellular ligand CD55. Detection of the CD97EGF expression in the tumor margin could be referred to as the molecular edge of gastric carcinomas.

Role of CD97(stalk) and CD55 as molecular markers for prognosis and therapy of gastric carcinoma patients.

OBJECTIVES: To explore the mechanism of development and aggressiveness in gastric carcinomas by investigating the expression and role of CD97 and its cellular ligand CD55 in gastric carcinomas. METHODS: Tumor and corresponding normal mucosal tissue, collected from 39 gastric carcinoma patients, were examined by immunohistochemistry and RT-PCR for the expression of CD97 and CD55. RESULTS: CD97(stalk) was strongly stained on scattered tumor cells or small tumor cell clusters at the invasion front of gastric carcinomas. The expression of CD97(stalk) was frequently observed in tumors of stage I and T1 gastric carcinoma patients. The expression of CD97(stalk) between Stage I and Stage II, III, IV specimens showed significant difference (P<0.05), between T1 and T2, T3, T4 specimens also showed significant difference (P<0.05). Specimens with tumor invasion depth limited in mucosa of T1 specimens showed higher positive CD55 expression than specimens with the same tumor invasion depth in T2, T3, T4 specimens, the expression of CD55 between T1 and T2, T3, T4 specimens was significantly different (P<0.05). There was strong correlation between the distribution patterns of CD97(stalk) and CD55 on tumor tissues (r=0.73, P<0.05). Signet ring cell carcinomas frequently contained strong CD97(stalk) and CD55-staining. CONCLUSIONS: Our results suggest that CD97(stalk) is probably involved in the growth, invasion and aggressiveness of gastric carcinomas by binding its cellular ligand CD55. CD97(stalk) and CD55 could be useful as molecular markers for prognosis and therapy of gastric carcinoma patients.

CD82, and CD63 in thyroid cancer.

CD82 (KAI1) and CD63 (ME491) are highly glycosylated proteins which belong to the transmembrane 4 superfamily (TM4SF). CD82 has been implicated as a possible prostate cancer metastasis suppressor gene, whereas CD63 is involved in the progression of human melanoma cancer. Down-regulation of both CD82 and CD63 expression has been associated with the metastatic potential of several solid tumors. Currently, information is lacking on the role of CD82 and CD63 during thyroid carcinogenesis. The aim of this study was to determine whether the expression of CD82 and CD63 is a useful prognostic indicator in patients with thyroid carcinoma. The expression of CD82 and CD63 was analysed by reverse transcriptase-PCR (RT-PCR) and immunohistochemistry in benign goiter (n=12) and 75 primary thyroid carcinoma tissue specimens (PTC: 33, FTC: 24, UTC: 18) out of which 36 were non-metastasized primary tumors and 39 were metastasized tumors (regional lymph node and/or distant metastases). All of the benign goiter tissues showed CD82 expression. By contrast, a significant decrease in CD82 mRNA and protein levels was detected in carcinoma tissues as compared to benign goiter tissues (p<0.001). A similar down-regulation was observed in metastasized tumor tissues when compared with non-metastasized tumors (all p<0.05). CD82 expression was correlated with pTNM status of differentiated and undifferentiated thyroid tumor and the pathologic stage of differentiated thyroid tumor. In contrast to CD82, CD63 mRNA and protein expression was unchanged in all thyroid carcinomas. Benign goiter tissues showed weak expression of CD63. There were no significant correlation between CD63 mRNA/protein expression and any clinical/pathological parameters. Our results support the hypothesis that down-regulation of CD82 expression may reflect an increased in vivo metastatic potential of thyroid cancer cells. CD82 may serve as a prognostic marker of metastasis in thyroid cancer. Constitutive expression of CD63 may indicate that this factor does not play a direct role in thyroid carcinogenesis.