Activating enhancer-binding protein-2α induces cyclooxygenase-2 expression and promotes nasopharyngeal carcinoma growth

Activating enhancer-binding protein-2α (AP-2α) regulates the expression of many cancer-related genes. Here, we demonstrated a novel mechanism by which AP-2α up-regulated cyclooxygenase-2 (COX-2) expression to promote the growth of nasopharyngeal carcinomas (NPCs). High expression of AP-2α in NPC cell lines and tumor tissues from NPC patients was detected and significantly correlated with COX-2 expression. Overexpression of AP-2α and COX-2 in tumor tissues was associated with advanced tumor stage, clinical progression, and short survival of patients with NPCs. Knockdown of AP-2α by siRNA markedly inhibited COX-2 expression and PGE2 production in NPC cells. Exogenous expression of AP-2α up-regulated the COX-2 and PGE2. Knockdown of AP-2α also significantly suppressed cell proliferation in NPC cells in vitro and tumor growth in a NPC xenograft mouse model. Moreover, we found that p300 played an important role in the AP-2α/COX-2 pathway. AP-2α could co-localize and interact with p300 in NPC cells. Overexpression of the p300, but not its histone acetyltransferase (HAT) domain deletion mutant, promoted the acetylation of AP-2α and its binding on the COX-2 promoter, thereby up-regulated COX-2 expression. Our results indicate that AP-2α activates COX-2 expression to promote NPC growth and suggest that the AP-2α/COX-2 signaling is a potential therapeutic target for NPC treatment.     

Inhibition of VEGF induces cellular senescence in colorectal cancer cells

Vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, have improved outcomes in metastatic colorectal cancer (CRC). Recent studies have suggested that VEGF can delay the onset of cellular senescence in human endothelial cells. As VEGF receptors are known to be upregulated in CRC, we hypothesized that VEGF inhibition may directly influence cellular senescence in this disease. In our study, we observed that treatment with bevacizumab caused a significant increase (p < 0.05) in cellular senescence in vitro in several CRC cells, such as MIP101, RKO, SW620 and SW480 cells, compared to untreated or human IgG-treated control cells. Similar results were also obtained from cells treated with a VEGFR2 kinase inhibitor Ki8751. In vivo, cellular senescence was detected in MIP101 tumor xenografts from 75% of mice treated with bevacizumab, while cellular senescence was undetectable in xenografts from mice treated with saline or human IgG (p < 0.05). Interestingly, we also observed that the proportion of senescent cells in colon cancer tissues obtained from patients treated with bevacizumab was 4.4-fold higher (p < 0.01) than those of untreated patients. To understand how VEGF inhibitors may regulate cellular senescence, we noted that among the two important regulators of senescent growth arrest of tumor cells, bevacizumab-associated increase in cellular senescence coincided with an upregulation of p16 but appeared to be independent of p53. siRNA silencing of p16 gene in MIP101 cells suppressed bevacizumab-induced cellular senescence, while silencing of p53 had no effect. These findings demonstrate a novel antitumor activity of VEGF inhibitors in CRC, involving p16.     

Apurinic/apyrimidinic endonuclease 1 regulates angiogenesis in a transforming growth factor β‐dependent manner in human osteosarcoma

Angiogenesis plays an important role in tumor growth and metastasis and has been reported to be inversely correlated with overall survival of osteosarcoma patients. It has been shown that apurinic/apyrimidinic endonuclease 1 (APE1), a dually functional protein possessing both base excision repair and redox activities, is involved in tumor angiogenesis, although these mechanisms are not fully understood. Our previous study showed that the expression of transforming growth factor β (TGFβ) was significantly reduced in APE1‐deficient osteosarcoma cells. Transforming growth factor β promotes cancer metastasis through various mechanisms including immunosuppression, angiogenesis, and invasion. In the current study, we initially revealed that APE1, TGFβ, and microvessel density (MVD) have pairwise correlation in osteosarcoma tissue samples, whereas TGFβ, tumor size, and MVD were inversely related to the prognosis of the cohort. We found that knocking down APE1 in osteosarcoma cells resulted in TGFβ downregulation. In addition, APE1‐siRNA led to suppression of angiogenesis in vitro based on HUVECs in Transwell and Matrigel tube formation assays. Reduced secretory protein level of TGFβ of culture medium also resulted in decreased phosphorylation of Smad3 of HUVECs. In a mouse xenograft model, siRNA‐mediated silencing of APE1 downregulated TGFβ expression, tumor size, and MVD. Collectively, the current evidence indicates that APE1 regulates angiogenesis in osteosarcoma by controlling the TGFβ pathway, suggesting a novel target for anti‐angiogenesis therapy in human osteosarcoma.     

Suppression of Cellular Apoptosis Susceptibility (CSE1L)Inhibits Proliferation and Induces Apoptosis in ColorectalCancer Cells

The cellular apoptosis susceptibility (CSE1L) gene has been demonstrated to regulate multiple cellularmechanisms including the mitotic spindle check point as well as proliferation and apoptosis. However, theimportance of CSE1L in human colon cancer is largely unknown. In the present study, we examined expressionlevels of CSE1L mRNA by semiquantitative RT-PCR. A lentivirus-mediated small interfering RNA (siRNA)was used to knock down CSE1L expression in the human colon cancer cell line RKO. Changes in CSE1L targetgene expression were determined by RT-PCR. Cell proliferation was examined by a high content screeningassay. In vitro tumorigenesis was measured by colony-formation assay. Cell cycle distribution and apoptosiswere detected by flow cytometric analysis. We found CSE1L mRNA to be expressed in human colon cancer cells.Using a lentivirus based RNAi approach, CSE1L expression was significantly inhibited in RKO cells, causingcell cycle arrest in the G2/M and S phases and a delay in cell proliferation, as well as induction of apoptosisand an inhibition of colony growth capacity. Collectively, the results suggest that silencing of CSE1L may be apotential therapeutic approach for colon cancer.     

Possible involvement of TGF beta 1 in the distinct tumorigenic properties of two rat colon carcinoma clones.

The presence in tumors of numerous cytokines suggests that they potentially modulate tumor cell activities and host tissue remodelling. To investigate the possible involvement of transforming growth factor type beta (TGF beta) in the metastatic process of cancer development, we have studied the effect of this factor on two rat colon carcinoma cell lines. These cell clones had been previously tested and selected for their ability to develop metastases in syngenic animals or lack of it. The two cell lines were characterized for their production of TGF beta. Production of active and latent forms of TGF beta 1 in the medium conditioned by the rat colon cancer cells were quantified using a bioassay. The presence of active TGF beta 1 was demonstrated in conditioned medium from the progressive tumor (PROb) cells and significant expression of latent forms of TGF beta 1 were found in the conditioned media from both cell clones. TGF beta 1 slightly inhibited proliferation of PROb cells which had been previously described as moderately differentiated, and significantly stimulated proliferation of the regressive (REGb) cells, described as poorly differentiated. On the basis of our observations, we suggest that this endogenous factor could be involved in autocrine regulation of tumor cell activities and in paracrine regulation of stroma cell and immune responses. Active and/or latent expression of TGF beta 1 by the two rat colon carcinoma cell lines, and their variable responses to the growth factor, strongly suggest that this polypeptide is involved in the regulation of tumorigenic expression of adenocarcinoma cells.     

TRAIL基因修饰联合阿霉素对大肠癌细胞株RKO作用的研究

目的:探讨肿瘤坏死因子(tumor necrsis factor,TNF)相关的凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)基因联合阿霉索后,应用于人大肠癌细胞株RKO基因治疗的实验研究.方法:将重组腺病毒载体(Ad)介导的TRAIL基因作用于大肠癌细胞株RKO,并联合低剂量的阿霉素协同作用.通过MTF比色法与流式细胞仪研究分析其对RKO细胞的作用效果,并以RT-PCR检测联合应用阿霉素前后TRAIL基因的表达水平.结果:病毒载体对RKO细胞的生长有轻微的抑制作用,作用4 d抑制率为11.9%,但不增加RKO细胞的凋亡率.TRAIL对RKO细胞的生长抑制率及凋亡诱导率分别为50.1%和19.8%.联合阿霉素后,TRAIL对RKO细胞株的生长抑制率及凋亡率均有显著的增强作用,分别达60.3%及49.0%.RT-PCR结果提示联合应用阿霉素后,TRAIL基因的表达并未增强.结论:TRAIL能有效抑制RKO的生长,联合阿霉素后,其对RKO的生长抑制作用及凋亡诱导作用均明显增强.阿霉素不是通过增加TRAIL基因的表达来实现上述作用的.     

TGF‐β induced PAR‐1 expression promotes tumor progression and osteoclast differentiation in giant cell tumor of bone

Although protease activated receptor‐1 (PAR‐1) has been confirmed as an oncogene in many cancers, the role of PAR‐1 in giant cell tumor (GCT) of bone has been rarely reported. The mechanism of PAR‐1 in tumor‐induced osteoclastogenesis still remains unclear. In the present study, we detected that PAR‐1 was significantly upregulated in GCT of bone compared to normal tissues, while TGF‐β was also overexpressed in GCT tissues and could promote the expression of PAR‐1 in a dose and time dependent manner. Using the luciferase reporter assay, we found that two downstreams of TGF‐β, Smad3 and Smad4, could activate the promoter of PAR‐1, which might explain the mechanism of TGF‐β induced PAR‐1 expression. Meanwhile, PAR‐1 was also overexpressed in microvesicles from stromal cells of GCT (GCTSCs), and might be transported from GCTSCs to monocytes through microvesicles. In addition, knockout of PAR‐1 by TALENs in GCTSCs inhibited tumor growth, angiogenesis and osteoclastogenesis in GCT in vitro. Using the chick CAM models, we further showed that inhibition of PAR‐1 suppressed tumor growth and giant cell formation in vivo. Using microarray assay, we detected a number of genes involved in osteoclastogenesis as the possible downstreams of PAR‐1, which may partly explain the mechanism of PAR‐1 in GCT. In brief, for the first time, these results reveal an upstream regulatory role of TGF‐β in PAR‐1 expression, and PAR‐1 expression promotes tumor growth, angiogenesis and osteoclast differentiation in GCT of bone. Hence, PAR‐1 represents a novel potential therapeutic target for GCT of bone.     

Gene Silencing of TGFβRII Can Inhibit Glioblastoma Cell Growth

Objective: Glioblastoma (GBM) is the most malignant and aggressive type of glioma, associated with a high rateof mortality. The transforming growth factor-β receptor II (TGFβ RII) is involved in glioma initiation and progression.On the other hand, TGFβ RII silencing is critical to the inhibition of GBM. Therefore, we aimed to determine theeffects of specific TGFβ RII siRNA on the survival of U-373MG cells. Methods: TGFβ RII siRNA was transfected,and qRT-PCR was performed to examine TGFβ RII mRNA expression. Cell survival was determined using colorimetricMTT assay, and platelet-derived growth factor-BB (PDGF-BB) level was measured in the culture supernatant usingELISA assay. Result: Our findings indicated that specific siRNAs could dose-dependently suppress TGFβ RII mRNAexpression after 48 hours. In addition, treatment with TGFβ RII siRNA significantly reduced tumor cell survival anddecreased the amount of PDGF-BB protein in the cell culture supernatant. Conclusion: Our results suggest that TGFβRII silencing can be a promising complementary treatment for glioma.     

Release of TGFβig-h3 by gastric myofibroblasts slows tumor growth and is decreased with cancer progression

Tumor progression has been linked to changes in the stromal environment. Myofibroblasts are stromal cells that are often increased in tumors but their contribution to cancer progression is not well understood. Here, we show that the secretomes of myofibroblasts derived from gastric cancers [cancer-associated myofibroblasts (CAMs)] differ in a functionally significant manner from those derived from adjacent tissue [adjacent tissue myofibroblasts (ATMs)]. CAMs showed increased rates of migration and proliferation compared with ATMs or normal tissue myofibroblasts (NTMs). Moreover, conditioned medium (CM) from CAMs significantly stimulated migration, invasion and proliferation of gastric cancer cells compared with CM from ATMs or NTMs. Proteomic analysis of myofibroblast secretomes revealed decreased abundance of the extracellular matrix (ECM) adaptor protein like transforming growth factor-β-induced gene-h3 (TGFβig-h3) in CAMs, which was correlated with lymph node involvement and shorter survival. TGFβig-h3 inhibited IGF-II-stimulated migration and proliferation of both cancer cells and myofibroblasts, and suppressed IGF-II activation of p42/44 MAPkinase; TGFβig-h3 knockdown increased IGF-II- and CM-stimulated migration. Furthermore, administration of TGFβig-h3 inhibited myofibroblast-stimulated growth of gastric cancer xenografts. We conclude that stromal cells exert inhibitory as well as stimulatory effects on tumor cells; TGFβig-h3 is a stromal inhibitory factor that is decreased with progression of gastric cancers.     

Inhibition of transforming growth factor-β signals suppresses tumor formation by regulation of tumor microenvironment networks

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-β (TGF-β) promotes tumor progression by inducing epithelial–mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-β receptor containing both TGF-β type I (TβRI) and type II (TβRII) receptors (TβRI-TβRII-Fc), which trapped all TGF-β isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TβRI-TβRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TβRI-TβRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TβRI-TβRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1β (IL-1β) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1β and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-β signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1β/EREG pathways and that TβRI-TβRII-Fc protein is a promising tool for targeting the TME networks.     

Selective activation of ceruloplasmin promoter in ovarian tumors: potential use for gene therapy

Gene therapy provides a novel treatment approach to cancer patients. Ideally, expression of therapeutic genes driven by cancer-specific promoters would only target tumors resulting in minimal toxicity to normal tissues. While there is a need of more effective and tolerable treatments for ovarian cancer patients, we aimed to identify gene promoters with high activity in ovarian tumors that can be potentially used in gene therapy to drive the expression of a therapeutic gene in tumors. To identify such promoters, a literature search was performed to reveal genes that are preferentially expressed in ovarian cancer compared with normal ovarian tissue. We found that the ceruloplasmin promoter drove up to 30-fold higher luciferase expression in ovarian cancer cells compared with immortalized normal cells. Furthermore, deletion studies revealed an activator protein-1 (AP-1) site in the ceruloplasmin promoter to be critical for optimal ceruloplasmin promoter activity. Ceruloplasmin promoter activity was significantly activated by 1-O-tetradecanoyl phorbol-13-acetate, a c-jun activator, and conversely suppressed by SP600125, a c-jun inhibitor. Consistently, the ceruloplasmin AP-1 site was specifically recognized by c-jun both in vitro and in vivo. Immunohistochemical analyses of human ovarian cancer specimens showed a direct correlation (r = 0.7, P = 0.007) between expression levels of c-jun and ceruloplasmin. In nude mice carrying SKOV3.ip1 xenografts, the ceruloplasmin promoter demonstrated significantly higher activities in tumors compared with normal organs. Together, these results suggest that the ceruloplasmin promoter activity is significantly enhanced in ovarian cancer and therefore may be exploited as a promising cancer-specific promoter in developing new gene therapy strategies for ovarian cancer.