SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells

Background

SULF2 is a 6-O-endosulfatase which removes 6-O sulfate residues from N-glucosamine present on heparan sulfate (HS). The sulfation pattern of HS influences signaling events mediated by heparan sulfate proteoglycans (HSPGs) located on cell surface, which are critical for the interactions with growth factors and their receptors. Alterations in SULF2 expression have been identified in the context of several cancer types but its function in cancer is still unclear where the precise molecular mechanism involved has not been fully deciphered. To further investigate SULF2 role in tumorigenesis, we overexpressed such gene in prostate cancer cell lines.

Methods

The normal prostate epithelial cell line RWPE-1 and the prostate cancer cells DU-145, and PC3 were transfected with SULF2-expressing plasmid pcDNA3.1/Myc-His(−)-Hsulf-2. Transfected cells were then submitted to viability, migration and colony formation assays.

Results

Transfection of DU-145 and PC3 prostate cancer cells with SULF2 resulted in increased viability, which did not occur with normal prostate cells. The effect was reverted by the knockdown of SULF2 using specific siRNAs. Furthermore, forced expression of SULF2 augmented cell migration and colony formation in both prostate cell lines. Detailed structural analysis of HS from cells overexpressing SULF2 showed a reduction of the trisulfated disaccharide UA(2S)-GlcNS(6S). There was an increase in epithelial-mesenchymal transition markers and an increase in WNT signaling pathway.

Conclusions

These results indicate that SULF2 have a pro-tumorigenic effect in DU-145 and PC3 cancer cells, suggesting an important role of this enzyme in prostatic cancer metastasis.     

Modulation of angiogenesis and tumorigenicity of human melanocytic cells by vascular endothelial growth factor and basic fibroblast growth factor

Human melanoma cells express two prominent angiogenic factors, e.g., vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF/fibroblast growth factor-2). In this study, we report on the relative contribution of these two factors to in vitro and in vivo growth of a tumorigenic melanoma cell line (WM164) and nontumorigenic, immortalized melanocytes (FM516SV). Overexpression of either cytokine significantly boosted tumorigenicity of WM164 cells in immunodeficient SCID mice. Attempting to overexpress bFGF antisense sequences produced no viable clones confirming earlier reports that autocrine bFGF is obligatory to melanoma cell survival and growth. By contrast, down-regulation of endogenous VEGF production did not affect growth of WM164 cells in vitro. In vivo expansion of WM164 cells expressing VEGF antisense was delayed but not abrogated. Forced expression of either bFGF or VEGF in immortalized but nontumorigenic melanocytes did not induce sustained tumor growth in vivo highlighting that neither of the two factors is sufficient for induction of tumorigenicity in this model system. Overexpression of either cytokine in WM164 cells led to the development of atypical large vessels but not to an increase in microvessel density. Taken together our results confirm an essential autocrine role of bFGF in human melanoma and indicate a beneficial but nonessential role of VEGF in the tumorigenic phenotype of human melanoma cells.     

Androgen-induced differentiation and tumorigenicity of human prostate epithelial cells

Androgen ablation is the primary treatment modality for patients with metastatic prostate cancer; however, the role of androgen receptor signaling in prostate cancer development remains enigmatic. Using a series of genetically defined immortalized and tumorigenic human prostate epithelial cells, we found that introduction of the androgen receptor induced differentiation of transformed prostate epithelial cells to a luminal phenotype reminiscent of organ-confined prostate cancer when placed in the prostate microenvironment. Moreover, androgen receptor expression converted previously androgen-independent, tumorigenic prostate epithelial cells into cells dependent on testosterone for tumor formation. These observations indicate that androgen receptor expression is oncogenic and addictive for the human prostate epithelium.     

E2F1对 TFDP3 表达的调控及其对前列腺癌PC3细胞凋亡的影响

目的：构建含TFDP3基因启动子和荧光素酶报告基因的重组载体pGL3-TFDP3-promoter,观察E2F1对TFDP3转录及表达的调控作用以及TFDP3对E2F1诱导肿瘤细胞凋亡的影响。方法：以人前列腺癌PC3细胞系基因组DNA为模板,PCR扩增TFDP3启动子序列并克隆入荧光素酶报告基因载体,与E2F1表达载体pCMV-E2F1-HA瞬时单独或共同转染PC3细胞,测定荧光素酶活性以观察E2F1对TFDP3启动子的调控作用,Western blotting检测pCMV-E2F1-HA转染对PC3细胞内TFDP3表达的影响,流式细胞术检测TFDP3与E2F1相互作用对前列腺癌细胞凋亡的影响。结果：成功构建TFDP3基因启动子重组质粒pGL3-TFDP3-promoter,与E2F1表达载体pCMV-E2F1-HA共转染PC3细胞后,TFDP3启动子诱导的荧光素酶活性较单独转染pGL3-TFDP3-promoter显著升高[（1.14±0.06）vs（0.61±0.05）, P<0.05]。转染pCMV-E2F1-HA的PC3细胞的TFDP3蛋白表达是未转染细胞的2.7倍[（0.24±0.03）vs（0.09±0.02）, P<0.05]。pCMV-E2F1-HA转染后PC3细胞凋亡率较未转染组显著上升[（7.10±0.003）% vs（2.66±0.001)%,P<0.05],而pCMV-E2F1-HA与pcDNA3.1-TFDP3共转染后细胞凋亡率较pCMV-E2F1-HA组显著下降[（4.92±0.002）% vs（7.10±0.003）%,P<0.05]。结论：E2F1可增强TFDP3启动子的活性,增加TFDP3蛋白的表达,其可能通过此机制抑制E2F1诱导的前列腺癌细胞凋亡。     

Claudin-2 expression increases tumorigenicity of colon cancer cells: role of epidermal growth factor receptor activation

Claudin-2 is a unique member of the claudin family of transmembrane proteins, as its expression is restricted to the leaky epithelium in vivo and correlates with epithelial leakiness in vitro. However, recent evidence suggests potential functions of claudin-2 that are relevant to neoplastic transformation and growth. In accordance, here we report, on the basis of analysis of mRNA and protein expression using a total of 309 patient samples that claudin-2 expression is significantly increased in colorectal cancer and correlates with cancer progression. We also report similar increases in claudin-2 expression in inflammatory bowel disease-associated colorectal cancer. Most importantly, we demonstrate that the increased claudin-2 expression in colorectal cancer is causally associated with tumor growth as forced claudin-2 expression in colon cancer cells that do not express claudin-2 resulted in significant increases in cell proliferation, anchorage-independent growth and tumor growth in vivo. We further show that the colonic microenvironment regulates claudin-2 expression in a manner dependent on signaling through the EGF receptor (EGFR), a key regulator of colon tumorigenesis. In addition, claudin-2 expression is specifically decreased in the colon of waved-2 mice, naturally deficient in EGFR activation. Furthermore, genetic silencing of claudin-2 expression in Caco-2, a colon cancer cell line, prevents the EGF-induced increase in cell proliferation. Taken together, these results uncover a novel role for claudin-2 in promoting colon cancer, potentially via EGFR transactivation.     

Interactions between MYC and transforming growth factor alpha alter the growth and tumorigenicity of liver progenitor cells

The MYC oncogene induces both cell proliferation and apoptosis. The apoptotic function of MYC is thought to inhibit carcinogenesis; thus, when disrupted, tumorigenic potential is increased. Both MYC and transforming growth factor alpha (TGFalpha) are commonly over-expressed in hepatocellular carcinomas, and transgenic mice expressing these genes rapidly develop tumors via the suppression of MYC-induced apoptosis by the growth factor. However, the nature of the interactions between MYC and TGFalpha are not well understood. Specifically, it is unclear whether TGFalpha acts only as an anti-apoptotic factor in its interactions with MYC or whether it has substantial effects on cell growth. We investigated whether TGFalpha can provide additional mitogenic signals if it is not required to act as an anti-apoptotic factor. We demonstrate that expression of MYC and TGFalpha in liver progenitor cells (known as oval cells) results in enhanced cell proliferation in culture and the generation of poorly differentiated tumors after inoculation into nude mice. We further demonstrate that while the apoptosis-deficient T58A and S71F alleles of MYC retain their ability to promote oval cell proliferation, they have opposite growth interactions with TGFalpha. The T58A allele has a stimulatory effect on both proliferation and tumorigenicity. In contrast, co-expression of the S71F allele reduces proliferation and slows tumor development. We conclude that the tumorigenic growth effects of MYC in TGFalpha-expressing liver progenitor cells are not solely dependent on its apoptotic activity.     

Sox2在人前列腺癌中的表达及其临床意义

目的 检测Sox2在人前列腺癌和癌旁组织中的表达情况,并分析其与前列腺癌病理分级和临床分期的关系.方法 分别采用免疫组织化学、Western杂交及反转录-聚合酶链反应(RT-PCR)检测86例前列腺癌组织标本和40例癌旁组织标本中Sox2的表达情况.分析其与前列腺癌病理分级和临床分期之间的相关性.结果 免疫组织化学检测结果显示前列腺癌组织中Sox2表达显著高于癌旁组织,阳性表达率分别为79.1％和7.5％,差异有统计学意义(x2=53.98,P＜0.05);在前列腺癌组织中Sox2的表达与Gleason评分之间相关性具有统计学意义(x2=19.49,P＜0.05);在淋巴结转移组和非淋巴结转移组Sox2的阳性表达率分别为92.3％和73.3％,差异有统计学意义(x2=3.95,P＜0.05);但在不同临床分期的前列腺癌组织中Sox2的表达差异无统计学意义(x2=0.16,P＞0.05).Western杂交及RT-PCR检测结果显示前列腺癌组织中Sox2表达显著高于癌旁组织,在淋巴结转移组和非淋巴结转移组之间Sox2表达差异具有统计学意义(t=-2.93,P＜0.05;t =3.29,P＜0.05).结论 Sox2的表达差异与前列腺癌的Gleason评分和淋巴结转移密切相关,而与临床分期无关,Sox2可能在前列腺癌的发生、分化和转移过程中起重要作用.     

The role of transforming growth factor alpha production and ErbB-2 overexpression in induction of tumorigenicity of lung epithelial cells.

Over-expression of erbB-2 is associated with shortened survival of patients with lung adenocarcinomas. We demonstrated that human lung epithelial cells, overexpressing erbB-2, formed tumours in nude mice only when high levels of transforming growth factor (TGF)-alpha were produced (E6T cells). To define the role that TGF-alpha production played in induction of tumorigenicity, a non-tumorigenic TGF-alpha-negative clone of ErbB-2 overexpressing cells (E2 cells) was transfected with an expression vector for TGF-alpha (E2alpha cells). Transfected clones produced TGF-alpha at 11-25% of the level produced by the E6T cell line. Tumorigenic E6T cells transfected with a TGF-alpha antisense vector (E6TA cells) expressed only 6% of the TGF-alpha level of the parental cells. Clones of E6T, E6TA, E2 and E2alpha were inoculated into athymic nude mice to measure tumorigenic potential. E6T cells formed tumours with a 70% efficiency. E2, E6TA and E2alpha cells failed to form tumours. The levels of EGFR were similar in non-tumorigenic E2 and tumorigenic E6T cells but higher in E2alpha and E6TA cells, and ErbB-2 were greatly overexpressed in an E2alpha clone. In vitro, ErbB-2 co-immunoprecipitated with EGFR in lysates of unstimulated E6T and E2alpha TGF-alpha-producing cells, indicating that the lower TGF-alpha levels were sufficient to induce in vitro heterodimerization. These studies suggest that induction of the tumorigenic phenotype depends on achieving a threshold level of TGF-alpha sufficient to activate downstream signalling by ErbB-2 containing active heterodimers.