Susu Xie

Deregulation of the HOXA10 Homeobox Gene in Endometrial Carcinoma: Role in Epithelial-Mesenchymal Transition

Homeobox genes encode transcription factors that control cell differentiation and play essential roles in developmental patterning. Increasing evidence indicates that many homeobox genes are aberrantly expressed in cancers, and that their deregulation significantly contributes to tumor progression. The homeobox gene HOXA10 controls uterine organogenesis during embryonic development and functional endometrial differentiation in the adult. We investigated whether HOXA10 expression is deregulated in endometrial carcinomas, and how counteracting this aberrant expression modifies tumor behavior. We found that down-regulation of HOXA10 expression in endometrial carcinomas strongly correlates with increased tumor grade and is associated with methylation of the HOXA10 promoter. Enforced expression of HOXA10 in endometrial carcinoma cells inhibited invasive behavior in vitro and tumor dissemination in nude mice. The inhibitory effect of HOXA10 on invasive behavior was attributable at least in part to the ability of HOXA10 to induce expression of the epithelial cell adhesion molecule E-cadherin by down-regulating expression of Snail, a repressor of E-cadherin gene transcription. These findings reveal a novel role for HOXA10 deregulation in the progression of endometrial carcinoma by promoting epithelial-mesenchymal transition.

Epigenetic alterations in neuroendocrine tumors: methylation of RAS-association domain family 1, isoform A and p16 genes are associated with metastasis

Well-differentiated neuroendocrine tumors including pancreatic endocrine tumors and carcinoid tumors are uncommon neoplasms that have site-specific differences in clinicopathological features, clinical course and genetic alterations. The epigenetic alterations in these tumors are not well characterized. We therefore compared methylation of the RAS-association domain family 1, isoform A (RASSF1A), p14, p16 and O6-methyl-guanine methyltransferase genes in neuroendocrine tumors from 47 patients including 16 pancreatic, 15 nonileal and 16 ileal neuroendocrine tumors. Methylation of the RASSF1A gene was present in 57% of tumors, p14 in 49%, p16 in 26% and O6-methyl-guanine methyltransferase in 13% of tumors. Ileal neuroendocrine tumors lacked methylation of O6-methyl-guanine methyltransferase gene (P=0.04). RASSF1A methylation was associated with histopathologic type of tumors (P=0.03) and lymph node metastasis (P=0.004), and p16 methylation with older patient age (P=0.002) and liver metastasis (P=0.04). Two or more genes were methylated in 53% of tumors, one gene was methylated in 30% of tumors, and all four genes were unmethylated in 17% of tumors. Methylation of one or more gene was associated with older age of patients (P=0.01), and methylation of two or more genes was associated with liver metastasis (P=0.044). Our study shows that in neuroendocrine tumors epigenetic alterations vary by tumor subsite and clinicopathologic features, including age of onset, histopatholoic type and metastasis status.

Hypomethylation-induced expression of S100A4 in endometrial carcinoma

Expression of various S100 genes has been associated with clinically aggressive subtypes in a variety of different cancers. We hypothesized that S100A4 would be overexpressed in endometrial carcinoma compared to benign endometrium. Quantitative real-time RT-PCR (qRT-PCR) was used to quantify the mRNA level of S100A4 in benign endometrium (n=19), endometrioid adenocarcinoma (n=87), and non-endometrioid tumors (n=21). Immunohistochemistry was used to verify the results of qRT-PCR and to assess protein localization. Possible mechanisms of S100A4 gene regulation were also examined. S100A4 was overexpressed in the grade 3 endometrioid tumors, uterine papillary serous carcinoma, and uterine malignant mixed müllerian tumor. Expression in grade 1 and grade 2 endometrioid tumors was comparable to that of normal endometrium, which was quite low. Expression was significantly higher in stage III and IV tumors compared with stage I. By immunohistochemistry, S100A4 was expressed in the tumor cell cytoplasm of poorly differentiated tumors, but was not detected in normal endometrial glandular epithelium. In benign endometrium, S100A4 expression was confined to stromal cells. S100A4 was not regulated by estrogen or progesterone, and its expression in tumors was not significantly correlated to estrogen receptor or progesterone receptor content. However, methylation of the S100A4 gene was detected in benign endometrium and grade 1 tumors with low S100A4 expression. In contrast, grade 3 endometrioid tumors with high S100A4 mRNA and protein expression showed no methylation of the gene. These methylation results were verified in endometrial cancer cell lines with differential baseline levels of S100A4 protein. These results suggest that hypomethylation is an important mechanism of regulating the expression of the S100A4 gene. These results support the emerging concept that hypomethylation may play a role in the upregulation of genes during later stages of tumorigenesis.

S100A4 mediates endometrial cancer invasion and is a target of TGF-β1 signaling

The molecular mechanisms of endometrial cancer invasion are poorly understood. S100A4, also known as FSP1 (fibroblast-specific protein 1), has long been known to be a molecular marker of fibrosis in a variety of different fibrotic diseases of the lungs, liver, kidney, and heart. We demonstrate here that increased expression of S100A4 is associated with advanced stage endometrial cancer and decreased recurrence free survival. To verify the essential role of S100A4 in invasiveness of endometrial cancer, S100A4 expression was downregulated by RNAi in HEC-1A cells, which resulted in undetectable S100A4 protein and significantly decreased migration and invasion. Owing to the established connection between TGF-β1 and S100A4 induction in experimental models of kidney and liver fibrosis, we next examined whether TGF-β1 could also regulate S100A4 in endometrial cancer cells. TGF-β1 stimulated endometrial cancer cell migration and invasion with a concomitant increase in S100A4 protein. Induction of S100A4 was associated with the activation of Smads. TGF-β1-mediated endometrial cancer cell motility was inhibited by S100A4 siRNA. In aggregate, these results suggest that S100A4 is a critical mediator of invasion in endometrial cancer and is upregulated by the TGF-β1 signaling pathway. These results also suggest that endometrial cancer cell invasion and fibrosis share common molecular mechanisms.

Abstract 1538: Inhibition of adenosine-mediated stabilization of intercellular adhesions promotes hypoxia-stressed endometrial carcinoma cell metastasis

Hypoxic microenvironments are commonly encountered in multiple epithelial malignancies. Epithelial cells classically up-regulate cellular adhesive mechanisms to counter the noxious effects of hypoxia. Inhibiting these tissue protective mechanisms is necessary for carcinoma cell invasion and metastasis. Our previous studies utilizing a transgenic model deficient for 5’nucleotidase (CD73), an enzyme responsible for adenosine biosynthesis, demonstrated that adenosine is required to circumvent hypoxia-mediated disruption of cellular adhesiveness in the normal endometrium. In endometrial carcinoma (EC), CD73 is down-regulated in poorly-differentiated, invasive, and metastatic ECs, but is expressed and functional in well-differentiated and non-metastatic disease. Based on these findings, we hypothesized that the malignant progression of EC is dependant on the loss of adenosine-mediated regulation of intercellular adhesions. Modified and unmodified Boyden chambers were used to assess migration and invasion of endometrial adenocarcinoma cells HEC-1A and HEC-1B. Hypoxia (5% or 1.5% O 2 ) significantly reduced HEC-1A and HEC-1B migration and invasion in vitro; this effect was abrogated by CD73 RNA interference and the CD73 enzymatic inhibitor, α, β- methylenediphosphate (AoPCP). The inhibitory effect of CD73 on EC cell migration and invasion was restored by the stable, receptor-targeting adenosine analog, 5’-N-ethylcarboxamido adenosine (NECA). Quantitative RT-PCR identified two adenosine receptors, A 1 R and A 2B R, to be expressed in HEC-1A and HEC-1B. Inhibition of the A 2B R by MRS1754 significantly increased HEC-1B migration and invasion, while A 1 R antagonism (DPCPX) had no effect. A 2B R inhibition in HEC-1A monolayers also resulted in increased paracellular permeability to FITC-labeled dextrans. Moreover, A 2B R antagonism lowered cellular levels of junctional adhesion proteins claudin-1 and claudin-2, and resulted in increased accumulation of β-catenin within the cytoplasm. Intracellular pools of β-catenin re-organized to the cell membrane with NECA application. In summary, we have shown that CD73-generated adenosine is necessary to maintain normal protective epithelial barrier function in the uterus. Our results demonstrate for the first time that inhibition of adenosine-mediated stabilization of intercellular adhesions promotes cancer cell invasion and metastasis in a hypoxic microenvironment. (NIH TL1RR024147 and NIH 1P50CA098258-01) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1538. doi:10.1158/1538-7445.AM2011-1538

Abstract 3384: Down-regulation of 5’-nucleotidase (CD73)-generated adenosine: A novel mechanism for regulating endometrial cancer metastasis

Metastasis remains a significant concern for women with advanced or recurrent endometrial cancer (EC), as adjuvant chemotherapy and hormonal therapy have little effect. Therefore, more attention recently has been focused on the development of targeted therapies. The purinergic pathway, a metabolic cascade responsible for adenosine biosynthesis and regulation, has been clinically targeted in chronic lung and neurodegenerative diseases. Of interest is the association of adenosine dysregulation with epithelial-to-mesenchymal transition (EMT) in chronic lung diseases, as EMT is an essential hallmark of tumor metastasis. Adenosine and the purinergic pathway9s involvement in cancer EMT are unknown. In this study, we evaluated the expression of purinergic pathway components (adenosine receptor subtypes, A 1 R, A 2A R, A 2B R, and A 3 R; adenosine biosynthesis enzyme, 5′-nucleotidase (CD73); nucleoside transporter, equilibrative nucleoside transporter 1 (ENT1); and adenosine metabolizing enzyme, adenosine deaminase (ADA)), by quantitative real-time RT-PCR in 12 normal endometrial tissues and 46 endometrial carcinomas of varying histotypes. CD73 was the single component significantly altered in EC. Specifically, CD73 was down-regulated in poorly-differentiated, invasive and metastatic ECs. Similar correlations were observed by immunoassays. CD73 was also evaluated in EC cell lines representative of various EMT stages (HEC-1A, well- and KLE, poorly-differentiated, primary tumor; AN3CA, poorly-differentiated, metastatic lesion). CD73 transcript levels were highest in HEC-1A, low in KLE, and undetectable in AN3CA. Immunoanalyses confirmed these findings and revealed CD73 to be primarily localized to extracellular membranes in direct contact with neighboring cells. Using reverse phase HPLC, we demonstrated CD739s potential to function catalytically, and in addition, confirmed a direct correlation between transcript expression and production of adenosine in endometrial tissues and cell lines (p 2 =0.9388; p 2 =0.9816, respectfully). Moreover, as seen by enzyme histochemistry and electron microscopy, CD73 activity occurred exclusively at established intercellular adhesions. Our results suggest a novel function for CD73-generated adenosine in maintaining endometrial epithelial adhesions, with its down-regulation being an essential event of EC EMT. For a variety of different cancer types, clinical correlations have been previously established between tumor cells retaining intercellular adhesions and therapeutic responsiveness, but factors regulating these adhesions have not yet been successfully directly targeted. The drugable nature of adenosine and the purinergic pathway and its likely role in mediating endometrial epithelial intercellular adhesions represents a potential therapeutic strategy for metastatic EC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3384.