Sialylation is catalyzed by sialyltransferases (STs) that adding sialic acids to the terminal positions of oligosaccharide of glycoproteins and glycolipids. This process is frequently enhanced in cancer and is associated with increased cancer metastasis. Recent studies demonstrated that over-expression of ST3Gal-I promotes mammary tumorigenesis. In our experiments, we also find overexpression of £\-2,3-ST in breast cancer cells. We previously synthesized a lithocholic acid-based ST inhibitor AL10 and demonstrated its anti-metastatic effect in vitro and in vivo. Our results showed that AL10 is an effective sialyltransferase inhibitor and exerts anti-metastatic effect in vivo via suppression of sialylation of beta1 integrin and CXCR4. Breast cancer cells expressing high level of chemokine receptors CXCR4 and CCR7 are prone to exhibit lymphatic metastasis because their cognate ligands CCL19, CCL21 and SDF-1 are continuously expressed by lymphatic endothelial cells. In this study, we demonstrate that AL10 can inhibit invasion, proliferation and induce anoikis of £\-2,3-ST-overexpressing MDA-MB231 human breast cancer cells. Our results indicate that inhibition of CCL19-induced invasion and CCL19-reduced anoikis by AL10 are associated with reduced sialylation of CCR7 and attenuated activation of the downstream signaling mediator ERK and p38. In addition, AL10 can inhibit proliferation by reducing activation of AKT via CCR7 sialylation independent pathway and p-38 via CCR7 sialylation dependent pathway which results in ubiquitin-dependent cyclin D1 degradation. Taken together, we conclude that sialylation of CCR7 is critical for CCL19-stimulated proliferation, invasion and anti-anoikis in breast cancer cells.

Estrogen receptor α (ERα) is a ligand activated transcription factor. Many widely used synthetic compounds and natural chemicals can activate ERα. The compounds investigated in this study include 17β-estradiol (E2), diethylstilbestrol (DES), antiestrogens ICI 182,780, 4-hydroxytamoxifen, the phytoestrogen resveratrol, and the xenoestrogens bisphenol A (BPA), nonylphenol (NP), octylphenol (OP), endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1- trichloroethane (HPTE) and 2,3,4,5-tetrachlorobiphenylol-4-ol (HO-PCB-Cl4). With the exception of the antiestrogens, all the compounds induced transactivation in MCF-7 or MDA-MB-231 cells transfected with wild-type ERα and a construct (pERE3) containing three tandem estrogen responsive elements (EREs) linked to a luciferase gene. However, these compounds differentially activated C-terminal deletion mutants of ERα. For example, neither E2 nor DES induced transactivation in MCF-7 transfected with ERα(1-537) due to partial deletion of helix 12 of ERα; however, OP, NP, resveratrol, kepone and HPTE induced this ERα mutant, demonstrating that the estrogenic activity of these synthetic compounds do not require activation function 2 (AF-2) of ERα. This study also investigated the effects of xenoestrogens on activation of reporter gene activity in MCF-7 and MDA-MB-231 cells transfected with a construct (pSp13) containing three tandem GC-rich Sp binding sites linked to the luciferase gene. In MCF-7 cells, antiestrogen-induced activation of ERα/Sp1 required the zinc finger motifs of ERα, whereas activation by estrogen and some xenoestrogens activation, such as endosulfan, NP and OP required the H12 of ERα. In contrast, xenoestrogens, such as HPTE, BPA, kepone and HO-PCBCl4, significantly induced transactivation of all four ERα deletion mutants tested in this study. Moreover, RNA interference assays demonstrated structuredependent differences in activation of ERα/Sp1, ERα/Sp3 and ERα/Sp4. The in vivo activities of E2, ICI 182,780, BPA and NP were further investigated in a transgenic mouse model containing pSp13 transgene. All the compounds induced luciferase activity in the mouse uterus; however activities observed in the penis and testis of male and stomach of both male and female mice were structure-dependent,. These results demonstrate that various ER ligands differentially activate ERα in breast cancer cells and transgenic mice, and their activities are dependent on ERα variants, promoter-, cell-context and selective use of different Sp proteins, suggesting these structurally diverse compounds are selective ER modulators (SERMs).

Virtually all signaling pathways that play key roles in development such as the transfroming growth factor (TGF)-beta, notch, and wnt pathways also influence tumor formation, implying that cancer is in a sense development gone awry. Therefore, identification and elucidation of developmental pathways has great potential for generating new diagnostic tools and molecular therapy targets. Singleminded-2s (SIM2s), a splice variant of the basic helilx-loop-helix / PER-ARNT-SIM (bHLH/PAS) transcriptional repressor Singleminded-2, is lost or repressed in approximately 70% of human breast tumors and has a profound influence on normal mammary development. In order to gain a better understanding of the mechanisms by which SIM2s restricts malignant transformation and progression in breast cancer, we depleted SIM2 RNA in MCF-7 cells using a retroviral shRNA system and examined gene expression and functional abilities of the SIM2-depleted MCF-7 cells (SIM2i) relative to a control MCF line expressing a non-specific “scrambled” shRNA (SCR). Depletion of SIM2 resulted in an epithelial-mesenchymal transition (EMT)-like effect characterized by increased migration and invasion, altered morphology, and loss of epithelial markers concomitant with gain of mesenchymal markers. The root of this effect may be loss of SIM2- mediated repression of the E-cadherin repressor slug, as SIM2 is able to bind and repress transcription from the slug promoter, and slug expression is dramatically elevated in SIM2i MCF-7 cells. Consistent with the previously established role of slug in resistance to various cancer therapies, SIM2i cells are resistant to the radiomimetic doxorubicin and appear to have elevated self-renewal capacity under certain conditions. Intriguingly, SIM2 protein levels are elevated by treatment with DNA damaging agents, and SIM2 interacts with the p53 complex via co-regulation of specific p53- target gene such as p21/WAF1/CIP1. These results provide a plausible mechanism for the tumor suppressor activity of SIM2, and provide insight into a novel tumor suppressive transcriptional circuit that may have utility as a therapeutic target.

The chaperone proteins designated 14-3-3 are expressed in all eukaryotic cells; they help to regulate signal transduction pathways controlling proliferation, differentiation, and survival. They associated directly or indirectly with proliferative signal-transducing proteins such as PKC, MEK kinases, PI3-kinase and Raf. In human, there are seven isotypes of 14-3-3 genes: £]¡]beta¡^¡B£^¡]gamma¡^¡B£`¡]epsilon¡^¡B£b¡]eta¡^¡B£m¡]sigma¡^¡B£n/£c¡]tau/theta¡^ and£a¡]zeta¡^, some of which would be pseudogenes, and yeast and plant each have two and fifteen genes. Althought these genes are diverse, all 14-3-3 isotypes share many conservation domains in amino acid sequences. The previous studies have suggested that 14-3-3 sigma is most directly linked to cancer because it is thought to function as a tumor suppressor by inhibiting cell-cycle progression. In tumor formation, inactivation of 14-3-3 sigma occurs with high frequency. More importantly, expression of 14-3-3 sigma is silenced in most breast cancer cells. The 14-3-3 sigma protein is associated with cyclin E-CDK2 complex as well as cyclin B-CDC2 complex and mediated their inactivation by cytoplasmic localization and causing cell-cycle arrest in G2 and G1. However, the roles of other 14-3-3 isotypes in the formation of breast cancer are controversial in published reference. The aim of this study was to determine the differential expressions of 14-3-3 gamma in non-tumor tissues and corresponding tumor tissues. Amplification and overexpression of 14-3-3 gamma in DNA, RNA, and protein of breast tumor tissues were found by experiments of RT-PCR, Western blot analysis, immunohistochemistry and Real-time PCR. However, the role of 14-3-3 gamma in the formation of breast cancer requires further study.

No description available.

MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.

Objective: The incidence rate and mortality of breast cancer are increasing in Taiwan during recent years. The incidence rate of breast cancer is ranked number one among top ten female cancers, and the mortality of breast cancer is ranked fourth among cause of death for female cancer sufferers. The most common age group for breast cancer is between 40 and 50 years old. Breast cancer causes huge disease burdens for individual, family and society. The breast sonography and fine needle aspiration cytology (FNAC) are common screening methods for breast cancer diagnosis. Nevertheless, little study has focused on the benefits of combing these two methods in clinical application. This study aims to fill such research gap. Method: This study conducted medical chart reviews and collected 2,776 observations that were under breast sonography and FNAC examination from a regional hospital locates in southern Taiwan. The diagnosis categories for sonography include: malignant, benign, and probably benign tumor. The diagnosis categories for FNAC include: malignant, benign, and suspicious for malignant. Results: Among 2,776 observations, there were 555 observations (20%) had operation in the studied hospital. The operation results indicated that 205 (36.9%) observations were with malignant status, and 350 (63.1%) observations were with benign status. The diagnosis categories of both sonography and FNAC were significantly associated with the operation results (p<0.001). The FNAC had specificity in 100%, false positive ratio in zero, and positive predictive value in 100%. The Odds ratios for sonography diagnosis categories, age groups, and tumor sizes were OR=4.132 (95%CI: 1.5¡V11.6), OR=31.957 (95% CI: 3.7¡V272.4), OR=0.457 (95% CI: 0.1¡V1.5), respectively. When combining sonography and FNAC in parallel tests, the diagnosis accuracy was 89.2%, sensitivity was 90.2%, specificity was 88.6%, positive predictive value was 82.2%, and negative predictive value was 93.9%. When combining sonography and FNAC in serial testing, the diagnosis accuracy was 88.1%, sensitivity was 67.8%, specificity was 100%, positive predictive value was 100%, and negative predictive value was 84.1%. Conclusion: Combining sonography and FNAC in breast cancer diagnosis can increase the accuracy, decrease false positive ratio and false negative ratio. These two methods can be conducted during outpatient visit and are fast, accurate and cost-effective tools for breast cancer diagnosis. These two methods particularly appropriate for younger female patients for early screening, early intervention, and may increase the survival rates.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences