miR-509 suppresses brain metastasis of breast cancer cells by modulating RhoC and TNF-α.

The median survival time of breast cancer patients with brain metastasis is less than 6 months, and even a small metastatic lesion often causes severe neurological disabilities. Because of the location of metastatic lesions, a surgical approach is limited and most chemotherapeutic drugs are ineffective owing to the blood brain barrier (BBB). Despite this clinical importance, the molecular basis of the brain metastasis is poorly understood. In this study, we have isolated RNA from samples obtained from primary breast tumors and also from brain metastatic lesions followed by microRNA profiling analysis. Our results revealed that the miR-509 is highly expressed in the primary tumors, whereas the expression of this microRNA is significantly decreased in the brain metastatic lesions. MicroRNA target prediction and the analysis of cytokine array for the cells ectopically expressed with miR-509 demonstrated that this microRNA was capable of modulating the two genes essential for brain invasion, RhoC and TNF-α that affect the invasion of cancer cells and permeability of BBB, respectively. Importantly, high levels of TNF-α and RhoC-induced MMP9 were significantly correlated with brain metastasis-free survival of breast cancer patients. Furthermore, the results of our in vivo experiments indicate that miR-509 significantly suppressed the ability of cancer cells to metastasize to the brain. These findings suggest that miR-509 has a critical role in brain metastasis of breast cancer by modulating the RhoC-TNF-α network and that this miR-509 axis may represent a potential therapeutic target or serve as a prognostic tool for brain metastasis.

Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells.

Epithelial-mesenchymal transition (EMT) is an essential step for tumor progression, although the mechanisms driving EMT are still not fully understood. In an effort to investigate these mechanisms, we observed that heregulin (HRG)-mediated activation of HER2, or HER2 overexpression, resulted in EMT, which is accompanied with increased expression of a known EMT regulator Slug, but not TWIST or Snail. We then investigated how HER2 induced Slug expression and found, for the first time, that there are four consensus HSF sequence-binding elements (HSEs), the binding sites for heat shock factor-1 (HSF-1), located in the Slug promoter. HSF-1 bound to and transactivated the Slug promoter independent of heat shock, leading to Slug expression in breast cancer cells. Mutation of the putative HSEs ablated Slug transcriptional activation induced by HRG or HSF-1 overexpression. Knockdown of HSF-1 expression by siRNA reduced Slug expression and HRG-induced EMT. The positive association between HSF-1 and Slug was confirmed by immunohistochemical staining of a cohort of 100 invasive breast carcinoma specimens. While investigating how HER2 activated HSF-1 independent of heat shock, we observed that HER2 activation resulted in concurrent phosphorylation of Akt and HSF-1. We then observed, also for the first time, that Akt directly interacted with HSF-1 and phosphorylated HSF-1 at S326. Inhibition of Akt using siRNA, dominant-negative Akt mutant, or small molecule inhibitors prevented HRG-induced HSF-1 activation and Slug expression. Conversely, constitutively active Akt induced HSF-1 phosphorylation and Slug expression. HSF-1 knockdown reduced the ability of Akt to induce Slug expression, indicating an essential role that HSF-1 plays in Akt-induced Slug upregulation. Altogether, our study uncovered the existence of a novel Akt-HSF-1 signaling axis that leads to Slug upregulation and EMT, and potentially contributes to progression of HER2-positive breast cancer.

Upregulation of VEGF-A and CD24 gene expression by the tGLI1 transcription factor contributes to the aggressive behavior of breast cancer cells.

The Hedgehog signaling pathway is one of the most dysregulated pathways in human cancers. The glioma-associated oncogene homolog 1 (GLI1) transcription factor is the terminal effector of the Hedgehog pathway, frequently activated in human breast cancer and an emerging target of breast cancer therapy. While somatic mutations in the human GLI1 gene have never been reported in any cell or tumor type, we recently uncovered the existence of a novel alternatively spliced, truncated GLI1 (tGLI1) that has an in-frame deletion of 41 codons spanning the entire exon 3 and part of exon 4 of the GLI1 gene. Using glioblastoma models, we showed that tGLI1 has gained the ability to promote glioblastoma migration and invasion via its gain-of-function transcriptional activity. However, the pathological impact of tGLI1 on breast cancer remains undefined. Here, we report that tGLI1 is frequently expressed in human breast cancer cell lines and primary specimens we have examined to date, but is undetectable in normal breast tissues. We found for the first time that tGLI1, but not GLI1, binds to and enhances the human vascular endothelial growth factor-A (VEGF-A) gene promoter, leading to its upregulation. Consequently, tGLI1-expressing MDA-MB-231 breast cancer cells secret higher levels of VEGF-A and contain a higher propensity, than the isogenic cells with control vector and GLI1, to stimulate in vitro angiogenesis of human vascular endothelial cells. We further showed that tGLI1 has gained the ability to enhance the motility and invasiveness of breast cancer cells in a proliferation-independent manner and that this functional gain is associated with increased expression of migration/invasion-associated genes, CD24, MMP-2 and MMP-9. tGLI1 has also acquired the property to facilitate anchorage-independent growth of breast cancer cells. Collectively, our results define tGLI1 as a gain-of-function GLI1 transcription factor and a novel mediator of the behavior of clinically more aggressive breast cancer.

Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival.

Emerging evidences suggest the existence of a new mode of epidermal growth factor receptor (EGFR) signalling pathway in which activated EGFR undergoes nuclear translocalization and subsequently regulates gene expression and potentially mediates other cellular processes. This signalling route is distinct from the better-characterized, traditional EGFR pathway that involves transduction of mitogenic signals through activation of multiple signalling cascades. Transcriptional activity of nuclear EGFR appears to depend on its C-terminal transactivation domain and its physical and functional interaction with other transcription factors that contain DNA-binding activity. Likely via its ability to upregulate gene expression, nuclear EGFR pathway is associated with major characteristics of more aggressive tumours: increased proliferative potential, nitric oxide synthesis, and accelerated G1/S cell cycle progression. A role of nuclear EGFR in prognostic prediction is further suggested in patients with breast carcinomas and oropharyngeal squamous cell carcinomas. It is noted that significant advances were made towards the knowledge of the nuclear EGFR pathway; however, many aspects of this new pathway remain unresolved and will be discussed in this review. As a number of other receptor tyrosine kinases (RTKs) and cytokine receptors also undergo similar nuclear translocalization, a better understanding of the physiological and malignant nature of the nuclear EGFR pathway will likely shed light into the biology of cancer with nuclear RTKs.

Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival.

Emerging evidences suggest the existence of a new mode of epidermal growth factor receptor (EGFR) signalling pathway in which activated EGFR undergoes nuclear translocalization and subsequently regulates gene expression and potentially mediates other cellular processes. This signalling route is distinct from the better-characterized, traditional EGFR pathway that involves transduction of mitogenic signals through activation of multiple signalling cascades. Transcriptional activity of nuclear EGFR appears to depend on its C-terminal transactivation domain and its physical and functional interaction with other transcription factors that contain DNA-binding activity. Likely via its ability to upregulate gene expression, nuclear EGFR pathway is associated with major characteristics of more aggressive tumours: increased proliferative potential, nitric oxide synthesis, and accelerated G1/S cell cycle progression. A role of nuclear EGFR in prognostic prediction is further suggested in patients with breast carcinomas and oropharyngeal squamous cell carcinomas. It is noted that significant advances were made towards the knowledge of the nuclear EGFR pathway; however, many aspects of this new pathway remain unresolved and will be discussed in this review. As a number of other receptor tyrosine kinases (RTKs) and cytokine receptors also undergo similar nuclear translocalization, a better understanding of the physiological and malignant nature of the nuclear EGFR pathway will likely shed light into the biology of cancer with nuclear RTKs.

Laparoscopic subsegmentectomy for hepatocellular carcinoma with cirrhosis.

BACKGROUND/AIMS: Laparoscopic liver resection is feasible for both benign and malignant disease with today's laparoscopic techniques and technology. Location of the tumor at the edge of segment 3, 4, 5, or 6 of our patients makes them an ideal candidate for laparoscopic resection. METHODOLOGY: There were 9 patients who underwent laparoscopic subsegmentectomy for hepatocellular carcinoma with cirrhosis. They were classified as Child A in 6 and B in 3 patients. Hepatitis B was found in 5 and Hepatitis C in 4 cases. Preoperative diagnosis of hepatocellular carcinoma was completed in 7 and definitive histologic diagnosis from frozen section in 2 cases. All 9 patients underwent subsegmentectomy and removal of the tumor with non-tumor cirrhotic liver with a distance of 10 mm at the least margin. Laparoscopic ultrasound allows exact localization of lesions and achievement of adequate resection margin. RESULTS: Those patients resumed a full diet on the 2nd-3rd day after the operation and were discharged home on day 4-7 with no complications but one had prolonging discharge due to ascitis from a drainage tube. Finally, the ascitis was controlled by medications for 1 week. All patients had high postoperative satisfaction. CONCLUSIONS: Laparoscopic liver resection is a procedure of significant risk and technically demanding. Therefore, it should be performed only by experienced liver surgeons with a high level of laparoscopic skill and in the carefully selected patient.

Laparoscopic subsegmentectomy for hepatocellular carcinoma with cirrhosis: a case report.

Laparoscopic liver resection is feasible for both benign and malignant disease with present laparoscopic techniques and technology. Laparoscopic liver tumor resection is indicated instead of the conventional hepatectomy if the tumor is located in the peripheral part of the liver. Here, we reported a case of a 73-year-old woman who accepted laparoscopic subsegmentectomy for hepatocellular carcinoma of segment 6. After traditional laparoscopic trocar was settled down under the low pneumoperitoneal pressure of 8 mm Hg, laparoscopic ultrasound allowed exact localization of lesions first and then transection line was marked. Then, dissection the liver parenchyma was carried out with laparoscopic microwave coagulator and ultrasonic aspirator gradually. After operation, she resumed full diet on the second day and was discharged on the 5th post-operative day with no complications and high patient satisfaction. She had follow-up study regularly in our clinic and was disease free at nine months. With the improvement of laparoscopic techniques and the development of new and dedicated technologies, laparoscopic hepatectomy has become feasible.

Role of angiogenesis in hepatitis and hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is usually a hypervascular tumor. Factor VIII-related antigens, including von Willebrand factor, are known to be expressed in HCC, which cause capillarization of the sinusoids of HCC. Capillarization of hepatic sinusoids may play a role in hepatocarcinogenesis and its metastasis. The aim of this study is to clarify the expression of Factor VIII in patients with hepatitis B or C (n = 18) and HCC (n = 16). METHODOLOGY: All specimens were sufficient for immunohistochemical study of the neo-angiogenesis with regard to clinical results. Microvessel count per square millimeter (MVC) and hot spot of microvessel per square millimeter (HSV) were measured from the histochemical study. RESULTS: In the patients with hepatitis group, the positive staining on the vessels of the portal triad was 11.1% (2/18) but in the non-neoplastic tissue of HCC patients the positive rate was 68.7% (11/16) showing a significant difference from the hepatitis group. The amount of vasculatures was easily found in the surrounding capsule of resected HCC. The MVC of the capsule was 10.17 +/- 2.78 and 13.66 +/- 5.42 for the HCC with non-direct invasion and direct invasion during operation, respectively. The HSV of capsules were 7.51 +/- 2.09 and 9.14 +/- 4.02 for the non-invasion and invasion, respectively. Therefore, in our study, it is clear that the high MVC or HSV scores were found in patients of direct invasion. However, there was no relation between hepatitis B or hepatitis C to the tumor invasiveness. The median survival times were 21.5 months for the non-invasive group and 14.5 months for the invasive group (p < 0.05). The positive rate of Factor VIII in the vessels of the portal triad were 60% and 83.3% for the non-invasive and invasive groups, respectively. However, the lower values of MVC and HSV showed a trend toward a longer recurrence time. CONCLUSIONS: It is pertinent to prove that the high score of neo-angiogenesis has a high risk of recurrence. In addition, it is wise to pay more attention to the interval of the follow-up study to detect the recurrent lesion earlier, where possible, in the patient with a high score of microvascularity.

Structure of the human allelic glutathione S-transferase-pi gene variant, hGSTP1 C, cloned from a glioblastoma multiforme cell line.

We recently reported the cloning of full-length cDNAs corresponding to mRNAs of three GST-pi genes, hGSTP1*A, hGSTP1*B and hGSTP1*C, as well as, the isolation of the full-length hGSTP1*C, of the human glutathione S-transferase-pi (GST-pi) gene that is characterized by a A-->G transition at +1404 in exon 5 and a C-->T transition at +2294 in exon 6. Although the promoter of the isolated gene was identical to that of the previously described GST-pi gene isolated from the MCF 7 and the HPB-ALL cell lines, both of which were hGSTP1*A, a number of structural differences were observed, including, nucleotide transitions, transversions, deletions and insertions, some of which created new restriction enzyme cleavage sites. A guanine insertion in the insulin response element, IRE, in intron 1 created an additional site for 5'-cytosine methylation. Seven repeat retinoic acid response element (RARE) consensus half sites, A(G)GG(T)TC(G)A at +1521 to +1644 were identified in the cloned hGSTP1*C. Five of the RARE half-sites had the minimal spacer nucleotide requirement for functionality and DNA mobility shift analysis with different pairs of the RARE half-sites and supershift studies using antibodies against RAR-beta showed significant binding of nuclear protein complexes from RA-treated cells to these RAREs. GST-pi gene expression was increased significantly in cells transfected with the GST-pi gene and treated with all-trans RA. These results contrast with those in a previous report in which RA was shown to suppress the GST-pi promoter, and indicate a complex mechanism of RA-mediated GST-pi gene regulation in tumor cells.

Genomic cloning of hGSTP1*C, an allelic human Pi class glutathione S-transferase gene variant and functional characterization of its retinoic acid response elements.

The complete hGSTP1*C, consisting of 7 exons and 6 introns contained in 3116 base pairs, was isolated from a cosmid genomic library of a glioblastoma multiforme cell line. Although the promoter of hGSTP1*C was identical to that of the previously reported GST-Pi gene, several of its structural features had not been previously described. These include several nucleotide transitions and transversions. Transitions of A --> G at +1404 and C --> T at +2294 in exons 5 and 6, respectively, changed codons Ile104 to Val104 and Ala113 to Val113. The gene also contained a guanine insertion at +51 in the insulin response element in intron 1 and six tandem repeats and one palindromic retinoic acid response element (RARE) consensus half-sites, A(G)GG(T)TC(G)A in intron 5. Retinoic acid (RA) treatment increased GST-Pi gene expression significantly in MGR3 cells. GST-Pi gene constructs with and without RARE deletion were used to show the RARE requirement for GST-Pi gene induction by RA. The isolation of the hGSTP1*C gene and the evidence that it contains functional RAREs should contribute to a better understanding of the molecular regulation of the GST-Pi gene in human cells.

A case of spindle cell sarcomatous change of hepatic ducts manifesting as obstructive jaundice.

Spindle cell carcinoma is a rare tumor commonly occurring in the upper aerodigestive tract. We report a 62-year-old male with spindle cell sarcomatous change located at the hepatic hilum, resulting in obstructive jaundice. The patient died after an extended resective operation. The rare disease and its histogenesis is discussed.

[A study about the desirable characteristics of the qualified physicians: the application of Delphi method].

The purpose of this study is to adapt the Delphi Study method in establishing a consensus of good doctor characteristics from a panel of department heads (N = 22) at four medical colleges in Taiwan within three given categories: 1. professional knowledge and techniques; 2. attitudes and standards; and 3. interpersonal relationship. In this study three rounds of questionnaires were administered to the panel to extract their opinions regarding the characteristics of good doctoring. 76 items of good doctoring characteristics were solicited from the department heads at four medical colleges to the opening questionnaire. The conclusive results indicated that 15 of these 58 items were ranked as the most important characteristics by panel members. The findings from this investigation will help facilitate the decision making process of officials regarding innovation in the training of good doctor. Moreover, the conclusions could be included in developing and planning of instruments for the future doctor evaluation methods.

Cholangiocarcinoma in hepatolithiasis.

During the period of 1979-1982, 10 cases of cholangiocarcinoma associated with hepatolithiasis were seen. We report the clinical features of 10 cases of cholangiocarcinoma in association with hepatolithiasis, along with detailed histopathology from the four resected specimens. Our observations suggest that whenever intrahepatic stones are encountered in patients over 50 years of age with a long history of recurrent cholangitis and intractable pain, further examination including echo-guided aspiration cytology, liver scanning, and peritoneoscopy should be performed to rule out a coexisting cholangiocarcinoma.