Role of Claudin-5 in the progression of human breast cancer

A key step in metastasis is the interaction and penetration of the vascular endothelium by cancer cells. Tight Junctions (TJ) are located between the cancer epithelial cells themselves functioning in an adhesive manner, and between the endothelial cells. They represent a critical barrier which the cancer cells must overcome in order to penetrate and initiate metastasis. The Claudin family are TJ proteins expressed in both endothelial and epithelial cells. They participate in the formation of tissue barriers between different tissue compartments by regulating the efflux of molecules through TJ complexes. This thesis examined the level of expression and distribution of Claudin-5 in human breast cancer tissues and analysed them against clinical parameters. The effect of knockdown and forced expression of Claudin-5 in the MDA-MB-231 aggressive breast cancer cell line and in the HECV human endothelial cell line was also examined. Results revealed that Claudin-5 is aberrantly expressed in human breast cancer and has a link to the clinical outcome of the patient. Patients who died from breast cancer had higher levels of Claudin-5 compared with patients who remained disease-free. Furthermore, patients whose tumours expressed high levels of Claudin-5 had shorter survival than those with low levels. Investigating in vitro the effect of altering levels of expression of Claudin-5 in MDA-MB-231and HECV cells revealed that the role of Claudin-5 was not primarily in keeping the cell barrier tight suggesting little function in the TJ of these cells, in fact a link was identified between Claudin-5 and cell motility. Furthermore, a possible link between Claudin-5 and N-WASP, and Claudin-5 and ROCK was demonstrated when interactions between these proteins were seen in both cell lines. Moreover, cell motility was assessed following treatment with N-WASP inhibitor, Arp2/3 inhibitor and ROCK inhibitor. Results show that the knockdown of Claudin-5 in HECV cells masked their response to N-WASP inhibitor, Arp2/3 inhibitor and ROCK inhibitor. A parallel response was observed in the knockdown of Claudin-5 in MDA-MB-231 after treatment with N-WASP inhibitor and Arp2/3 inhibitor however treatment with ROCK inhibitor did not reveal any differences in motility in this particular cell line. This study portrays a very new and interesting role for Claudin-5 in cell motility involving the N-WASP and ROCK signalling cascade indicating a possible role for Claudin-5 in the metastasis and progression of human breast cancer

Claudin-5 is involved in breast cancer cell motility through the N-WASP and ROCK signalling pathways

Background: Recent studies have shown dysregulation in TJ structure of several cancers including breast. Claudin-5 is a protein member of the TJ structure expressed in both endothelial and epithelial cells. This study examined the level of expression and distribution of Claudin-5 in human breast cancer tissues and the effect of knockdown and forced expression of Claudin-5 in the MDA-MB-231 breast cancer cell line. Methods: Immunohistochemistry and quantitative-PCR were used to analyse patient tissue samples. The Claudin-5 gene was cloned and overexpressed or knocked down using ribozyme technology in human breast cancer cells. Changes in function were assessed using in vitro assays for invasion, growth, adhesion, wounding, motility, transepithelial resistance and electric cell-substrate impedance sensing. Changes in cell behaviour were achieved through the use of Hepatocyte Growth factor (HGF) which we have shown to affect TJ function and expression of TJ proteins. In addition, an in vivo model was used for tumour growth assays. Results data was analyzed using a Students two sample t-test and by Two-way Anova test when the data was found to be normalized and have equal variances. In all cases 95% confidence intervals were used. Results: Patients whose tumours expressed high levels of Claudin-5 had shorter survival than those with low levels (p = 0.004). Investigating in vitro the effect of altering levels of expression of Claudin-5 in MDA-MB-231cells revealed that the insertion of Claudin-5 gene resulted in significantly more motile cells (p < 0.005). Low levels of Claudin-5 resulted in a decrease in adhesion to matrix (p < 0.001). Furthermore, a possible link between Claudin-5 and N-WASP, and Claudin-5 and ROCK was demonstrated when interactions between these proteins were seen in the cells. Moreover, followed by treatment of N-WASP inhibitor (Wiskostatin) and ROCK inhibitor (Y-27632) cell motility was assessed in response to the inhibitors. Results showed that the knockdown of Claudin-5 in MDA-MB-231 masked their response after treatment with N-WASP inhibitor; however treatment with ROCK inhibitor did not reveal any differences in motility in this cell line. Conclusions: This study portrays a very new and interesting role for Claudin-5 in cell motility involving the N-WASP signalling cascade indicating a possible role for Claudin-5 in the metastasis of human breast cancer

Complement inhibitor CSMD1 acts as tumor suppressor in human breast cancer

Human CUB and Sushi multiple domains 1 (CSMD1) is a membrane-bound complement inhibitor suggested to act as a putative tumor suppressor gene, since allelic loss of this region encompassing 8p23 including CSMD1 characterizes various malignancies. Here, we assessed the role of CSMD1 as a tumor suppressor gene in the development of breast cancer in vitro and in vivo. We found that human breast tumor tissues expressed CSMD1 at lower levels compared to that in normal mammary tissues. The decreased expression of CSMD1 was linked to a shorter overall survival of breast cancer patients. We also revealed that expression of CSMD1 in human breast cancer cells BT-20 and MDA-MB-231 significantly inhibited their malignant phenotypes, including migration, adhesion and invasion. Conversely, stable silencing of CSMD1 expression in T47D cells enhanced cancer cell migratory, adherent and clonogenic abilities. Moreover, expression of CSMD1 in the highly invasive MDA-MB-231 cells diminished their signaling potential as well as their stem cell-like properties as assessed by measurement of aldehyde dehydrogenase activity. In a xenograft model, expression of CSMD1 blocked the ability of cancer cells to metastasize to secondary sites in vivo, likely via inhibiting local invasion but not the extravasation into distant tissues. Taken together, these findings demonstrate the role of CSMD1 as a tumor suppressor gene in breast cancer

Transcript analyses of stromal cell derived factors (SDFs): SDF-2, SDF-4 and SDF-5 reveal a different pattern of expression and prognostic association in human breast cancer

Stromal derived factors, SDFs, are a loosely defined group of molecules that may be generated by stromal cells. Two of the stromal derived factors, SDF-1 and SDF-4 belong to the chemokine family. Other SDFs, such as SDF-2 and SDF-5 are not well defined and their biological functions are less known. Although SDF-1 and its receptor have been strongly indicated in the progression of various cancers including breast cancer, little is known with regard to the role of other SDFs in malignant conditions including breast cancer. In the present study, we analysed the pattern of expression of SDF-2, SDF2-like-1, SDF-4 and SDF-5 in breast cancer tissues and cells, at transcript and protein levels. It was found that SDF-2, SDF2-L1, SDF-4, and SDF-5 were ubiquitously expressed in various cancer cell lines. However, in clear contrast to SDF-1 whose over-expression has been shown to be linked to a poor clinical outcome, the present study provides evidence that the opposite appear to be true for SDF-2/SDF2-L1, SDF-4 and SDF-5. Significantly low levels of SDF-2 and SDF-4 were seen in patients with poor clinical outcome (with metastatic disease and death as a result of breast cancer, p<0.05, and p<0.01 respectively), when compared with patients who remained disease-free. SDF2-L1 and SDF-5 showed a similar trend. SDF-2 and SDF-L1 were also independent prognostic indicators (p=0.047 and p=0.012, respectively). It is concluded that SDF-2, SDF-4 and SDF-5 are expressed in mammary tissues and cells and that a reduced level of SDF-2, SDF2-L1 and SDF-4 are associated with a poor clinical outcome. These SDFs thus have prognostic value and warrant further investigation in their biological functions and clinical value

PGF isoforms, PLGF-1 and PGF-2 and the PGF receptor, neuropilin, in human breast cancer: prognostic significance

Placenta growth factor (PLGF) is a member of the vascular endothelial growth factor (VEGF) family, a group of angiogenic growth factors. Recently, isoforms have been identified. This study examined PLGF-1, PGF-2 and its receptor neuropilin-1 levels in human breast cancer in relation to patient's clinical parameters and how changes in expression may be linked to prognosis of the disease. PLGF-1, PGF-2 and neuropilin-1 transcript expression and distribution were examined quantitatively using real-time quantitative polymerase chain reaction (Q-PCR) on a cohort of human breast cancer (n=114) and background breast tissue (n=30) with a 10-year follow-up. Protein expression was assessed by an immunohistochemical method. We demonstrate that PLGF-1 transcript levels were significantly elevated when comparing tumours from patients with poor outcome and patients who remained disease-free (P=0.03), indicating a potential prognostic value. Immunohistochemistry demonstrated a marked increased in PGF-2 expression in tumour section compared with normal tissues (P<0.05). PGF-2 transcripts, showed little change in expression between tumour and background. High levels of PLGF-1 and PGF-2 were seen in ERβ-negative breast tumour tissues. Neuropilin transcript was below detection in substantial portion of the samples and was more frequently detected in high grade tumours (P=0.008 vs. low grade) and in tumours from patients who died of breast cancer (P<0.001 vs. those who remained disease-free). Our study shows that PLGF isoforms PLGF-1 and PGF-2 and indeed their receptor neuopilin, have an aberrant pattern of expression and that high levels of the PLGF-1 and neuropilin are linked to a poor prognosis

Correction: Complement inhibitor CSMD1 acts as tumor suppressor in human breast cancer

This article has been corrected: In Figure 4B, the image of MDA-MB-231 cells expressing CSMD1 is an accidental duplicate of the image showing invaded BT-20 cells expressing CSMD1 in Figure 4A. The correct Figure 4, produced using the original data, is shown below. The authors declare that these corrections do not change the results or conclusions of this paper. Original article: Oncotarget. 2016; 7:76920–76933. https://doi.org/10.18632/oncotarget.1272

膀胱癌における抗クローディン4細胞外ドメイン中和抗体の化学療法増感効果

Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in three-dimensional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy.博士（医学）・甲第649号・平成28年3月15日Copyright © 2015 Elsevier Ireland Ltd. All rights reserved

Coordinating the impact of structural genomics on the human α-helical transmembrane proteome

Given the recent successes in determining membrane-protein structures, we explore the tractability of determining representatives for the entire human membrane proteome. This proteome contains 2,925 unique integral α-helical transmembrane-domain sequences that cluster into 1,201 families sharing more than 25% sequence identity. Structures of 100 optimally selected targets would increase the fraction of modelable human α-helical transmembrane domains from 26% to 58%, providing structure and function information not otherwise available