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.

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients.

BACKGROUND: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. METHODS: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. RESULTS: Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. CONCLUSION: Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.

The novel complement inhibitor human CUB and Sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly.

CUB and Sushi multiple domains 1 (CSMD1) is a transmembrane protein containing 15 consecutive complement control protein (CCP) domains, which are characteristic for complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized complement inhibitors were used as positive controls, whereas Coxsackie adenovirus receptor, a protein with no effect on complement, was a negative control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the controls. Furthermore, by expressing a soluble 17-21 CCP fragment of CSMD1, we found that CSMD1 inhibits complement by promoting factor I-mediated C4b/C3b degradation and inhibition of MAC assembly at the level of C7. Our results revealed a novel complement inhibitor for the classical and lectin pathways.

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-231 cells 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.

Claudin-5 participates in the regulation of endothelial cell motility.

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 and between the endothelial cells functioning in an adhesive manner. They represent a critical barrier which the cancer cells must overcome in order to penetrate and initiate metastasis. Claudin-5 is a protein member of the Claudin family, a group of TJ proteins expressed in both endothelial and epithelial cells. This study examined in vitro the effect of altering levels of expression of Claudin-5 in HECV cells. Insertion of Claudin-5 gene in HECV cells resulted in cells that were significantly less motile and less adhesive to matrix (P < 0.001). These cells also exhibited a significant decreased in the angiogenic potential (P < 0.001). Results also revealed a link 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 the cell line. Moreover, followed by treatment of N-WASP inhibitor (Wiskostatin) and ROCK inhibitor (Y-27632), cell motility and angiogenic potential were assessed in response to the inhibitors. Results showed that the knockdown of Claudin-5 in HECV cells masked their response to both N-WASP and ROCK inhibitors. In conclusion, this study portrays a new and interesting role for Claudin-5 in cell motility involving the N-WASP and ROCK signalling cascade which is beyond the primarily role of Claudin-5 in keeping the cell barrier tight as it was originally reported.

The Claudin family and its role in cancer and metastasis.

Tight Junctions are the most apical element of the junctional complex in epithelial and endothelial cells. Tight Junctions form a barrier to paracellular movement of substances separating the apical and basolateral fluid compartments on opposite sides of the epithelial cell layer. The Claudin family are Tight Junction proteins expressed in both endothelial and epithelial cells. They participate in the development of tissue barriers between different tissue compartments by regulating the efflux of molecules through Tight Junction complexes. At least 24 different Claudin members are known today, all of which are thought to vary in expression depending on location and cell type. Relatively little is know about Claudins and their role in carcinogenesis and progression to metastasis. Recently, this new area of research has become very promising as a result of the frequent existence of altered Claudin expression in cancer. That Claudins are pivotal in the maintenance of Tight Junctions function begs investigation into the changes that can occur during the metastatic process.

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 ERbeta-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.

PGF isoforms, PLGF-1 and PGF-2, in colorectal cancer and the prognostic significance.

AIM: This study examined the expression of PLGF-1 and PGF-2 in human colorectal cancer and how changes in expression may be linked to prognosis of the disease. MATERIALS AND METHODS: Expression of PLGF transcripts in primary colorectal tumours (n=94) and background tissue (n=80) were analysed by Q-PCR. Immunohistochemistry was carried out on a number of matched background and tumour sections. RESULTS: There was a progressive increase in Dukes A to C (430+/-134 versus 674+/-2680) and with TNM 1 to 3 as compared to background tissues (479+/-152 versus 777+/-327 p=0.032). Interestingly, there was an increase in PLGF-1 expression in patients with poor outcome compared with patients remaining disease-free. PGF-2 expression was significantly elevated in tumours compared to normal (4558+/-240 versus 859+/-67.3 p<0.0001). Moreover, expression was increased in patients with poor prognosis compared to those remaining disease free (4585+/-285 versus 3909+/-446). CONCLUSION: There is increased expression of the PLGF isoforms in human colorectal 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.