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The aim of this study was to investigate the expression of bone morphogenetic protein 7 (BMP7), in human pulmonary cancer tissues/cells and to evaluate the cellular impact of bone morphogenetic proteins on pulmonary cancer cells. BMP7 expression was determined in human lung cancer cell lines. The invasiveness and growth of cells transfected with BMP7, in vitro, were evaluated using the in vitro invasion assay and in vitro tumour models. Cellular migration was analysed using wounding assays. BMP7-positive tumours correlated with the absence of bone metastasis (P=0.040). In this analysis, we identified that 4 of 4 small cell lung cancer (SCLC) tissue specimens had no BMP7 expression, which illustrated that BMP7 may have no role in SCLC. BMP7 expression was not correlated with the overall survival time in lung cancer patients. Downregulation of BMP7 expression significantly inhibited the invasiveness of SPC-A1 cells (P<0.001) and forced-expression of BMP7 dramatically increased the motility of A549 cells. Overexpression of BMP7 in A549 cells and its knockdown in SPC-A1 cells did not significantly alter proliferation compared with the control cells (P>0.5 respectively). In conclusion, we have demonstrated that BMP7 has an important role in controlling lung cancer cell motility and invasiveness, without affecting the growth process, cell proliferation and cell apoptosis. A higher BMP7 expression may be an indicator for bone metastasis. The therapeutic role of BMP7 warrants further investigation.

Growth differentiation factor-9 (GDF-9) is a family member of bone morphogenetic proteins (BMPs), which belong to the TGF-β superfamily. There has been a recent surge of interest in the role of growth differentiation factors and other BMPs in the development and spread of cancer. However, the role of GDF-9 in bladder cancer remains unknown. The present study investigated the expression of GDF-9 in normal and malignant human bladder tissue and its molecular interactions within bladder cancer cells. The expression of GDF-9 in human bladder tissues and bladder cancer cell lines was assessed at both the mRNA and protein levels using RT-PCR and immunohistochemistry, respectively. Full-length GDF-9 cDNA was amplified from normal mammary tissues. GDF-9 was overexpressed in bladder cancer cell lines using a mammalian expression construct. The effect of GDF-9 on cellular functions, was examined in bladder cancer cells overexpressing GDF-9 using a variety of in vitro assays. In normal bladder tissues, stronger staining of GDF-9 was seen in transitional cells, both in the cytoplasm and in the nucleus. In contrast, the staining of GDF-9 was notably weak or absent in cancer cells of tumour tissues. Similarly, the bladder cancer cell lines RT112 and EJ138, expressed very low levels of GDF-9. Moreover, overexpression of GDF-9 reduced the growth, adhesion and migration of bladder cell lines in vitro. However, the overexpression of GDF-9 had little bearing on the invasion of bladder cell lines in vitro. In conclusion, GDF-9 is expressed at lower levels in human bladder cancer cells compared with normal transitional cells of the bladder. GDF-9 levels are inversely correlated with the growth, adhesion and migration of bladder cancer cells in vitro. The results of the present study suggest that GDF-9 is a potential tumour suppressor in human bladder cancer.

Occludin is an integral membrane protein localised at tight junctions (TJs). It has become clear that the TJ is an important structure that cancer cells must overcome in order to metastasize successfully. The purpose of this study was to elucidate the importance of the expression of occludin in human breast cancer. Human tissues and breast cancer cell lines were amplified for functional regions of occludin. Tumour tissues showed truncated and/or variant signals. There was also considerable variation in the expression of occludin in the 10 human breast cancer cell lines investigated. Western blotting demonstrated that variants in the MDA-MB-231 and MCF-7 human breast cancer cell lines did not fit the expected occludin signals for changes in phosphorylation status. Immunostaining showed similarly disparate levels of expression. Ribozyme knockdown resulted in increased invasion, reduced adhesion and significantly reduced TJ functions. Q-RT-PCR analysis of 124 tumour and 33 background human breast tissues showed occludin to be significantly decreased in patients with metastatic disease. Immunohistochemical staining showed a decreased expression of occludin in the tumour sections. This study demonstrates for the first time that occludin is differentially expressed in human breast tumour tissues and cell lines. This loss of or aberrant expression has clear repercussions as to the importance of occludin in maintaining TJ integrity in breast tissues. Such inappropriate expression could play a part in breast cancer development.

The metastatic lymph node 64 (MLN64) gene was initially identified as highly expressed in the metastatic lymph node from breast cancer. It is localized in q12-q21 of the human chromosome 17 and is often co-amplified with erbB-2. However, the role played by MLN64 in breast cancer remains unclear. In the present study, the expression of MLN64 was examined in a breast cancer cohort using quantitative real-time PCR and immunohistochemical staining. It demonstrated that MLN64 was highly expressed in breast tumours compared to corresponding background tissues at both transcript level and protein level. The elevated level of MLN64 transcripts was correlated with the poor prognosis and overall survival of the patients. A panel of breast cancer cell sublines was subsequently developed by knockdown of MLN64 expression. Loss of MLN64 expression in MCF-7 cells resulted in a significant reduction of cell growth (absorbance for MCF-7DeltaMLN64 being 0.87+/-0.07, P<0.01 vs. wild-type control (MCF-7WT 1.13+/-0.06) and transfection control (MCF-7pEF 1.27+/-0.05). In cell-matrix adhesion assay, MDA-MB-231DeltaMLN64 cells showed a significant increase in adhesion (86+/-14), p<0.01 compared with both MDA-MB-231WT (61+/-20) and MDA-MB-231pEF (45+/-27). Further investigations demonstrated an increase in protein level of the focal adhesion kinase (FAK) in MDA-MB-231DeltaMLN64 cells using Western blot analysis and immunofluorescent staining of FAK. Moreover, addition of FAK inhibitor to these cells diminished the effect of MLN64 on cell-matrix adhesion, suggesting that FAK contributed to the increased adhesion in MDA-MB-231DeltaMLN64 cells. In conclusion, MLN64 is overexpressed in breast cancer, and its level correlates with poor prognosis and patient survival. MLN64 contributes to the development and progression of breast cancer through the regulation of cell proliferation and adhesive capacity.

Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily, which plays important roles in foetal and postnatal development and also maintains the homeostasis of various tissues and organs. Due to the critical role played by BMPs in bone formation and bone turnover, the implication of these molecules in bone metastasis has been intensively studied over the past decade. BMPs have been implicated in the development and progression of solid tumours, particularly the disease-specific bone metastasis. In breast cancer, a tumour type which most commonly metastasizes to bones, aberrations of both BMP expression and their signalling have been recently demonstrated. These aberrations have certain correlations with the development and progression of the disease. Recent in vitro studies have also demonstrated that BMPs can regulate a range of biological functions of breast cancer cells. Targeting BMPs or BMP signalling may provide novel therapeutic approaches for breast cancer. In the current review, we discuss the present knowledge on BMP abnormalities and their implication in the development and progression of breast cancer, particularly in the disease-specific bone metastasis.

Vascular endothelial growth inhibitor (VEGI), also known as tumour necrosis factor superfamily member 15 (TNFSF15) and TNF ligand related molecule 1 (TL1), is a recently identified anti-angiogenic cytokine that belongs to the TNF superfamily. Three isoforms of VEGI, VEGI 174, 192, and 251 have been documented, all sharing 151 common C-terminal amino acids but differing in their N-terminal regions. The investigations into the biological functions of VEGI have pointed to a potential cancer inhibitory role for the cytokine. The inhibitory effects of VEGI on cancer are manifested in three main areas, the direct effect on cancer cells, the anti-angiogenic effects on endothelial cells, and stimulation of maturation of dendric cells. The clinical aspect of VEGI in cancer is also being explored in recent years. The present article overviews the recent progress on this molecule and discusses the value of VEGI as a potential therapeutic target in cancer therapy.

KAI1, also known as CD82, has been shown to have a potential impact on the invasiveness of cancer cells. In the present study, expression pattern of KAI1, both at transcription and translation levels and the potential clinical value of the expression were explored in a cohort of normal and ductal mammary cancer tissues (n=71). A marked reduction of KAI1 transcript was observed in invasive ductal breast tumours as compared to normal tissues. Expression of KAI1 protein was higher in normal tissues as compared to tumour samples. Though no significant difference of KAI1 expression between different grades of tumour was observed (p=0.064), significant correlation of TNM staging with KAI1 expression has been observed in invasive ductal breast cancer patients (p=0.045). Additionally, it was also observed that patients showing higher expression of KAI1 had a longer 10-year survival rate as compared to a low level or completely negative expression KAI1 (p=0.0136). KAI1 inverse correlation with tumour progression may be used as a strong prognostic marker.

Cell migration is critical during the metastatic spread of cancer cells. Metastases, rather than primary tumours, are responsible for most cancer-related deaths. Invasive cancer cells acquire a migratory phenotype which is associated with an increased expression of several genes involved in cell motility. Actin, which is the most abundant protein in most eukaryotic cells, is necessary for whole cell locomotion. Reorganisation of actin filaments is regulated by a highly integrated signalling cascade governed by 'molecular switches' which belong to the Rho GTPase family. WASP family proteins are downstream molecules which form a link between the GTPases and the actin cytoskeleton. The WASP family includes 5 members and is structurally divided into 2 groups: Wiskott-Aldrich Syndrome proteins (WASPs) and WASP verprolin homologous proteins (WAVEs). Current evidence suggests that WAVEs are crucial for cell motility and metastasis. This is a review on the possible role of WAVEs in cancer and the clinical associations found in human cancer.

Lymphangiogenesis is key to the lymphatic spread of cancer cells. The current study examined the potential effect of hepatocyte growth factor (HGF), a factor known to have strong biological effects on endothelial cells, on the lymphangiogenic function of endothelial cells and the formation of lymphatic vessels using both in vitro and in vivo models. Human endothelial cells that have lymphatic characteristics, human prostate and breast cancer cells PC-3 and MDA MB 231, were used. Expression of lymphatic markers, podoplanin, Prox-1, vascular endothelial growth factor receptor 3 (VEGF-R3) and LYVE-1 was determined using reverse transcription polymerase reaction and quantitative PCR. In nude mice prostate and breast xenograft tumour models, either HGF or an HGF-producing fibroblast cell line MRC-5 was given with or without the HGF antagonist, NK4. The lymphangiogenic marker and lymphatic vessels in tumour tissues were also assessed using quantitative PCR and immunohistochemistry, respectively. In the mice tumour models, infusion of rhHGF significantly increased the levels of podoplanin and LYVE-1 in the tumour (p=0.05 for podoplanin and p<0.05 for LYVE-1 vs. without HGF in the prostate tumour model, p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF for the breast tumour model; p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF in the breast tumour model). The increased level of LYVE-1 transcript was supported by an increase in the number of LYVE-1-positive lymphatic vessels in tumours, using immunohistochemical analysis. Co-injection of MRC5 cells also increased the levels of LYVE-1 and number of LYVE-1-positive vessels in tumour tissues. The effects of HGF and MRC5 were significantly reduced by the HGF antagonist, NK4. In the in vitro models, rhHGF significantly increased the level of both podoplanin and LYVE-1, as shown by quantitative PCR analysis. Hepatocyte growth factor has potential lymphangiogenic activities, and this may have important implications in the nodal spread of cancer cells.