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Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily which has been indicated in the pathophysiology of the nervous system and is important in a number of neurological and psychological conditions. Recently, BDNF was also shown to play a role in the development and progression of solid tumour myeloma. It has been reported that BDNF is aberrantly expressed in human breast cancer and that a raised level of BDNF is associated with poor clinical outcome and reduced survival. The present study investigated the role of BDNF in human breast cancer. A panel of human breast cancer cells was used and the expression profile of BDNF was evaluated using RT-PCR. We constructed a set of anti-BDNF transgenes which were used to transfect breast cancer cells in order to generate BDNF knocked down cells. The impact of BDNF knockdown on growth and apoptosis was evaluated. Statistical analysis was performed using SPSS. P<0.05 was considered statistically significant. BDNF gene transcripts were successfully detected in the breast cancer cell lines MCF-7, MDA-MB-231 and ZR75-1 MDA-MB-231 and MCF-7 wild-type cells were subject to transfection of anti-BDNF transgenes, followed by the establishment of BDNF knocked down sublines. Knockdown of BDNF in MDA-MB-231 and MCF-7 cell lines resulted in decreased rates of growth and proliferation. Analysis of apoptosis showed that cell apoptosis was increased in cells stably transfected with ribozymes for BDNF compared with the vector control. It is concluded that BDNF, a neurotrophic growth factor aberrantly expressed in cancers such as breast cancer, has a profound impact on the cellular behaviour of breast cancer cells and that BDNF is associated with a reduction of the apoptosis of breast cancer. BDNF is, therefore, a potential therapeutic target in breast cancer and its effect in human breast cancer requires further investigation.

Repulsive guidance molecule (RGM) family members RGMA, RGMB and RGMC are GPI-linked membrane proteins recently identified as co-receptor of bone morphogenetic proteins (BMPs). BMPs are a group of proteins enriched in bone and play important roles in prostate cancer. The current study aimed to investigate roles played by RGMs in prostate cancer. Expression of RGMs was examined in prostate cancer cell lines and prostate cancer tissues using RT-PCR and immunohistochemical staining. Knockdown of each RGM in prostate cancer cells was performed using the respective anti-RGMA, RGMB and RGMC transgenes. A variety of in vitro function tests were employed to analyze the influence on cancer cell functions by RGM knockdown. The implications of RGM knockdown in BMP signalling were also examined using both Western blot and real-time quantitative PCR. Knockdown of RGMA had no effect on cell growth, migration and invasion, but promoted cell-matrix adhesion. Knockdown of RGMB and RGMC increased growth and adhesion, but only RGMB knockdown increased capacities of migration and invasion in PC-3 cells. Further investigations showed an increase in Smad-3 activation and reduced levels of Smad-1 in PC-3 cells by RGMB and RGMC knockdown, and also an up-regulation of ID1, a BMP target gene particularly in exposure to BMP7. RGMs play inhibitory roles in prostate cancer by suppressing cell growth, adhesion, migration and invasion. RGMs can coordinate Smad-dependent signalling of BMPs in prostate cancer cells.

The prostate transglutaminase, TGase-4, is a member of the transglutaminase family and is uniquely expressed in the prostate gland. The function of the protein is largely unknown, although an influence on cell motility and adhesion has been indicated. The present study investigated the impact of the differential expression of TGase-4 in human prostate cancer cells on RON, the hepatocyte growth factor-like/macrophage-stimulating protein (HGF-L/MSP) receptor, mediated cellular functions. Using human prostate cancer cell lines and prostate tissues, we demonstrated that human TGase-4 had a high degree of co-localisation with RON, primarily at the cell periphery and cell-cell adhesion region. High levels of TGase-4 expression in CAHPV10 cells and in PC3 cells engineered to over-express TGase-4 were associated with significantly increased cell motility in response to HGF-L, a clear contrast to wild-type and control cells. Neutralising antibody to RON and rhHGFL/MSP had no further bearing on the increased motility in TGase-4 over-expressing cells, although they had profound effect on the control cells. Akt pathway inhibitor significantly diminished the effect induced by HGF-L in the cells. Finally, over-expression of TGase-4 in prostate cancer cells resulted in autophosphorylation of RON. It is concluded that TGase-4 expression is intrinsically linked to the activation of RON in prostate cancer cells and that this autoactivation of RON contributes to the increased cell motility in TGase-4 expressing cells.

The WISPs (Wnt-inducted secreted proteins, WISP-1, WISP-2 and WISP-3) are part of the CCN family. These molecules are known to play a diverse role in cells but their role in cancer cells remains controversial. We analysed the expression of the three WISP molecules at the mRNA and protein levels in a cohort of 94 human colorectal tumours and 80 normal colorectal tissues and correlated the results with the pathological features and clinical outcome of the patients. WISP-1 transcripts were found at higher levels in the tumour samples than in the normal tissue (p=0.0015); higher in patients with Dukes stage B and C compared to Dukes A (p=0.017 and p=0.024, respectively); higher in patients with moderately and poorly differentiated cancers compared to the well differentiated cancers (p=0.020 and p=0.076, respectively and p=0.0035 when combined); higher in node positive tumours compared with the node negative (p=0.11) and in the patients with higher TNM staging (TNM 2, 3 and 4 compared to TNM 1 p=0.037). WISP-2 showed the opposite pattern with lower levels of expression in cancer cells compared to normal (p=0.082). Although no significant differences were found within the cancer group when indices of a more aggressive tumour were compared to the normal tissue a significant reduction in expression was found (Dukes C p=0.044, poorly differentiated p=0.019, TNM 3 p=0.020 and node positive disease p=0.048). WISP-3 transcript levels showed no significant differences between groups. WISPs may play important but contrasting roles in colorectal cancer with WISP-1 appearing to act as a factor stimulating aggressiveness, WISP-2 as a tumour suppressor and WISP-3 having no definable beneficial or detrimental role.

Vascular endothelial growth inhibitor (VEGI) has been associated with tumour-related vasculature in certain maligancies. However, its implication in prostate cancer remains unknown. We investigated the expression pattern and role of VEGI in prostate cancer and prostate cancer cells. The expression of VEGI was examined in human prostate tissue and prostate cancer cell lines. The biological impact of modifying the expression of VEGI in prostate cancer cells was evaluated using in vitro models. VEGI mRNA was expressed in a wide variety of human prostate cancer cell lines and most prostate specimens. VEGI protein was seen in normal prostate epithelium, but was decreased or absent in prostate cancer specimens, particularly in tumours with high Gleason scores. Moreover, forced-expression of VEGI led to a decrease in the motility and adhesion of prostate cancer cells in vitro. In contrast, knocking down VEGI in the cells resulted in an increase in motility and adhesion. Interestingly, both forced-expression and knocking down of VEGI had no bearing on growth and invasive capacity of prostate cells. In conclusion, the expression of VEGI is decreased in prostate cancer and is almost absent in tumours with high Gleason scores. Together with its inhibitory effect on cellular motility and adhesion, this suggests that VEGI functions as a negative regulator for aggressiveness during the development and progression of prostate cancer.

Pigment epithelium-derived factor (PEDF) is an endogenous protein factor that has been shown to act as anti-angiognesis factor. The present study aimed to determine the direct biological effects of PEDF on lung cancer cells and deduce a clinical relevance in patients with lung cancer, major cause of death worldwide in which the knowledge of PEDF remains poor. We constructed a mammalian expression system for human PEDF produced recombinant PEDF (rhPEDF) protein from 3T3 cells. The expression of PEDF was examined using SDS-PAGE and Western blot analysis. Using the rhPEDF protein, we investigated the biological function of PEDF in the lung cancer cells as well as endothelial cells. PEDF expression levels were assessed in a cohort of human lung cancer specimen (77 pairs of matched normal and tumour tissues), in association with patient clinical variables and survival, using quantitative analysis of PEDF. In vitro, we found that administration of rhPEDF on two lung cancer cell lines (A549 and SK-MES1) significantly reduced tumour cell growth (P<0.05) with no significant effect on the growth of vascular cell line (HECV). We also found that rhPEDF significantly decreased lung cancer motility and adhesion to extracellular matrix (Matrigel) when compared with the control cells (P<0.05). We showed that reduced PEDF levels in lung cancer tissues significantly correlated with lymph node metastasis and an overall poor prognosis in the lung cancer patients. PEDF suppresses the growth and motility of lung cancer cells and has a significant correlation with the clinical outcome of the patients. These results contribute to our understanding of the molecular mechanisms of PEDF and indicates a potential prognostic and therapeutic impact of PEDF in lung 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.

The role and expression of ROCKI and ROCKII in human breast cancer was investigated and the effect on clinical outcome assessed. ROCK knockdown cells (MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII were tested for their in vitro invasiveness, motility and in vivo tumour growth. Samples of fresh frozen breast tumour tissue (n=113) and normal background tissue (n=30) were processed for immunohistochemical and quantitative RT-PCR analyses. MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII cells showed significantly decreased invasiveness compared with control cells (mean +/- SEM 4.33+/-0.84 MDA-MB-231DeltaROCKI vs. 18.4+/-1.4 control, p<0.001; 6.8+/-1.2 MDA-MB-231DeltaROCKII vs. 18.4+/-1.4 control, p<0.001). Similarly, both exhibited reduced motility compared with control cells (p<0.001) and lost their response to HGF. Importantly, no significant difference existed between knockdown and control cells in in vivo tumour growth. ROCKI was significantly higher in human mammary tumours than normal background tissue (2.9+/-1.1 vs. 0.29+/-0.13, p=0.023), although expression of ROCKII was fairly consistent in both (2050+/-646 vs. 2303+/-2079). ROCKI expression was greater in patients who died from breast cancer than in patients who remained disease free (11.6+/-7.1 vs. 1.95+/-0.95). Higher levels of ROCKI were associated with increased grade (0.95+/-0.73 grade-1; 2.11+/-1.72 grade-2; and 4.06+/-1.99 grade-3). Levels of ROCKI, but not ROCKII, were significantly correlated with overall survival of patients (p=0.004, Univariate analysis, median follow-up 120 month). These results show that ROCKI and possibly ROCKII are key factors in regulation of motility/invasion of breast cancer cells. This, together with significant correlation between ROCKI and poor outcome in clinical breast cancer, indicates that it is a potential target in human breast cancer.

Hepatocyte growth factor (HGF) plays multiple roles in cancer, by acting as a motility, invasion and angiogenesis stimulating factor, which promotes metastasis and tumour growth. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily. The effects of BMPs are mediated by two subgroups of receptors, type I and type II. Recent studies have shown that some BMPs, via their signaling pathways, affect the growth of prostate cancer cells. BMPR-IB and BMPR-II have been reported to be expressed at low levels in prostate cancer. However, little is known about the crosstalk between HGF and BMP pathways. In this study, prostate cancer cells (PC-3 and DU-145) were exposed to HGF at different concentrations (1-75 ng/ml) for 18 h, or were treated with HGF at 40 ng/ml over various time periods (up to 24 h). The effect of HGF on BMP receptor expression was further investigated in a nude mouse PC-3 xenograft model. Mice were treated with either HGF, the HGF antagonist NK4, or a combination of both. The expression of BMPR-IB and BMPR-II mRNA was up-regulated by HGF, as shown by both conventional PCR and quantitative PCR. An elevation of BMPR-IB and BMPR-II at the protein level was confirmed by both Western blot analysis and immunocytochemical staining. In a murine prostate tumour model, infusion of recombinant HGF resulted in an increase in the levels of both BMPR-IB and BMPR-II transcript in prostate tumours. Concomitant delivery of NK4, an HGF antagonist, prevented this effect. In conclusion, HGF up-regulates the expression of the bone morphogenetic protein receptors, BMPR-IB and BMPR-II, in prostate cancer cells, both in vitro and in vivo. This may have important implications in the development of bone metastasis in prostate cancer.

Lymphangiogenesis (growth of new lymphatic vessels) is thought to play an important role in cancer lymphatic spread to the regional lymph nodes. However, the molecular pathways involved in lymphangiogenesis and their regulation are still unclear. Recently, there has been a significant advance in the studies of the lymphatic system and lymphangiogenesis as several novel specific lymphatic markers are discovered. Here, the effects of several cytokines on the lymphatic expression of human endothelial cells were studied. Amongst these cytokines, interleukin-7 (IL-7) was found to have significant impact on the lymphatic expression as it induced the expression of podoplanin, prox-1 and LYVE-1 in endothelial cells. Furthermore, IL-7 enhanced endothelial cell growth, migration and generation of lymphatic tubules in vitro via upregulating the expression of the lymphangiogenic growth factor and vascular endothelial growth factor-D. The specificity of these effects of IL-7 was confirmed using blocking anti-bodies and ribozyme transgene technology. These effects of IL-7 were totally abolished when IL-7R null endothelial cell mutants were used. IL-7 activated its transmembrane receptor, IL-7R, on endothelial cells, as well as its downstream signalling intermediates, Jak-1, Jak-3, PI3-K and Stat-5. Selective inhibition of these intermediates using specific inhibitors showed that IL-7 induced the afore-mentioned effects via a Wortmannin sensitive pathway. Collectively, these results demonstrate, for the first time, that IL-7 is a lymphangiogenic growth factor by inducing the lymphangiogenic properties of endothelial cells. This might have a significant impact on the lymphatic spread of solid tumours. Furthermore, interruption of IL-7 signalling might provide an attractive therapeutic option in cancer lymphatic metastasis.