The Presence of (1→3)-ß-D-Glucan as Prognostic Marker in Patients After Major Abdominal Surgery.

BACKGROUND: While the serological detection of (1→3)-ß-D-glucan (BDG) can indicate invasive fungal disease (IFD), false positivity occurs. Nevertheless, the presence of BDG can still be recognized by the host's innate immune system and persistent BDG antigenemia, in the absence of IFD, can result in deleterious proinflammatory immune responses. METHODS: During the XXX (INTENSE) study into the preemptive use of micafungin to prevent invasive candidiasis (IC) after abdominal surgery, the serum burden of BDG was determined to aid diagnosis of IC. Data from the INTENSE study were analyzed to determine whether BDG was associated with organ failure and patient mortality, while accounting for the influences of IC and antifungal therapy. RESULTS: A BDG concentration >100 pg/mL was associated with a significantly increased Sequential Organ Failure Assessment score (≤100 pg/mL: 2 vs >100 pg/mL: 5; P < .0001) and increased rates of mortality (≤100 pg/mL: 13.7% vs >100 pg/mL: 39.0%; P = .0002). Multiple (≥2) positive results >100 pg/mL or a BDG concentration increasing >100 pg/mL increased mortality (48.1%). The mortality rate in patients with IC and a BDG concentration >100 pg/mL and ≤100 pg/mL was 42.3% and 25.0%, respectively. The mortality rate in patients without IC but a BDG concentration >100 pg/mL was 37.3%. The use of micafungin did not affect the findings. CONCLUSIONS: The presence of persistent or increasing BDG in the patient's circulation is associated with significant morbidity and mortality after abdominal surgery, irrespective of IC. The potential lack of a specific therapeutic focus has consequences when trying to manage these patients, and when designing clinical trials involving patients where host-associated BDG concentrations may be elevated. CLINICAL TRIALS REGISTRATION: NCT01122368.

Boswellia frereana suppresses HGF-mediated breast cancer cell invasion and migration through inhibition of c-Met signalling.

BACKGROUND: Hepatocyte growth factor (HGF) plays a pivotal role in breast cancer cell motility, invasion and angiogenesis. These pro-metastatic events are triggered through HGF coupling and activation of the c-Met receptor. Reports have demonstrated that HGF/c-Met signalling plays an important part in breast cancer progression and that their expression is linked to poor patient outcome. In the present study, we investigated the anti-metastatic potential of an extract from traditional Somalian frankincense, Boswellia frereana, on human breast cancer cells. In addition, we also examined the effect of this Boswellia frereana extract (BFE) upon HGF-mediated stimulation of the c-Met receptor. METHODS: Two triple negative human breast cancer cell lines, BT549 and MDA-MB-231, were utilised in the study to examine the effect of BFE on tumour cell proliferation, migration, matrix-adhesion, angiogenesis and invasion. Cell migration was investigated using a Cell IQ time-lapsed motion analysis system; while tumour cell-matrix adhesion, angiogenesis and invasion were assessed through Matrigel-based in vitro assays. Breast cancer cell growth and spheroid formation was examined through proliferation assay and 3D non-scaffold cell culture techniques. Western Blotting was employed to determine the phosphorylation status of the c-Met receptor tyrosine kinase following BFE treatment and subsequent HGF stimulation. RESULTS: Following HGF treatment, the breast cancer cells displayed a significant increase in migration, matrix adhesion, vessel/tubule formation, invasion and c-Met activation. HGF did not appear to have any bearing on the proliferation rate or spheroid formation of these breast cancer cells. The addition of the BFE extract quenched the HGF-enhanced migratory, angiogenic and invasive potential of these cells. Further study revealed that BFE inhibited c-Met receptor tyrosine kinase phosphorylation within these breast cancer cells. CONCLUSIONS: Our findings reveal that BFE was able to significantly suppress the influence of HGF in breast cancer cell motility and invasion in vitro, through the ability of BFE to reduce HGF/c-Met signalling events. Therefore, these results indicate that BFE could play a novel role in the treatment of breast cancer.

Predicting Invasive Aspergillosis in Hematology Patients by Combining Clinical and Genetic Risk Factors with Early Diagnostic Biomarkers.

Personalized medicine provides a strategic approach to the management of IA. The incidence of IA in high-risk hematology populations is relatively low (<10%), despite unavoidable Aspergillus exposure in patients with a potentially similar clinical risk. Nonclinical variables, including genetic mutations that increase susceptibility to IA, could explain why only certain patients develop the disease. This study screened for mutations in 322 hematology patients classified according to IA status and developed a predictive model based on genetic risk, established clinical risk factors, and diagnostic biomarkers. Genetic markers were determined by real-time PCR and, with clinical risk factors and Aspergillus PCR results, subjected to multilogistic regression analysis to identify a best-fit model for predicting IA. The probability of IA was calculated, and an optimal threshold was determined. Mutations in dectin-1 (rs7309123) and DC-SIGN (rs11465384 and rs7248637), allogeneic stem cell transplantation, respiratory virus infection, and Aspergillus PCR positivity were all significant risk factors for developing IA and were combined in a predictive model. An optimal threshold requiring three positive factors generated a mean sensitivity/specificity of 70.4%/89.2% and a probability of developing IA of 56.7%. In patients with no risk factors, the probability of developing IA was 2.4%, compared to >79.1% in patients with four or more factors. Using a risk threshold of 50%, preemptive therapy would have been prescribed for 8.4% of the population. This pilot study shows that patients can be stratified according to risk of IA, providing personalized medicine based on strategic evidence for the management of IA. Further studies are required to confirm this approach.

Evaluation of real-time PCR, galactomannan enzyme-linked immunosorbent assay (ELISA), and a novel lateral-flow device for diagnosis of invasive aspergillosis.

Diagnosis of invasive aspergillosis (IA) remains challenging. With a relatively low incidence of disease, the use of expensive empirical antifungal therapy exposes many patients to unnecessary toxicity. Diagnosis places emphasis on specific but temporal radiological evidence. Circulating biomarker diagnosis has shown potential, but assays show variable performance, take several hours to perform, and require a degree of technical ability. A novel and simple lateral-flow device (LFD) using monoclonal antibody JF5, which targets an extracellular glycoprotein, has been developed and potentially removes any technical requirements, reducing processing time considerably. In this study, we evaluate the performance of this LFD compared to real-time PCR (targeting the 28S rRNA gene) and galactomannan (GM) detection when testing serum from a European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG)-defined hematological population. In a proven/probable-IA population versus a no-IA population, the LFD performance was comparable to that of both PCR and galactomannan enzyme immunoassay. Specificity (98.0%) was similar to that of PCR (96.6%) and slightly superior to that of GM (91.5%). Sensitivity (81.8%) was inferior to that of PCR (95.5%) but better than that of GM (77.3%). In combination with PCR, it provided both 100% sensitivity and 100% specificity. The LFD permits rapid testing of easily obtainable specimens, to be used as an adjunct test, before confirmation by other investigations. Its simplicity provides centers without specialist diagnostics with a test with clinical performance superior to that of classical microbiological approaches and results that can be used to direct antifungal management. In summary, microbiological diagnosis of IA is difficult and options are limited, with variable performance. An LFD assay targeting a novel specific biomarker has been developed, one which is methodologically simple and provides good clinical performance, particularly if combined with PCR.

The type II transmembrane serine protease, matriptase-2: Possible links to cancer?

Matriptase-2 is a newly identified member of the Type II Transmembrane Serine Protease (TTSP) family. The expression profile of many members of this family of proteases is frequently altered in cancerous cells and tissues and a number of TTSPs have been linked to cancer progression and development. Matriptase-2 is structurally similar to matriptase-1, a TTSP which has gained recent interest due to its potential to enhance the aggressive nature of cancer cells and its links with a variety of human cancers. Recently, matriptase-2 has been functionally linked to the regulation of iron metabolism; however, there is also evidence to suggest that, as with other members of the TTSPs, matriptase-2 may have a role in cancer development and progression. This article reviews the current literature on matriptase-2, together with its potential roles in physiological and disease states particularly focusing on cancer.

Hepatocyte growth factor activation inhibitors - therapeutic potential in cancer.

Hepatocyte growth factor (HGF) plays a plethora of roles in the progression of many invasive and metastatic cancers. The interaction between tumour cells and their surrounding stromal environment remains a crucial factor governing tumour invasion and metastasis. HGF is primarily synthesised by stromal fibroblasts as an inactive precursor known as pro-HGF. A number of proteases have demonstrated the ability to convert pro-HGF into the biologically active form of HGF, although the two main factors responsible are HGF activator (HGFA) and matriptase. The HGF activation inhibitors (HAI-1 and HAI-2) are two novel Kunitz-type serine protease inhibitors that regulate HGFA and matriptase activity to govern the influence of HGF within the body. Deregulation of HAI expression can lead to shift in the HGF activation/inhibition balance ratio in favour of enhanced HGF production. Therefore, these HGF activation inhibitors may have a direct bearing on cancer invasion and metastasis. This review examines the accumulating evidence on the emerging role and therapeutic potential of HAI-1 and HAI-2 in cancer.

Metastasis suppressor 1 (MTSS1) demonstrates prognostic value and anti-metastatic properties in breast cancer.

Metastasis suppressor 1 (MTSS1) may play an important role in cancer metastasis. Firstly, this study assessed MTSS1 expression levels within breast cancer patients to reveal any clinical relevance. Secondly, we aimed to clarify the cellular function of MTSS1 in breast cancer cells. MTSS1 expression levels were assessed in a cohort of breast cancer specimens (normal n=33; cancer n=127), through quantitative PCR analysis and immuno-histochemical techniques. The influence of MTSS1 was further examined via biological overexpression and knockdown within breast cancer cell lines. We report that patients with tumours expressing reduced levels of MTSS1 had a poorer prognosis (p=0.042). High levels of MTSS1 correlated with an increased patient overall survival (p=0.0108) and disease-free survival (p=0.012). Furthermore, overexpression of MTSS1 significantly suppressed (p<0.01) the invasive, migratory, growth and adherence properties of a human breast cancer cell line. In contrast, knockdown of MTSS1 dramatically enhanced these properties. We conclude that MTSS1 is a prognostic indicator of disease-free survival in breast cancer patients and demonstrates the ability to play a role in governing the metastatic nature of breast cancer cells.

Genetic upregulation of matriptase-2 reduces the aggressiveness of prostate cancer cells in vitro and in vivo and affects FAK and paxillin localisation.

The cellular function and the role of matriptase-2 in cancer progression are poorly understood. This study assesses the importance of this protease in prostate cancer cell lines. Two prostate cancer cell lines, PC-3 and DU-145, previously displaying minimal expression of matriptase-2, were forced to over-express matriptase-2 using a human mammalian expression construct. Over-expression of matriptase-2 significantly reduced the invasive capacity and significantly slowed the migration rates of PC-3 and DU-145 cells in vitro. Similarly, PC-3 cells containing the matriptase-2 expression plasmid were dramatically less able to survive, grow and develop into noticeable tumours, compared to control PC-3 cells containing an empty plasmid alone, following subcutaneous inoculation into CD1 nude mice. This trend was observed throughout the experiment, becoming apparent after the initial reading on day 7 (P = 0.0002) and continuing to the experimental end point at day 27 (P = 0.0002). Enhanced matriptase-2 levels were also seen to correlate with increased fluorescent staining of the paxillin and FAK adhesion molecules, where a greater extent of these molecules were localised to the focal adhesion complexes. This data suggests a suppressive role for matriptase-2 in the invasion and migration of prostate cancer cells in vitro and also in their development and growth in vivo, highlighting the potential of this molecule to interfere with key stages of metastasis. Furthermore, the data presented implies a possible connection between matriptase-2 and the paxillin and FAK adhesion molecules which may ultimately contribute to the reduced migration rates seen in this study.

Suppression of hepatocyte growth factor activator inhibitor-1 leads to a more aggressive phenotype of prostate cancer cells in vitro.

The hepatocyte growth factor (HGF) pathway has been well documented as playing a vital role in the progression and development of many different types of human cancers; as such this pathway is usually tightly regulated. In cancer cells, the regulation of this pathway has been shown to be disrupted, allowing an increase in activation of pro-HGF to active HGF. There are a number of molecules capable of activating pro-HGF, such as matriptase-1, a type II transmembrane serine protease, or hepatocyte growth factor activator, and in turn, these are also subject to regulation. In the current study we examined the importance of hepatocyte growth factor activator inhibitor-1 (HAI-1) which is known to inhibit a number of HGF-activating molecules. We reduced the expression of this molecule in both PC-3 and DU-145 cell lines using hammerhead ribozyme technology, and we examined various important characteristics associated with cancer progression and development in vitro. Prostate cancer cells, after loss of HAI-1, had a significantly increased in vitro invasiveness together with an increase in cellular motility. Notably, loss of HAI-1 resulted in a slower rate of cell growth over a prolonged period (5 days). This in vitro evidence collectively suggests that the suppression of HAI-1 expression gives rise to a more aggressive cancer cell phenotype. This implies that therapies inducing the overexpression of HAI-1 or delivering an exogenous source of HAI-1 protein may hold potential as a treatment to slow the progression of prostate cancer.

Matriptase-2 inhibits breast tumor growth and invasion and correlates with favorable prognosis for breast cancer patients.

PURPOSE: The type II transmembrane serine proteases are cell surface proteolytic enzymes that mediate a diverse range of cellular functions, including tumor invasion and metastasis. Matriptase (matriptase-1) and matriptase-2 belong to the type II transmembrane serine protease family. Matriptase-1 is known to play a role in breast cancer progression, and elevated levels of matriptase-1 correlate with poor patient outcome. The role of matriptase-2 and its cellular function in cancer is unknown. This study aimed to provide new insights into the significance of matriptase-2 in cancer. EXPERIMENTAL DESIGN: Matriptase-2 expression levels were assessed in a cohort of human breast cancer specimens (normal, n = 34; cancer, n = 95), in association with patient clinical variables, using both quantitative and qualitative analysis of the matriptase-2 transcript along with immunohistochemical techniques. Matriptase-2 was also experimentally overexpressed in the MDA-MB-231 human breast cancer cell line. The effects of matriptase-2 overexpression were examined through a series of in vitro and in vivo studies. RESULTS: Here, we show that reduced matriptase-2 levels in breast cancer tissues correlate with an overall poor prognosis for the breast cancer patient. This study also reveals that matriptase-2 overexpression in breast cancer cells significantly suppressed tumorigenesis in CD1 athymic mice (P = 0.000003). Furthermore, we report that matriptase-2 overexpression dramatically reduced the invasive (P = 0.0001) and migratory properties (P = 0.01) of the breast cancer cells. CONCLUSIONS: Matriptase-2 suppresses breast tumor development in vivo, displays prognostic value for breast cancer patients, inhibits both breast cancer cell invasion and motility in vitro, and may play a contrasting role to matriptase-1 in breast cancer.

Reduced vascular endothelial growth inhibitor (VEGI) expression is associated with poor prognosis in breast cancer patients.

Vascular endothelial growth inhibitor (VEGI) is a novel anti-angiogenic cytokine that belongs to the tumour necrosis factor (TNF) superfamily. Very little is known about the significance of VEGI in cancer. Our study analysed VEGI expression in relation to breast cancer patient clinical parameters. The VEGI expression profile was assessed qualitatively (RT-PCR), quantitatively (real-time Quantitative-PCR), and immuno-histochemically (IHC), in a panel of 24 human normal and cancer cell lines and in a cohort of 151 mammary tissue samples (n = 33 normal breast tissue; n = 118 breast cancer tissue) with a 6-year median follow-up. Patients who had died of breast cancer or had local recurrence of the disease expressed significantly lower levels of VEGI in comparison to the elevated levels in the disease free patients. High levels of VEGI were associated with an increased chance of patient survival. Importantly, patients with breast tumours expressing reduced levels of VEGI had a poorer prognosis than those patients expressing high levels of VEGI. However, no significant correlations were observed between VEGI expression and tumour grade, TNM classification, or nodal involvement. In conclusion, VEGI is aberrantly expressed in human breast cancer tissues. VEGI displays prognostic relevance as breast cancer patients with an overall poor prognosis express significantly lower levels of VEGI compared to those with a favourable prognosis.

Decreased pigment epithelium-derived factor expression in human breast cancer progression.

PURPOSE: The aim of this study was to correlate the expression of pigment epithelium-derived factor (PEDF), a potent endogenous antiangiogenic molecule, with severity and prognosis in breast cancer. EXPERIMENTAL DESIGN: To investigate the gene expression profile of PEDF in human breast cancer in relation to a patient's clinical variables, we examined human breast cancer tissue (n = 119), background breast tissue (n = 33), and a range of cell lines for mRNA and protein levels of PEDF by using reverse transcription PCR, real-time quantitative PCR, immunohistochemistry, and ELISA. RESULTS: By using reverse transcription PCR, real-time quantitative PCR, immunohistochemistry, and ELISA, PEDF expression was found to be dramatically decreased in breast cancer. An overall outlook for the patients inversely correlated with PEDF mRNA levels. Exogenous PEDF inhibits endothelial tubule formation induced by breast cancer cell-conditioned medium, in vitro. CONCLUSION: These observations collectively support the hypothesis that a lack of PEDF expression is a potent factor for the enhancement of tumor growth and angiogenesis in breast cancer.

Expression of membrane type-1 matrix metalloproteinase, MT1-MMP in human breast cancer and its impact on invasiveness of breast cancer cells.

MT1-MMP (membrane type-1 matrix metalloproteinase), otherwise known as MMP14 is a proteolytic enzyme known to be involved in degradating extracellular matrix and assist progression of cancer invasion and progression. We investigated the impact of targeting the expression of MT1-MMP in breast cancer and its clinical relevance. Human breast cancer cell line MDA-MB-231 was used. Expression of MT1-MMP in the breast cancer cell line was manipulated by way of retroviral ribozyme transgene. The in vitro invasion, growth and cell migration were determined on cell lines transfected with either the transgene or control plasmid. Protein and message levels of MMP14 was also assessed using immunohistochemistry and real-time quantitative analysis, and correlated with clinical and pathological information of the patients. Retroviral ribozyme transgene to human MT1-MMP successfully knocked down the levels of MT1-MMP mRNA from MDA-MB-231 cells. Reduction of MT1-MMP from the breast cancer cells resulted in significant reduction of in vitro invasiveness and loss of response to an invasion stimulus, HGF, compared with control and wild-type cells. The invasion index for MT1-MMP knockdown cells were 13+/-3.1 (without HGF) and 16.4+/-2.3 (with HGF, p=0.14), and the index for transfection control cells 25.3+/-4.3 (without HGF) and 40.4+/-4.1 (with HGF, p=0.0049). Transfection with the transgenes did not change the rate of cell growth. In clinical breast cancer, MT1-MMP staining was both membranous and cytoplasmic. Tumour cells displayed stronger staining compared with normal mammary epithelial cells. Tumour tissues had a marginally higher levels of the MMP14 transcript (8.6+/-1.9), compared with normal tissues (4.7+/-1.4), p=0.13. No significant difference was observed between node positive and node negative tumours (9.0+/-2.2 vs 8.7+/-3.1, p=0.24). Marginally higher levels of the MMP14 transcript were seen in tumours which developed metastasis and local recurrence. However, tumours from patients who died of breast cancer related causes had significantly higher levels of the transcript, compared with tumours from patients who remained disease-free 10 years after initial surgery (12.2+/-2.5 vs 6.3+/-1.2, p=0.0091). MT1-MMP is a proteolytic enzyme that is pivotal in controlling the invasiveness of breast cancer cells. It is highly expressed in aggressive breast tumours and is associated with clinical outcome. The enzyme is a potential therapeutic target in breast cancer.

Hepatocyte growth factor activation inhibitors (HAI-1 and HAI-2) regulate HGF-induced invasion of human breast cancer cells.

Hepatocyte growth factor (HGF) plays a plethora of roles in cancer metastasis and tumour growth. The interaction between tumour cells and their surrounding stromal environment is a crucial factor regulating tumour invasion and metastasis. Stromal fibroblasts are the main source of HGF in the body, and release HGF as an inactive precursor (pro-HGF). HGF activator (HGFA), matriptase, urokinase-type plasminogen activator and hepsin are the main factors responsible for converting pro-HGF into active HGF. HAI-1 and HAI-2 are 2 novel Kunitz-type serine protease inhibitors that regulate HGF activity through inhibition of HGFA, matriptase and hepsin action. Recent studies demonstrate that HAI-1 and HAI-2 may also potently inhibit a number of other pro-metastatic serine proteases and therefore have direct bearing on the spread of tumours. Our study examined the potential of these HAI's to suppress the influence of HGF and regulate cancer metastasis. We generated a retroviral expression system that induced HAI expression in a human fibroblast cell line. Forced expression of either HAI-1 or HAI-2 in these fibroblasts resulted in a dramatic decrease in the production of bioactive hepatocyte growth factor (HGF). This reduction in HGF activity subsequently suppressed HGF's metastatic influence on breast cancer cells. To further assess the anti-cancer properties of HAI-1 and HAI-2 we generated recombinant HAI proteins. These recombinant HAI proteins possessed the ability to potently quench HGF activity. We also demonstrate that these recombinant HAI's suppressed fibroblast-mediated breast cancer invasion. An additional ribozyme transgenes study revealed that elimination of HAI-1 and HAI-2 expression, in an MDA-MB-231 breast cancer cell line, significantly enhanced the migratory, proliferative and invasive nature of these breast cancer cells. Overall, our data demonstrates the important roles of HAI-1 and HAI-2 in cancer metastasis, and reveals that these serine protease inhibitors display strong therapeutic potential.

Genetic reduction of matriptase-1 expression is associated with a reduction in the aggressive phenotype of prostate cancer cells in vitro and in vivo.

PURPOSE: Matriptase-1 has been implicated as playing an important role in various types of cancer progression through many different cancer related pathways. In the current study we assessed the efficacy of targeting matriptase-1 using ribozyme technology in vitro and in vivo. EXPERIMENTAL DESIGN: Matriptase-1 expression was reduced in the PC-3 and DU-145 cell line using hammerhead ribozyme transgenes. In vitro assays were set up to assess changes in growth, invasion, adhesion and migration in these cells. In vivo tumour development model was also used to examine the efficacy of targeting matriptase-1 in a living environment. RESULTS: The in vitro results suggest an overall reduction in the aggressive nature of the two cell lines (PC-3 and DU-145) when matriptase-1 levels are reduced, with properties such as growth, invasiveness and migration all being reduced (in most cases a greater than 50% reduction in migration and invasion compared to the control was observed), though strangely an increase in adhesion is seen in the PC-3 knockout. The in vitro data is strongly backed up by the results of the in vivo work which demonstrates matriptase-1 deficient cells have a substantially reduced ability to grow and develop in vivo compared to control cells when explanted into nude mice, with significant differences in growth and development (P < or = 0.05) being seen after 7 days, and highly significant differences (p < or = 0.001) after 15 days. CONCLUSIONS: Together this data strongly implicates matriptase-1 as playing a vital role in the aggressive nature and progression of prostate cancer.

Placenta growth factor is over-expressed and has prognostic value in human breast cancer.

Placenta growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family, a group of angiogenic factors that are crucial for tumour angiogenesis. Very little is known about the significance of PlGF in human cancer. We hypothesise that PlGF may have a potent influence in breast cancer. This study examined PlGF levels in human breast cancer in relation to patient's clinical parameters. PlGF expression and distribution was examined quantitatively using real-time quantitative polymerase chain reaction (Q-PCR) on a cohort of human breast cancer tissue (n = 119) and background breast tissue (n = 33), qualitatively using reverse transcriptase polymerase chain reaction (RT-PCR) on a range of cell lines, and immunohistochemically on patient samples. All these techniques revealed that PlGF expression was dramatically increased (P = 0.028) in breast cancer tissues compared with normal breast tissue. We demonstrate that PlGF displays prognostic value through analysis of patient survival status (6-year follow-up), as elevated levels of PlGF were significantly associated (P = 0.017) with recurrence, metastasis and patient mortality. Our study has shown that PlGF is over-expressed in breast cancer tissues and correlates with patient prognosis, and is likely to play a major role in the pathogenesis of tumours.

The potential lymphangiogenic effects of hepatocyte growth factor/scatter factor in vitro and in vivo.

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.

Targeting matrilysin and its impact on tumor growth in vivo: the potential implications in breast cancer therapy.

INTRODUCTION: Matrilysin (MMP-7) is a metalloproteinase that is involved in the degradation of extracellular matrix, invasion, and tumor progression. The current study examined if targeting matrilysin using retroviral ribozyme transgenes may have an impact on breast cancer cells and may have clinical implications. EXPERIMENTAL DESIGN: Retroviral hammerhead ribozyme transgenes were designed to specifically target human matrilysin mRNA. The breast cancer cell MDA-MB-231 was transfected with either a retroviral matrilysin transgene or a control retroviral transgene. Stably transfected cells were tested for their invasiveness and migratory properties in vitro. The cells were also used in creating a tumor model in athymic nude mice in which the growth of tumors and levels of matrilysin were assessed. In addition, levels of both protein and mRNA of matrilysin were investigated in a cohort of human breast tumors. RESULTS: Expression of matrilysin in MDA-MB-231 was successfully eliminated by the retroviral hammerhead ribozyme transgene for matrilysin as revealed by reverse transcription-PCR. Matrilysin transgene-transduced cancer cells (MDA-MB-231DeltaMatrilysin) exhibited a significantly lower degree of invasion (number of invading cells 16.0 +/- 2.5) compared with wild type (MDA-MB-231(WT); 26.2 +/- 6.2, P < 0.05) or control transgene-transduced cancer cells (MDA-MB-231pRevTRE; 25.3 +/- 4.2, P < 0.01). However, the rate of growth of the cells in vitro was not significantly affected. In the in vivo tumor model, MDA-MB-231DeltaMatrilysin tumors, which had very low levels of immunoreactive matrilysin, grew at a significantly lower rate (0.24 +/- 0.03 cm3, 4 weeks after inoculation) compared with the wild-type MDA-MB-231(WT) (1.46 +/- 0.04 cm3) and MDA-MB-231pRevTRE (1.12 +/- 1.0 cm3) tumors. In human breast tumors, breast cancer cells stained matrilysin at a significantly higher density, compared with normal mammary epithelium. The highest level of matrilysin was seen in high-grade tumors and that from patients with moderate and poor prognosis. Finally, high levels of matrilysin were significantly linked with a poor long-term survival (P = 0.0143). CONCLUSION: Matrilysin, which is aberrantly expressed in human breast tumors, can be effectively eliminated from breast cancer cells by way of hammerhead ribozyme transgene. Elimination of matrilysin is associated with low invasiveness and slow tumor growth. Taken together, the study suggests that targeting matrilysin may have important therapeutic implications.

Stromal cell derived factor-1: its influence on invasiveness and migration of breast cancer cells in vitro, and its association with prognosis and survival in human breast cancer.

INTRODUCTION: Stromal cell-derived factor (SDF)-1 (CXC chemokine ligand-12) is a member of the CXC subfamily of chemokines, which, through its cognate receptor (CXC chemokine receptor [CXCR]4), plays an important role in chemotaxis of cancer cells and in tumour metastasis. We conducted the present study to evaluate the effect of SDF-1 on the invasiveness and migration of breast cancer cells, and we analyzed the expression of SDF-1 and its relation to clinicopathological features and clinical outcomes in human breast cancer. METHOD: Expression of SDF-1 mRNA in breast cancer, endothelial (HECV) and fibroblast (MRC5) cell lines and in human breast tissues were studied using RT-PCR. MDA-MB-231 cells were transfected with a SDF-1 expression vector, and their invasiveness and migration was tested in vitro. In addition, the expression of SDF-1 was investigated using immunohistochemistry and quantitative RT-PCR in samples of normal human mammary tissue (n = 32) and mammary tumour (n = 120). RESULTS: SDF-1 expression was identified in MRC5, MDA-MB-435s and MDA-MB-436 cell lines, but CXCR4 expression was detected in all cell lines and breast tissues. An autocrine loop was created following transfection of MDA-MB-231 (which was CXCR4 positive and SDF-1 negative) with a mammalian expression cassette encoding SDF-1 (MDA-MB-231SDF1+/+) or with control plasmid pcDNA4/GFP (MDA-MB-231+/-). MDA-MB-231SDF1+/+ cells exhibited significantly greater invasion and migration potential (in transfected cells versus in wild type and empty MDA-MB-231+/-; P < 0.01). In mammary tissues SDF-1 staining was primarily seen in stromal cells and weakly in mammary epithelial cells. Significantly higher levels of SDF-1 were seen in node-positive than in node-negative tumours (P = 0.05), in tumours that metastasized (P = 0.05), and tumours from patients who died (P = 0.03) than in tumours from patients who were disease free. It was most notable that levels of SDF-1 correlated significantly with overall survival (P = 0.001) and incidence-free survival (P = 0.035). CONCLUSION: SDF-1 can increase the invasiveness and migration of breast cancer cells. Its levels correlated with node involvement and long-term survival in patients with breast cancer. SDF-1 may therefore have potential value in assessing clinical outcomes of patients with breast cancer.

Hepatocyte growth factor, its receptor, and their potential value in cancer therapies.

Hepatocyte growth factor plays multiple roles in cancer, by acting as a motility and invasion stimulating factor, promoting metastasis and tumour growth. Furthermore, it acts as a powerful angiogenic factor. The pivotal role of this factor in cancer has indicated HGF as being a potential target in cancer therapies. The past few years have seen rapid progress in developing tools in targeting HGF, in the context of cancer therapies, including development of antagonists, small compounds, antibodies and genetic approaches. The current article discusses the potential value of HGF and its receptor as targets in cancer therapies, the current development in anti-HGF research, and the clinical value of HGF in prognosis and treatment.