Gaynor Davies

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-231ΔMatrilysin) exhibited a significantly lower degree of invasion (number of invading cells 16.0 ± 2.5) compared with wild type (MDA-MB-231WT; 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-231ΔMatrilysin 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-231WT (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.

Levels of expression of endothelial markers specific to tumour-associated endothelial cells and their correlation with prognosis in patients with breast cancer.

Tumour endothelial markers (TEMs) are a newly discovered family of endothelial markers associated with tumour specific angiogenesis. This study sought to examine the levels of expression for TEMs in human breast cancer. Breast cancer tissues (n=120) together with normal background tissues (n=33) were obtained after surgery. RNA was extracted from frozen sections for gene amplification. The expression of TEMs was assessed using RT-PCR and the quantity of their transcripts was determined using real-time-quantitative PCR (Q-RT-PCR). TEM-7R (P=0.05) and TEM-8 (P<0.01) were significantly raised in breast cancer tissues compared with the levels detected in normal background tissues. After a median follow-up of 72.2 months it was found that patients who had recurrent disease and/or who had died from breast cancer had a significantly (P<0.05) elevated level of TEM-1 compared to those patients who were disease free. In addition, elevated levels of TEM-4, TEM-5, TEM-6, TEM-7 and TEM-7R were also raised in breast cancer tissues. Patients who had developed nodal involvement exhibited significantly (P<0.05) high levels of TEM-1 and TEM-7R compared to patients who were node negative. Furthermore, the levels of TEMs did not correlate with tumour or histological grade. We conclude that elevated levels of TEM-1, TEM-7R and TEM-8 (but not TEM-2, 4, 5, 6 and 7) are associated with either nodal involvement, and/or disease progression, and may therefore, have a prognostic value in breast cancer.

The HGF/SF antagonist NK4 reverses fibroblast- and HGF-induced prostate tumor growth and angiogenesis in vivo.

Our study examined the in vitro and in vivo responses of a newly discovered HGF/SF antagonist, NK4, on HGF/SF‐promoted growth of human prostate cancer cells (PC‐3). Nude mice were s.c. injected with either PC‐3‐ and/or HGF/SF‐producing fibroblasts (MRC5), and tumor size was measured over a 4‐week period. rh‐HGF/SF and/or NK4 were introduced by osmotic minipumps. An in vitro study found that NK4 significantly suppressed HGF/SF‐induced invasion (HGF/SF; p < 0.01 vs. HGF/SF+NK4) and migration (HGF/SF; p < 0.05 vs. HGF/SF+NK4). Similarly, NK4 also suppressed the invasion (MRC5; p < 0.01 vs. MRC5+NK4) and migration (MRC5; p < 0.05 vs. MRC5+NK4) induced by MRC5 cells. NK4 also suppressed HGF/SF‐ and MRC5‐induced tyrosine phosphorylation of the HGF/SF receptor Met as assessed by immunoprecipitation. Using a nude mouse model, prostate tumor volume (mm3) was significantly increased in both HGF/SF‐ (HGF/SF; p < 0.05 vs. control) and MRC5‐ (MRC5; p < 0.01 vs. control) treated groups compared to the control. In contrast, NK4 alone significantly reduced the growth of prostate tumors (NK4; p < 0.01 vs. control). In addition, NK4 also suppressed both HGF/SF‐ (HGF/SF; p < 0.01 vs. HGF/SF+NK4) and MRC5‐ (MRC5; p < 0.05 vs. MRC5+NK4) induced tumor growth in vivo by significantly reducing (p < 0.05) the degree of tumor angiogenesis using a recently discovered family of tumor endothelial markers (TEMs) by Q‐RT‐PCR analysis. In conclusion, NK4 suppresses both HGF/SF‐ and MRC5‐induced invasion/migration of PC‐3 cells in vitro. Furthermore, the HGF/SF antagonist NK4 significantly reduces prostate tumor growth in vivo by inhibiting the degree of tumor angiogenesis as determined by TEM‐1 and TEM‐8. Finally, our study provides evidence of the therapeutic potential of NK4 in prostate cancer development by antagonising HGF/SF‐mediated events.

CELL-CELL ADHESION MOLECULES AND SIGNALING INTERMEDIATES AND THEIR ROLE IN THE INVASIVE POTENTIAL OF PROSTATE CANCER CELLS

PURPOSE The highly variable natural history of prostate carcinoma may be reflected in heterogeneity of invasive potential between tumors. MATERIALS AND METHODS We have examined two prostate cancer cell lines of low invasive potential (CAHPV10 and PZHPV7) and three cell lines of high invasive potential (DU-145, PC-3, LNCapFGC), to determine whether specific adhesion molecule profiles correlated with their invasive behavior. RESULTS Using an in vitro invasion assay, we demonstrated that DU-145, LNCapFGC and PC-3 cells were highly invasive compared with CA-HPV-10 and PZ-HPV-7 cells. LNCapFGC cells expressed high levels of E-cadherin, alpha-, beta- and gamma-catenin, desmoglein, desmoplakin and GSK3beta using immunoblotting. This was, in general, comparable to immunohistochemical staining. PC-3 cells had no E-cadherin or alpha-catenin, but expressed a high level of the HGF/SF receptor c-Met. In contrast, DU-145 cells were found to express E-cadherin and low levels for all other protein molecules, except c-Met. The DU-145 cell line also lacked alpha-catenin expression. In CA-HPV-10 and PZ-HPV-7 cells, there was no detection of APC, PECAM-1, P-cadherin or Wnt-1. DU-145, LNCapFGC and PC-3 cells formed cell-cell aggregates, which were reduced by inclusion of anti-E-cadherin antibody and the motogen HGF/SF. CONCLUSION These results show that prostate cancer cells exhibit a diverse expression of cell-cell adhesion molecules and their signaling intermediates. The expression of these adhesion molecules bears an important relationship with the invasive phenotype of these cells.

Cell adhesion molecules and adhesion abnormalities in prostate cancer

Prostate cancer, the leading male cancer in Western countries, has accelerated in its incidence in the past decade. Patients with prostate cancer frequently have a poor prognosis as a result of local or distant spread of cancer. This review summarises some of the recent progress made in understanding the biology of cancer metastasis with a special emphasis on the role of cell adhesion molecules and adhesion abnormalities. The molecular and cellular function of cell adhesion molecules, their role in cancer and cancer progression, the clinical impact of these molecules, and therapeutic considerations are also discussed.

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.

NAPHTHYL PHOSPHORAMIDATE DERIVATIVES OF BVdU AS POTENTIAL ANTICANCER AGENTS: DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION

The phosphoramidate technology we have developed has been recently applied to BVdU, leading to NB1011 (NewBiotics Inc., California), a novel potential anticancer compound recently entered into phase 2 of the clinical trials for colon cancer. We report in this work a new series of derivatives containing naphthol as aryl masking group on the phosphate moiety, which has shown a significant increase in anticancer activity in preliminary biological evaluations.

Phospholipase-C gamma-1 (PLCγ-1) is critical in hepatocyte growth factor induced in vitro invasion and migration without affecting the growth of prostate cancer cells

BACKGROUND Phospholipase C gamma-1 (PLCgamma-1) is an intracellular signalling molecule regulating a number of biological processes including transporter mechanisms, transcription factors, and scaffolding proteins mediating cytoskeleton and membrane trafficking. Hepatocyte growth factor (HGF) is a pleiotrophic factor and a mediator of metastatic spread. This study sought to determine the effect of HGF on the invasive and migratory potential of prostate cancer cells targeted by a ribozyme transgene to PLCgamma-1. METHODS A ribozyme transgene consisting of hammerhead ribozyme and antisense specific to PLCgamma-1 was cloned into a PEF6 expression vector and transfected into PC-3 cells. RT-PCR and Western blotting confirmed knock down of PLCgamma-1. In vitro invasion and a cytodex-2 bead motility assays with in vitro and in vivo growth models were used to assess the impact of PLCgamma-1 manipulation. RESULTS PC-3 cells stably transfected with PLCgamma-1 ribozyme transgene (PC-3(DeltaPLC)gamma) manifested a reduction of PLCgamma-1 expression at mRNA/protein levels. HGF/SF increased invasiveness (P < 0.01) and motility (P < 0.0001) of PC-3(WT) and PC-3(PEF6) cells. In contrast, PLCgamma-1 knock down PC-3(DeltaPLC)gamma cells had reduced invasiveness (P < 0.05) and motility (P < 0.01) compared with PC-3(WT) and PC-3(PEF6) cells. Although there was a marginal change of cell growth in vitro, there was no difference in the rate of growth between PC-3(DeltaPLC)gamma, PC-3(WT), and PC-3(PEF6) cells. CONCLUSION Targeting PLCgamma-1 by way of a hammerhead ribozyme to PLCgamma-1 is an effective method in reducing invasive phenotype of prostate cancer. PLCgamma-1 is a signalling intermediate that has prime influence on HGF induced cellular invasion and migration without affecting the growth of the cells.

Rapid Molecular and Morphological Responses of Prostate Cell Lines to Cell–Cell Contact

Cell–cell adhesion in 2-D PZ-HPV-7 prostate epithelial and DU-145 prostate cancer cell aggregates (monolayers), synchronously and rapidly (within 30 s) formed in suspension in an ultrasound trap has been examined over 60 min. The intracellular distributions of the cadherin/catenin complex components for both cell lines were time-dependent and were clearly identifiable as early as 150 s following cell–cell contact in the trap, while equilibrium positions were reached within 60 min following cell–cell contact. The accumulation of E-cadherin at the cell–cell interface was greater for PZ-HPV-7 than for DU-145 cells over 60 min in the trap, with the apparent formation of adherens junctions over that time scale in PZ-HPV-7 but not in DU-145 cells. The amounts of F-actin, α-, β-, and γ-catenins recruited to the cell–cell interface of PZ-HPV-7 cells were on average 2.4 times higher than those of DU-145 cells. The ability of different cell types to spread along neighboring cells was 1.5-fold greater for the PZ-HPV-7 than for the DU-145 cells. These results, discussed also in the context of earlier studies of cell adhesion in an ultrasound trap, characterize a reduced adhesiveness of DU-145 cells compared to PZ-HPV-7 cells.

HEPATOCYTE GROWTH FACTOR/SCATTER FACTOR AND PROSTATE CANCER METASTASIS

Metastasis is a life-threatening event in tumor bearing patients resulting in total organ failure and to subsequent death. Understanding the fundamental processes involved in how cancer cells detach from the primary tumor in order to enter the metastatic cascade is a key factor in delivering therapeutic starategies to prevent metatasis from occuring. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is an unique growth factor capable of inducing a number of biological responses in a wide variety of normal and neoplastic cells, including invasion, cell spreading, scattering, motility and shedding of cell-cell adhesion molecules. This review is intended to highlight some of the more recent discoveries made in scientific and clinical research, with particular emphasis on the effect of HGF/SF on protsate cancer metastasis and invasion.