ATP‐P2Y2‐β‐catenin axis promotes cell invasion in breast cancer cells

Extracellular adenosine 5′‐triphosphate (ATP), secreted by living cancer cells or released by necrotic tumor cells, plays an important role in tumor invasion and metastasis. Our previous study demonstrated that ATP treatment in vitro could promote invasion in human prostate cancer cells via P2Y2, a preferred receptor for ATP, by enhancing EMT process. However, the pro‐invasion mechanisms of ATP and P2Y2 are still poorly studied in breast cancer. In this study, we found that P2Y2 was highly expressed in breast cancer cells and associated with human breast cancer metastasis. ATP could promote the in vitro invasion of breast cancer cells and enhance the expression of β‐catenin as well as its downstream target genes CD44, c‐Myc and cyclin D1, while P2Y2 knockdown attenuated above ATP‐driven events in vitro and in vivo. Furthermore, iCRT14, a β‐catenin/TCF complex inhibitor, could also suppress ATP‐driven migration and invasion in vitro. These results suggest that ATP promoted breast cancer cell invasion via P2Y2‐β‐catenin axis. Thus blockade of the ATP‐P2Y2‐β‐catenin axis could suppress the invasive and metastatic potential of breast cancer cells and may serve as potential targets for therapeutic interventions of breast cancer.     

The loss of E-cadherin, alpha- and beta-catenin expression is associated with metastasis and poor prognosis in invasive breast cancer

Loss of E-cadherin and catenin expression may be associated with distant and lymph node metastases in breast cancer. Heterogeneity of E-cadherin expression is associated with poor prognosis, suggesting that E-cadherin and catenins may serve as useful prognostic markers for invasive breast carcinoma. Reduction or loss of expression of either E-cadherin or catenins is associated with invasion, metastasis and poor prognosis in several types of human malignancies. We investigated the expression of E-cadherin, and alpha- and beta-catenins by immunohistochemistry in 171 cases of primary invasive breast cancer, and compared the expression with clinicopathological parameters to define the relationship between expression and prognosis. E-cadherin immunoreactive protein was shown to be expressed in 97 cases. Reduction or lack of expression of E-cadherin was associated with distant metastasis. Based on immunohistochemical heterogeneity, E-cadherin-positive tumors were classified into heterogeneous, homogeneous and intermediate types. Interestingly, although patients with heterogeneous type demonstrated the lowest incidence of distant metastasis at diagnosis, they showed a higher incidence of subsequent distant metastasis, after surgery, and a lower survival rate than those with homogeneous type (p<0.05). E-cadherin expression was reduced or negative in metastatic axillary lymph nodes regardless of the expression in the primary tumor, suggesting that changes in E-cadherin expression are associated with not only distant metastasis but also lymph node metastasis. Tumors negative for either alpha- or beta-catenin expression demonstrated a higher incidence of distant metastasis than those expressing both catenins, suggesting that the expression of catenins is involved in breast cancer metastasis. Reduction or loss of E-cadherin and catenin expression may be associated with distant and lymph node metastases in invasive breast cancer, and the heterogeneous type may be associated with poor prognosis.     

Hsp27 regulates EGF/β‐catenin mediated epithelial to mesenchymal transition in prostate cancer

Increased expression of the molecular chaperone Hsp27 is associated with the progression of prostate cancer (PCa) to castration‐resistant disease, which is lethal due to metastatic spread of the prostate tumor. Metastasis requires epithelial to mesenchymal transition (EMT), which endows cancer cells with the ability to disseminate from the primary tumor and colonize new tissue sites. A wide variety of secreted factors promote EMT, and while overexpression and constitutive activation of epidermal growth factor (EGF) signaling is associated with poor prognosis of PCa, a precise role of EGF in PCa progression to metastasis remains unclear. Here, we show that Hsp27 is required for EGF‐induced cell migration, invasion and MMPs activity as well as the expression of EMT markers including Fibronectin, Vimentin and Slug with concomitant decrease of E‐cadherin. Mechanistically, we found that Hsp27 is required for EGF‐induced AKT and GSK3β phosphorylation and β‐catenin nuclear translocation. Moreover, silencing Hsp27 decreases EGF dependent phosphorylation of β‐catenin on tyrosine 142 and 654, enhances β‐catenin ubiquitination and degradation, prevents β‐catenin nuclear translocation and binding to the Slug promoter. These data suggest that Hsp27 is required for EGF‐mediated EMT via modulation of the β‐catenin/Slug signaling pathway. Together, our findings underscore the importance of Hsp27 in EGF induced EMT in PCa and highlight the use of Hsp27 knockdown as a useful strategy for patients with advanced disease.     

Expression of E-cadherin/catenin complex in nasopharyngeal carcinoma: correlation with clinicopathological parameters.

The E-cadherin/catenin complex plays an essential role in maintaining intimate intercellular associations and is considered to be involved in tumor metastasis and suppressing invasion by cancer cells. We have analyzed the expression of E-cadherin/catenin complex in a series of nasopharyngeal carcinoma (NPC) specimens using immunohistochemistry and immunoblotting. Data are correlated with the patients' clinicopathological parameters, including the clinical stage, presence of intracranial invasion, presence of lymph node or distant metastasis, and histological grading. The E-cadherin/catenin complex is down-expressed in most of the samples examined. Correlation with clinicopathological parameters shows that expression of alpha- and beta-catenin is associated with the occurrence of intracranial invasion.     

Distribution and expression of CD44 isoforms and Ezrin during prostate cancer-endothelium interaction

CD44 is a multifunctional cell surface adhesion molecule that has been implicated in tumour cell invasion and metastasis. Many cancer cell types as well as their metastases express high levels of CD44. Furthermore, the expression of certain CD44 variants has been linked with metastasis and tumour progression. It is known that ezrin, a member of the ERM family of proteins, can bind to CD44 and thus raises the possibility that it is involved in cell migration and metastasis. Therefore we examined the expression and distribution of CD44, its co-localisation and translocation with ezrin in prostate cancer cell lines as they interact with endothelial cells. Experimental results indicate prostate cancer cells express multiple CD44 isoforms that co-localise with ezrin in DU-145 and PC-3 prostate cancer cells. Treatment with hepatocyte growth factor (HGF/SF) resulted in up-regulation of CD44 and its co-translocation with ezrin during tumour-endothelial cell interactions. In addition, tumour cell adhesion to endothelial cells and their invasiveness was increased after exposure to HGF/SF, and can be blocked by the presence of anti-CD44 antibodies. It is concluded that CD44 and ezrin interact in endothelial cells and that they co-localise in the areas of tumour-endothelial contact. The CD44/ezrin complex plays a pivotal role in the capture and invasion of endothelial cells by prostate cancer cells.     

Src family kinase inhibitor PP2 restores the E-cadherin/catenin cell adhesion system in human cancer cells and reduces cancer metastasis.

The E-cadherin/catenin cell adhesion system is often down-regulatedin epithelial tumors. This is thought to play an important role in cancer invasion and metastasis. Restoring this system may enable suppression of the metastatic spread of cancer. This study examined the effect of Src family kinase inhibitor PP2 on E-cadherin-mediated cell-cell adhesion and metastatic potentials. In cell aggregation assays, PP2 stimulated the aggregation of colon, liver, and breast cancer cells. In vitro cultures of cancer cells showed that PP2 induced strong cell-cell contact. Immunoblot analysis showed that PP2 enhanced E-cadherin/catenin expression and that increased E-cadherin/catenin proteins were strongly associated with the actin cytoskeleton. Northern blot studies indicated that the observed increase of E-cadherin/catenin protein content was due to their increased gene expression. After the spleens of severe combined immunodeficient mice were inoculated with cancer cells, treatment with PP2 for 3 weeks markedly reduced the rate of liver metastasis, compared with the control counterparts. Our data demonstrate that PP2 can activate the functioning of the E-cadherin-mediated cell adhesion system, which is associated with the suppression of metastasis in cancer cells. Thus, selective inhibition of Src activation may be potentially useful in the prevention of cancer metastasis.     

The influence of CD44v3-v10 on adhesion, invasion and MMP-14 expression in prostate cancer cells

The expression of certain CD44 variants has been linked with metastasis and tumour progression. In particular, high molecular weight forms of CD44 show restricted expression in tumours and may correlate with tumour development and metastasis. In this study, we examined the expression of CD44 variants in prostate cancer cell lines: the invasive PC-3 and DU-145, low invasive LNCaP, and two non-invasive prostate epithelial cell lines. PC-3 prostate cancer cells were transfected with a high molecular weight CD44 variant isoform, CD44v3-v10, isolated from non-invasive prostate epithelial cell lines. These transfected cells (PC-NIVO) were assessed using in vitro invasion, tumour-endothelial, growth, and migration assays. The expression of MMP-14 was examined using SDS-PAGE and Western blot analysis. Transfected PC-3 cells (PC-NIVO) were found to be less adherent to endothelial cells and had significantly reduced invasiveness compared to wild-type PC-3 or control cells. In addition, tumour cell adhesion to endothelial cells and invasiveness was increased after exposure to HGF/SF, and can be blocked by the presence of anti-CD44 antibodies. Further investigation revealed a reduction in the expression of MMP-14 in PC-NIVO cells, but not in PC-3 or control cells. In conclusion, non-invasive prostate epithelial cells express a high molecular weight CD44 isoform, CD44v3-v10, which may counteract the standard isoform function of CD44 by reducing adhesion and invasion of endothelium by prostate tumour cells through negation of the MMP-14 function.     

Immunohistological analysis of E-cadherin, alpha-, beta- and gamma-catenin expression in colorectal cancer: implications for cell adhesion and signaling

Intercellular adhesion mediated by the E-cadherin/catenin complex is a prerequisite for epithelial integrity and differentiation. In carcinomas, E-cadherin function is frequently disturbed, and has been suggested to increase invasion and metastasis of tumour cells. beta-catenin has also been implicated in signaling pathways essential for tumour formation. We analysed the E-cadherin/catenin adhesion system of colorectal tumours at different clinical stages. In primary carcinomas (n = 91), there was a frequent reduction in E-cadherin (44%) and alpha-catenin expression (36%). In contrast, beta-catenin and gamma-catenin expression were seldom reduced (4% and 15%, respectively). Similar expression patterns were observed in liver metastases from unrelated colorectal tumours (n = 27). There was a significant relationship between loss of E-cadherin and alpha-catenin expression and poorly differentiated (G3-4) tumours. Our results suggest that reduction of E-cadherin/alpha-catenin expression is a frequent event in primary and metastatic colorectal carcinomas. Furthermore, beta-catenin expression remains normal in colorectal cancer, suggesting the essential role of beta-catenin in signaling pathways.     

Ras Farnesylation Inhibitor FTI-277 Restores the E-Cadherin/Catenin Cell Adhesion System in Human Cancer Cells and Reduces Cancer Metastasis

The E-cadherin/catenin cell adhesion system is often down-regulated in epithelial tumors. This is thought to play an important role in cancer invasion and metastasis, and restoration of this system may suppress metastatic spread of cancer. In this study, the effects of a Ras farnesylation inhibitor (FTI-277) on E-cadherin-mediated cell-cell adhesion and metastatic potential were examined. In cell aggregation assays, FTI-277 stimulated aggregation of colon, liver and breast cancer cells. In vitro cultures of cancer cells showed that FIT-277 induced strong cell-cell contact. Immunoblotting analysis showed that FTI-277 increased E-cadherin/catenin (α, β and γ) expression and strongly stabilized E-cadherin/catenin with the actin cytoskeleton. Northern blotting studies indicated that the observed increase in the E-cadherin/catenin protein content was due to increased expression of their genes. After inoculation of the spleens of mice with severe combined immunodeficiency (SCID) with cancer cells, FTI-277 treatment for 3 weeks markedly reduced splenic primary tumor growth and the rate of liver metastasis compared with control counterparts. Our data demonstrate that FTI-277 can activate functioning of the E-cadherin-mediated cell adhesion system, which is associated with suppression of cancer cell metastasis. Therefore, selective inhibition of Ras activation may be useful for preventing cancer metastasis.     

HER2 and COX2 expression in human prostate cancer

COX2 and HER2 expression are associated with a poor prognosis in prostate cancer and HER2 has been linked to COX2 expression in colorectal cancer. The association between COX2 and HER2 expression was investigated in 117 patients with prostate cancer (89) or Benign prostatic hyperplasia (BPH) (28). Tissue was analysed for HER2 amplification by fluorescent in situ hybridisation, and HER2 and COX2 protein expression by immunohistochemistry (IHC). All tumours analysed expressed COX2 at a significantly higher level than BPH tissue (P=0.041). Only low levels of HER2 gene amplification (8%, 7/89) and HER2 protein expression (12%, 11/89) were observed. HER2 protein expression was rarely observed and did not correlate with HER2 amplification or COX2 expression. Although HER2 does not drive COX2 expression in prostate cancer, this study identified high levels of COX2 expressed in locally advanced prostate cancer, suggesting COX2 could be a potential therapeutic target. COX2 inhibitors are currently being used in clinical trials for the treatment of other tumour types.     

Ras farnesylation inhibitor FTI-277 restores the E-cadherin/catenin cell adhesion system in human cancer cells and reduces cancer metastasis.

The E-cadherin/catenin cell adhesion system is often down-regulated in epithelial tumors. This is thought to play an important role in cancer invasion and metastasis, and restoration of this system may suppress metastatic spread of cancer. In this study, the effects of a Ras farnesylation inhibitor (FTI-277) on E-cadherin-mediated cell-cell adhesion and metastatic potential were examined. In cell aggregation assays, FTI-277 stimulated aggregation of colon, liver and breast cancer cells. In vitro cultures of cancer cells showed that FTI-277 induced strong cell-cell contact. Immunoblotting analysis showed that FTI-277 increased E-cadherin/catenin (alpha, beta and gamma) expression and strongly stabilized E-cadherin/catenin with the actin cytoskeleton. Northern blotting studies indicated that the observed increase in the E-cadherin/catenin protein content was due to increased expression of their genes. After inoculation of the spleens of mice with severe combined immunodeficiency (SCID) with cancer cells, FTI-277 treatment for 3 weeks markedly reduced splenic primary tumor growth and the rate of liver metastasis compared with control counterparts. Our data demonstrate that FTI-277 can activate functioning of the E-cadherin-mediated cell adhesion system, which is associated with suppression of cancer cell metastasis. Therefore, selective inhibition of Ras activation may be useful for preventing cancer metastasis.     

Prostate carcinoma and stem cells

Stem cells, as classically defined, are cells with a capacity to self-renew and to generate daughter cells that can differentiate down several cell lineages to form all of the cell types that are found in the mature tissue. Stem cells and tumour cells have many similar features, including infinite lifespan, self-renewal, multidrug resistance, telomerase expression and, in the instance of the prostate, androgen independence. Evidence supports a role for stem cells in the etiology of many types of cancer. The evolution of androgen-independent prostate carcinoma may reflect the emergence of stemlike prostate tumour cells. Because cancer may be a disease of stem cell lineages and Shh-Gli signalling controls the behaviour of precursors and of cells with stem cell properties in the mammalian tissues, prostate cancer might derive from inappropriate expansion of prostatic epithelial stem cell lineages caused by abnormal Shh-Gli function. This review attempts to integrate these recent results.     

Metformin inhibits prostate cancer cell proliferation,migration, and tumor growth through upregulation of PEDF expression

Metformin has been reported to inhibit the growth of various types of cancers, including prostate cancer. Yet the mode of anti-cancer action of metformin and the underlying mechanisms remain not fully elucidated. We hypothesized that the antitumorigenic effects of metformin are mediated through upregulation of pigment epithelium-derived factor (PEDF) expression in prostate cancer cells. In this report, metformin treatment significantly inhibited the proliferation and colony formation of prostate cancer cells, in a dose- and time-dependent manner. Meanwhile, Metformin markedly suppressed migration and invasion and induced apoptosis of both LNCaP and PC3 cancer cells. Metformin also reduced PC3 tumor growth in BALB/c nude mice in vivo. Furthermore, metformin treatment was associated with higher PEDF expression in both prostate cancer cells and tumor tissue. Taken together, metformin inhibits prostate cancer cell proliferation, migration, invasion and tumor growth, and these activities are mediated by upregulation of PEDF expression. These findings provide a novel insight into the molecular functions of metformin as an anticancer agent.     

Hyaluronan expression in gastric cancer cells is associated with local and nodal spread and reduced survival rate

Hyaluronan (HA), an extracellular high-molecular-mass polysaccharide, is supposed to be involved in the growth and progression of malignant tumours. We studied the cellular expression of HA in 215 operated stage I-IV gastric cancer patients using a specific biotinylated probe. Most (93%) of the tumours showed HA staining in their parenchyma, whereas the stroma inside and around the tumour was stained in every case. When HA expression was compared with the clinical and histological features of the tumours, a strong staining intensity in the tumour parenchyma was found to be associated with deep tumour invasion (pT3 or 4) and with mixed type of Laurén. A high proportion of HA-positive cells of all neoplastic cells was significantly associated with deep tumour invasion, nodal metastasis, positive lymphatic invasion, poor differentiation grade, as well as with inferior prognosis in univariate survival analysis. However, in multivariate analysis, only pT, pN, and vascular and lymphatic invasion emerged as independent predictors of survival in gastric cancer. The results indicate that ectopic HA expression is a frequent feature of gastric adenocarcinoma, and is associated with tumour progression and poor survival rate.     

MUC-1 gene is associated with prostate cancer death: a 20-year follow-up of a population-based study in Sweden

Anti-adhesion mucins have proven to play an important part in the biology of several types of cancer. Therefore, we test the hypothesis that altered expression of MUC-1 is associated with prostate cancer progression. We retrieved archival tumour tissue from a population-based cohort of 195 men with localised prostate cancer (T1a-b, Nx, M0) that has been followed for up to 20 years with watchful waiting. Semi-automated, quantitative immunohistochemistry was undertaken to evaluate MUC-1 expression. We modelled prostate cancer-specific death as a function of MUC-1 levels accounting for age, Gleason grade and tumour extent, and calculated age-adjusted and multivariate adjusted hazard ratios (HR). Men that had tumours with an MUC-intensity lower or higher than normal tissue had a higher risk of dying in prostate cancer, independent of tumour extent and Gleason score (HR 5.1 and 4.5, respectively). Adjustment for Gleason grade and tumour stage did not alter the results. Men with a Gleason score > or =7 and MUC-1 deviating from the normal had a 17 (RR=17.1 95% confidence interval=2.3-128) times higher risk to die in prostate cancer compared with men with Gleason score <7 and normal MUC-1 intensity. In summary, our data show that MUC-1 is an independent prognostic marker for prostate cancer death.     

Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy

High activity of histone deacetylases (HDACs) causes epigenetic alterations associated with malignant cell behaviour. Consequently, HDAC inhibitors have entered late-phase clinical trials as new antineoplastic drugs. However, little is known about expression and function of specific HDAC isoforms in human tumours including prostate cancer. We investigated the expression of class I HDACs in 192 prostate carcinomas by immunohistochemistry and correlated our findings to clinicopathological parameters including follow-up data. Class I HDAC isoforms were strongly expressed in the majority of the cases (HDAC1: 69.8%, HDAC2: 74%, HDAC3: 94.8%). High rates of HDAC1 and HDAC2 expression were significantly associated with tumour dedifferentiation. Strong expression of all HDACs was accompanied by enhanced tumour cell proliferation. In addition, HDAC2 was an independent prognostic marker in our prostate cancer cohort. In conclusion, we showed that the known effects of HDACs on differentiation and proliferation of cancer cells observed in vitro can also be confirmed in vivo. The class I HDAC isoforms 1, 2 and 3 are differentially expressed in prostate cancer, which might be important for upcoming studies on HDAC inhibitors in this tumour entity. Also, the highly significant prognostic value of HDAC2 clearly deserves further study.     

Epigenetic DNA-methylation regulation of genes coding for lipid raft-associated components: a role for raft proteins in cell transformation and cancer progression (review)

Metastatic progression is the cause of most cancer deaths. Host tumour cell separation (fission) is accompanied by simultaneous acquisition of migrating capability of cancer cells, remodeling of cellular architecture and effective 'homing' in body host environment. Cell remodeling involves cytoskeletal protein-protein and lipid-protein interaction together with altered signaling. Alteration of signaling in tumour cells may affect expression of many genes also by DNA-methylation/demethylation. This would alter the steady-state intracellular level of structural proteins or metabolic enzymes, and notably enzymes involved in the biosynthesis of lipids, affecting the composition of membranes. Lipid rafts are small, heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can be stabilized to form larger platforms through protein-protein and protein-lipid interactions. Lipid rafts play an important role in intracellular protein transport, membrane fusion and trans-cytosis, also being platforms for cell surface antigens and adhesion molecules which are crucial for cell activation, polarization and signaling. Detachment of individual tumour cells from the host tumour lump requires lipid-protein-lipid raft (LPLR) reordering. Lipid rafts are also involved in angiogenesis and local invasion, which occurs within the host tumour vicinity by exchange of enzymes, cytokines and motility factors that modify the surrounding extracellular matrix (ECM). Many cell surface adhesion, ECM, and signaling proteins (such as E-cadherin, catenin, CD44, MMP-9 and caveolin-1) are known to be absent or reduced following gene promoter-CpG-island hypermethylation in mid-stage growing tumours, but re-expressed (by gene promoter-mCpG-DNA demethylation) in carcinomas such as metastasized lung, prostate and sarcomas. The recent research acquisitions on lipid rafts have tremendous implications in understanding the genetic and biochemical bases of metastatic diffusion of cancer.