Restoration of TGF-beta signalling reduces tumorigenicity in human lung cancer cells

Members of the transforming growth factor-beta (TGF-beta) family regulate a wide range of biological processes including cell proliferation, migration, differentiation, apoptosis, and extracellular matrix deposition. Resistance to TGF-beta-mediated tumour suppressor function in human lung cancer may occur through the loss of type II receptor (TbetaRII) expression. In this study, we investigated the expression pattern of TbetaRII in human lung cancer tissues by RT-PCR and Western blot analyses. We observed downregulation of TbetaRII in 30 out of 46 NSCLC samples (65%) by semiquantitative RT-PCR. Western blot analyses with tumour lysates showed reduced expression of TbetaRII in 77% cases. We also determined the effect of TbetaRII expression in lung adenocarcinoma cell line (VMRC-LCD) that is not responsive to TGF-beta due to lack of TbetaRII expression. Stable expression of TbetaRII in these cells restored TGF-beta-mediated effects including Smad2/3 and Smad4 complex formation, TGF-beta-responsive reporter gene activation, inhibition of cell proliferation and increased apoptosis. Clones expressing TbetaRII showed reduced colony formation in soft-agarose assay and significantly reduced tumorigenicity in athymic nude mice. Therefore, these results suggest that reestablishment of TGF-beta signalling in TbetaRII null cells by stable expression of TbetaRII can reverse malignant behaviour of cells and loss of TbetaRII expression may be involved in lung tumour progression.     

Poly(A) RT-PCR measurement of diagnostic genes in pancreatic juice in pancreatic cancer

Background:

The last decade has seen significant progress in understanding the molecular biology of pancreatic adenocarcinoma. There is now an urgent need to translate these molecular techniques to clinical practice in order to improve diagnosis and prediction of response to treatment. The objectives of this study are to utilise poly(A) RT–PCR to measure expression levels of diagnostic Indicator genes, selected from microarray studies, of RNA from intraoperatively sampled pancreatic ductal juice and to correlate these expression levels with those in matched pancreatic tissue resection samples.

Methods:

Intraoperative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreaticoduodenectomy for suspected tumour. RNA was isolated and poly(A) PCR and real-time PCR used to measure expression levels of 30 genes. Spearman''s rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue.

Results:

Of the 30 Indicator genes measured, just one, ANXA1, showed a significant correlation of expression level between pancreatic juice and tissue samples, whereas three genes, IGFBP3 (P⩽0.035), PSCA (P⩽0.001) and SPINK1 (P⩽0.05), showed significantly different expression between cancerous and benign pancreatic tissue samples.

Conclusions:

These results demonstrate that RNA analysis of pancreatic juice is feasible using the poly(A) cDNA technique, that correlation of gene expression exists between pancreatic juice and tissue for very few genes and that gene expression profiling can distinguish between benign and malignant pancreatic tissue. This indicates possible use of the technique for measurement of Indicator genes in pancreatic tissue for diagnosis of pancreatic cancer from very small tissue samples.     

Cyclooxygenase-2 overexpression abrogates the antiproliferative effects of TGF-beta

The influence of cyclooxygenase-2 (COX-2) overexpression on the development of tumours has been well documented. The underlying mechanism however has still not been completely elucidated. An escape of proliferating cells from the regulatory influence of TGF-beta for example in the intestine has been discussed as well as a preponderance or prolongation of growth factor stimulation. The experiments presented here demonstrated that COX-2 transfection of a TGF-beta-sensitive cell line abrogates the growth inhibitory effects of TGF-beta. However, analysis of the TGF-beta/Smad-signalling pathway clearly revealed that COX-2 overexpression did not interfere with that. Neither TGF-receptor expression nor Smad phosphorylation and signal transfer into the nucleus were influenced by COX-2 overexpression. In addition, a TGF-beta reporter assay revealed no difference between controls and COX-2-transfected cells. Thus, the proliferation inhibiting effects must have been well compensated by growth-inducing stimuli. Indications for this came from experiments showing an induction of TGF-alpha expression and secretion with a higher and prolonged stimulation of the ERK 1/2 (p42/44) pathway in COX-2 transfectants. This effect could have been triggered by direct prostaglandin receptor stimulation or changes in intracellular lipid mediators. An increase in PPAR signalling as proven by a reporter assay is indication for the latter. Therefore, inhibiting both COX-2 as well as the PPAR and TGF/EGF pathway could be effective in the inhibition of adenoma or even carcinoma development in the intestine.     

TGF-beta1 genotype and phenotype in breast cancer and their associations with IGFs and patient survival

Transforming growth factor-beta (TGF-beta)-mediated signals play complicated roles in the development and progression of breast tumour. The purposes of this study were to analyse the genotype of TGF-beta1 at T29C and TGF-beta1 phenotype in breast tumours, and to evaluate their associations with IGFs and clinical characteristics of breast cancer. Fresh tumour samples were collected from 348 breast cancer patients. TGF-beta1 genotype and phenotype were analysed with TaqMan and ELISA, respectively. Members of the IGF family in tumour tissue were measured with ELISA. Cox proportional hazards regression analysis was performed to assess the association of TGF-beta1 and disease outcomes. Patients with the T/T (29%) genotype at T29C had the highest TGF-beta1, 707.9 pg mg(-1), followed by the T/C (49%), 657.8 pg mg(-1), and C/C (22%) genotypes, 640.8 pg mg(-1), (P=0.210, T/T vs C/C and C/T). TGF-beta1 concentrations were positively correlated with levels of oestrogen receptor, IGF-I, IGF-II and IGFBP-3. Survival analysis showed TGF-beta1 associated with disease progression, but the association differed by disease stage. For early-stage disease, patients with the T/T genotype or high TGF-beta1 had shorter overall survival compared to those without T/T or with low TGF-beta1; the hazard ratios (HR) were 3.54 (95% CI: 1.21-10.40) for genotype and 2.54 (95% CI: 1.10-5.89) for phenotype after adjusting for age, grade, histotype and receptor status. For late-stage disease, however, the association was different. The T/T genotype was associated with lower risk of disease recurrence (HR=0.13, 95% CI: 0.02-1.00), whereas no association was found between TGF-beta1 phenotype and survival outcomes. The study suggests a complex role of TGF-beta1 in breast cancer progression, which supports the finding of in vitro studies that TGF-beta1 has conflicting effects on tumour growth and metastasis.     

Enhanced VEGF production and decreased immunogenicity induced by TGF-beta 1 promote liver metastasis of pancreatic cancer

TGF-betas are multifunctional polypeptides that regulate cell growth and differentiation, extracellular matrix deposition, cellular adhesion properties, angiogenesis and immune functions. In this study, we investigated the effect of TGF-beta1 on liver metastasis and its mechanism by using human pancreatic cancer cell lines Panc-1, Capan-2, and SW1990. Capan-2 and SW1990 cells demonstrated enhanced liver metastatic potential by in vivo splenic injection with TGF-beta1. Consequently, we examined the role of TGF-beta1 on in vitro angiogenesis and received cytotoxicity by peripheral blood mononuclear leukocytes (PBMLs). While TGF-beta1 slightly decreased cell proliferation, it also upregulated VEGF production in all cancer cells examined. The binding of PBMLs to cancer cells and cancer cell cytotoxicity during co-culture with PBMLs were remarkably decreased by treatment with TGF-beta1. Panc-1 cells revealed no liver metastasis despite their high immunogenetic and angiogenetic abilities, which was attributed to a lack of expression of the cell surface carbohydrates that induce attachment to endothelial cells. We concluded that the presence of TGF-beta1 in the microenvironment of tumour site might play an important role in enhancing liver metastasis of pancreatic cancer by modulating the capacity of angiogenesis and immunogenicity.     

Expression of PPARdelta in multistage carcinogenesis of the colorectum: implications of malignant cancer morphology

Whether peroxisome proliferator-activated receptor (PPAR) delta is a good target for the chemoprevention and/or treatment of colorectal cancer (CRC) remains controversial. Our goal was to examine PPARdelta expression in multistage carcinogenesis of the colorectum and to assess the relevance of PPARdelta in CRC. Immunohistochemical analysis indicated that PPARdelta expression increased from normal mucosa to adenomatous polyps to CRC. In cancer tissues, the PPARdelta protein was accumulated only in those cancer cells with highly malignant morphology, as represented by a large-sized nucleus, round-shaped nucleus, and presence of clear nucleoli. Interestingly, the cancer tissue often contained both PPARdelta-positive and -negative areas, each retaining their respective specific morphological features. Moreover, this pattern persisted even when PPARdelta-positive and -negative cells were aligned next to each other within a single cancer nest or gland and was present in the majority of CRC cases. Immunohistochemistry for Ki-67 proliferation marker showed no significant correlation between Ki-67 and PPARdelta in CRC samples. Based on Western blot analysis and quantitative RT-PCR, high PPARdelta protein expression correlated with high PPARdelta mRNA levels. Peroxisome proliferator-activated receptor delta may have a supporting role in tumorigenesis, and the close association between PPARdelta expression and malignant morphology of CRC cells suggests a pivotal role in cancer tissue.     

Prognostic value of maspin mRNA expression in ER alpha-positive postmenopausal breast carcinomas

Maspin, a member of the serpin family, has a role in cell migration, angiogenesis and apoptosis. Little is known of the clinical significance of maspin gene expression in human cancers. We developed a real-time quantitative RT-PCR assay to quantify the full range of maspin mRNA copy numbers in a series of 10 ER alpha-positive and 10 ER alpha-negative breast tumours. We observed a statistical link between low maspin mRNA levels and positive oestrogen status (P=0.0012). In consequence, to better assess the prognostic value of maspin gene expression in breast cancer, we then quantified maspin mRNA content in an additional independent well-defined cohort of 105 ER alpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. Maspin expression varied widely in tumour tissues (by nearly four orders of magnitude), being underexpressed in 33 out of 105 tumours (31.4%) and overexpressed in 24 out of 105 tumours (22.9%) relative to normal breast tissues. Immunohistochemical studies demonstrated that maspin protein was strictly expressed in myoepithelial cells of normal breast tissue and in tumour epithelial cells, exclusively in maspin-overexpressing tumours. Patients with tumours overexpressing the maspin gene had significantly shorter relapse-free survival after surgery than patients whose tumours normally expressed or underexpressed maspin (P=0.0011). The prognostic significance of maspin overexpression persisted in Cox multivariate regression analysis (P=0.0024). These findings show that the maspin mRNA level can have important prognostic significance in human breast cancer, and point to the maspin gene as a putative molecular predictor of hormone responsiveness in breast cancer.     

DNA topoisomerase IIalpha expression and the response toprimary chemotherapy in breast cancer

The alpha isoform of Topoisomerase IIalpha (Topo IIalpha) is a proliferation marker as well as a target for several chemotherapeutic agents such as anthracyclines. In vitro studies have demonstrated the relationship between the Topo IIalpha expression level and chemosensitivity of target cancer cells. To verify this effect in vivo, we selected 125 patients presenting with T(2)>3 cm and T(3) N(0-1) M(0) breast tumours who were treated by six cycles of primary chemotherapy, including epirubicin before any surgery. Therapy response was assessed by clinical and X-ray mammogram measurements of tumour shrinkage. The pretherapeutic core biopsies were immunostained with a monoclonal antibody (Ki-S7) against Topo IIalpha. Ki-S7 positivity ranged from 0 to 50% (median, 15%). A high percentage of Ki-S7-positive cells (>15%) was associated with tumour regression under chemotherapy (OR=2.88, CI: 1.3-6.4, P=0.004). Ki-S7 further emerged as an independent predictor of tumour regression (OR=3.34, CI: 1.41-7.93, P=0.006), together with tumour size of less than 40 mm (OR=3.82, CI: 1.58-9.25, P=0.002) and negative oestrogen receptor (ER) status (OR=3.35, CI: 1.43-7.86, P=0.005), in a multivariate analysis including tumour size, SBR grade, ER and PR status, Ki-67, p53 and Her-2/neu. Our clinical results confirm in vitro data on the relationship between Topo IIalpha expression and tumour chemosensitivity and thus may have important practical implications.     

MUC1 expression in primary and metastatic pancreatic cancer cells for in vitro treatment by (213)Bi-C595 radioimmunoconjugate

Control of micrometastatic pancreatic cancer remains a major objective in pancreatic cancer treatment. The overexpression of MUC1 mucin plays an important role in cancer metastasis. The aim of this study was to detect the expression of MUC1 in human primary tumour tissues and three pancreatic cancer cell lines (CAPAN-1, CFPAC-1 and PANC-1), and target MUC1-positive cancer cells in vitro using (213)Bi-C595 alpha-immunoconjugate (AIC). The expression of MUC1 on pancreatic tumour tissues and cancer cell lines was performed by immunohistochemistry and further confirmed by confocal microscope and flow cytometry analysis on the cell surface. Cytotoxicity of (213)Bi-C595 was tested by MTS assay. Apoptosis was documented using TUNEL assay. Overexpression of MUC1 was found in approximately 90% of tested tumour samples and the three pancreatic cancer cell lines. (213)Bi-C595 is specifically cytotoxic to pancreatic cancer cells in a concentration-dependent fashion. These results suggest that overexpression of MUC1 in pancreatic cancer is a useful target, and that the novel (213)Bi-C595 AIC selectively targets pancreatic cancer cells in vitro. (213)Bi-C595 may be a useful agent for the treatment of micrometastases or minimal residual disease (MRD) in pancreatic cancer patients with overexpression of MUC1 antigen.     

Endostatin expression in a pancreatic cell line is modulated by a TNFalpha-dependent elastase

Endostatin, an inhibitor of angiogenesis, is a 20 kDa fragment of the basement membrane protein, collagen XVIII. The formation of endostatin relies upon the action of proteases on collagen XVIII. TNFalpha, produced by activated macrophages, is a multifunctional proinflammatory cytokine with known effects on endothelial function. We postulated that TNFalpha may modulate the activities of proteases and thus regulate endostatin formation in pancreatic cells. Collagen XVIII/endostatin mRNA was expressed in one pancreatic cell line, SUIT-2, but not in BxPc-3. The 20 kDa endostatin was found in the cell-conditioned medium of SUIT-2 cells. Precursor forms only were found in the cells. Exogenous endostatin was degraded by cellular lysates of SUIT-2 cells. Elastase activity was found in cell extracts but not the cell-conditioned media of SUIT-2 cells. Incubation of SUIT-2 cells with TNFalpha increased intracellular elastase activity and also increased secretion of endostatin into the medium. We conclude that endostatin is released by SUIT-2 cells and that increases in intracellular elastase, induced by TNFalpha, are correlated with increased secretion. Endostatin is however susceptible to degradation by intracellular proteases and if tissue injury accompanies inflammation, endostatin may be degraded, allowing angiogenesis to occur.     

The role of versican isoforms V0/V1 in glioma migration mediated by transforming growth factor-beta2

Versican is a large chondroitin sulphate proteoglycan produced by several tumour cell types, including high-grade glioma. The increased expression of certain versican isoforms in the extracellular matrix (ECM) plays a role in tumour cell growth, adhesion and migration. Transforming growth factor-beta2 (TGF-beta2) is an important modulator of glioma invasion, partially by remodeling the ECM. However, it is unknown whether it interacts with versican during malignant progression of glioma cells. Here, we analysed the effect of TGF-beta2 on the expression of versican isoforms. The expression of versican V0/V1 was upregulated by TGF-beta2 detected by quantitative polymerase chain reaction and immunoprecipitation, whereas V2 was not induced. Using time-lapse scratch and spheroid migration assays, we observed that the glioma migration rate is significantly increased by exogenous TGF-beta2 and inhibited by TGF-beta2-specific antisense oligonucleotides. Interestingly, an antibody specific for the DPEAAE region of glycosaminoglycan-beta domain of versican was able to reverse the effect of TGF-beta2 on glioma migration in a dose-dependent manner. Taken together, we report here that TGF-beta2 triggers the malignant phenotype of high-grade gliomas by induction of migration, and that this effect is, at least in part, mediated by versican V0/V1.     

Down-regulation of TGF-beta1 production restores immunogenicity in prostate cancer cells

The objective of this study is to determine if a non-immunogenic Dunning's rat prostate cancer cell line, MATLyLu, can become immunogenic by reducing the endogenous production of TGF-beta1. An expression construct containing a DNA sequence in an antisense orientation to TGF-beta1 (TGF-beta1 antisense) was stably transfected into MATLyLu cells. Following transfection, cellular content of TGF-beta1 reduced from 70 to 10 pg per 2x10(4) cells and the rate of in vitro 3H-thymidine incorporation increased 3-5-fold. After subcutaneous injection of tumour cells into syngeneic male hosts (Copenhagen rats), the tumour incidence was 100% (15/15) for the wild type MATLyLu cells and cells transfected with the control construct, but only 43% (9/21, P< or =0.05) for cells transfected with TGF-beta1 antisense. However, when cells were injected into immunodeficient hosts (athymic nude rats), the incidence of tumour development was 100% (10/10) for both the wild type MATLyLu cells and cells transfected with the control construct and 90% (9/10) for cells transfected with TGF-beta1 antisense. These observations support the concept that MATLyLu cells are immunogenic, when the endogenous production of TGF-beta1 is down-regulated.     

Smad7 induces hepatic metastasis in colorectal cancer

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-beta type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-beta/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.