Interleukin-6, tumour necrosis factor alpha and interleukin-1beta in patients with renal cell carcinoma

As regulators of malignant cell behaviour and communication with stroma, cytokines have proved useful in understanding cancer biology and developing novel therapies. In renal cell carcinoma, patients with inflammatory reactions are known to have poor prognosis. In order to elucidate the relation between renal cell carcinoma and the host, serum levels of inflammatory cytokines, interleukin-6, tumour necrosis factor alpha, interleukin-1beta, were measured. One hundred and twenty-two patients with renal cell carcinoma and 21 healthy control subjects were studied, and serum cytokine levels were measured using a highly sensitive ELISA kit. As a result, in the control group, interleukin-6, tumour necrosis factor alpha and interleukin-1beta levels were 1.79+/-2.03, 2.74+/-0.94 and 0.16+/-0.17 pg ml(-1), respectively. In the renal cell carcinoma patients, they were 8.91+/-13.12, 8.44+/-4.15 and 0.53+/-0.57 pg ml(-1), respectively, and significantly higher. In the comparison of stage, interleukin-6 level was significantly higher in the stage IV group compared to the other stage groups including the control group, while tumour necrosis factor alpha level was significantly higher in each stage group compared to the control group. As for grade, interleukin-6 level was significantly higher in the grade 3 group compared to the control, grade 1 and grade 2 groups, while tumour necrosis factor alpha level was significantly higher in each grade group compared to the control group. All cytokines had a positive correlation with tumour size. In regard to the correlation with CRP, all cytokines had a positive correlation with CRP, while interleukin-6 had a particularly strong correlation. In conclusion, interleukin-6 may be one of the factors for the poor prognosis of patients with renal cell carcinoma. In addition, tumour necrosis factor alpha may be useful in the early diagnosis of renal cell carcinoma and post-operative follow-up.     

Autocrine and paracrine motility factors and their involvement in invasiveness in a human oral carcinoma cell line.

Invasive potentials of malignant cancer cells are regulated by cell motility factors. To examine the regulation of motility and invasiveness in oral squamous carcinoma, we investigated autocrine- and/or paracrine-acting cell motility factors, using a newly established human cell line (IF cells) from oral squamous cell carcinoma, which has highly invasive and metastatic characteristics. Conditioned medium derived from IF cells stimulated cell scattering and migration of GB-d1 gallbladder carcinoma cells, indicating that IF cells secreted cell motility factors. Using antibodies, IF-derived cell motility factors proved to be transforming growth factor (TGF)-alpha and TGF-beta1. Antibodies against TGF-alpha and TGF-beta1 inhibited autonomous migration of the IF cells. On the other hand, in vitro invasion of IF cells was strongly enhanced by hepatocyte growth factor (HGF) but only slightly by TGF-alpha and TGF-beta1. The conditioned medium from fibroblasts enhanced in vitro invasion of IF cells, an event abrogated by anti-HGF antibody, but not by antibodies against TGF-alpha and TGF-beta1. Importantly, IF cells secreted a factor inducing HGF production in fibroblasts and the factor was identified as interleukin-1, which means that a mutual interaction exists between tumour cells and fibroblasts, as mediated by the HGF/HGF-inducer loop. These results indicate that IF cells utilize TGF-alpha and TGF-beta1 as autocrine-acting motility factors and HGF as a paracrine-acting motility factor, and that invasiveness of IF cells is particularly stimulated by HGF derived from stromal fibroblasts. Utilization of multiple cell motility/invasion factors that act in distinct pathways may confer highly invasive and metastatic potentials in IF oral squamous carcinoma cells.     

Prostaglandin production in mouse mammary tumour cells confers invasive growth potential by inducing hepatocyte growth factor in stromal fibroblasts.

Interactions between stromal and mammary tumour cells play a crucial role in determining the malignant behaviour of tumour cells. Although MMT mouse mammary tumour cells do not produce hepatocyte growth factor (HGF), addition of conditioned medium (CM) from MMT cells to cultures of human fibroblasts derived from skin and breast tissues stimulated the production of HGF, thereby indicating that MMT cells secrete an inducing factor for HGF. This HGF-inducing factor, purified from MMT-derived CM, proved to be prostaglandin E2 (PGE2). Consistently, treatment of MMT cells with indomethacin, an inhibitor of cyclooxygenase, abolished this HGF-inducing activity in MMT-derived CM, while treatment of MMT cells with HGF stimulated cell growth and cell motility. Likewise, HGF strongly enhanced urokinase-type plasminogen activator activity and invasion of MMT cells through Matrigel: a 15-fold stimulation in the invasion of MMT cells was seen by HGF. Finally, MMT cells in the upper compartment were co-cultivated with fibroblasts in the lower compartment of the Matrigel chamber, HGF levels in the co-culture system exceeded the level in fibroblasts alone and suppression occurred with exposure to indomethacin. Together with increase in the HGF level, the invasion of MMT cells was enhanced by co-cultivation with fibroblasts, whereas the increased invasion of MMT cells was significantly inhibited by an anti-HGF antibody and by indomethacin. These results indicate mutual interactions between MMT cells and fibroblasts: MMT-derived PGE2 plays a role in up-regulating HGF production in fibroblasts, while fibroblast-derived HGF leads to invasive growth in MMT cells. The mutual interactions mediated by HGF and prostaglandins may possibly be a mechanism regulating malignant behaviour of mammary tumour cells, through tumour-stromal interactions.     

In vitro Prostaglandin E2 production by glioblastoma cells and its effect on interleukin-2 activation of oncolytic lymphocytes

Summary Glioblastoma cells constitutively produce various amounts of PGEZ (prostaglandin E₂)in vitro. The amounts of PGE₂ found in the conditioned medium of the glioblastoma cultures (less than 5 ng/ml) were not enough to inhibit the IL-2 (interleukin-2) activation of peripheral blood lymphocytes. However the amount of PGE₂ produced by approximately 1 x 10⁷ of the glioblastoma cells can be assumed to suppress the generation of IL-2-induced killing activity against glioblastoma cells.     

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma

Background:

Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3β positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-κB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3β and to assess the anti-cancer effect of GSK-3β inhibition in RCC.

Methods:

Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3β in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT–PCR, BrDU incorporation and MTS assays to study the effect of GSK-3β inactivation on renal cancer cell proliferation and survival.

Results:

We detected aberrant nuclear accumulation of GSK-3β in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-κB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells.

Conclusions:

Our results show nuclear accumulation of GSK-3β as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.     

Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells

Fas ligand (FasL/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E(2) (PGE(2)), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE(2) increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E(2)-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE(2) positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE(2).     

Interleukin-2/interferon-alpha2a/13-retinoic acid-based chemoimmunotherapy in advanced renal cell carcinoma: results of a prospectively randomised trial of the German Cooperative Renal Carcinoma Chemoimmunotherapy Group (DGCIN)

We performed a prospectively randomised clinical trial to compare the efficacy of four subcutaneous interleukin-2-(sc-IL-2) and sc interferon-alpha2a (sc-IFN-alpha2a)-based outpatient regimens in 379 patients with progressive metastatic renal cell carcinoma. Patients with lung metastases, an erythrocyte sedimentation rate < or =70 mm h(-1) and neutrophil counts < or =6000 microl(-1) (group I) were randomised to arm A: sc-IL-2, sc-IFN-alpha2a, peroral 13-cis-retinoic acid (po-13cRA) (n=78), or arm B: arm A plus inhaled-IL-2 (n=65). All others (group II) were randomised to arm C: arm A plus intravenous 5-fluorouracil (iv-5-FU) (n=116), or arm D: arm A plus po-Capecitabine (n=120). Median overall survival (OS) was 22 months (arm A; 3-year OS: 29.7%) and 18 months (arm B; 3-year OS: 29.2%) in group I, and 18 months (arm C; 3-year OS: 25.7%) and 16 months (arm D; 3-year OS: 32.6%) in group II. There were no statistically significant differences in OS, progression-free survival, and objective response between arms A and B, and between arms C and D, respectively. Given the known therapeutic efficacy of sc-IL-2/sc-INF-alpha2a/po-13cRA-based outpatient chemoimmunotherapies, our results did not establish survival advantages in favour of po-Capecitabine vs iv-5-FU, and in favour of short-term inhaled-IL-2 in patients with advanced renal cell carcinoma receiving systemic cytokines.     

Expression of E-cadherin,alpha-catenin,and beta-catenin in the process of lymph node metastasis in oral squamous cell carcinoma

Regional lymph node metastasis is a very important prognostic indicator. In the metastatic process, reduction in cell to cell adhesion including E-cadherin-catenin cell adhesion complex is an essential step. We investigated immunohistochemical expression of E-cadherin, alpha-catenin and beta-catenin in 159 tissue samples from patients with oral squamous cell carcinoma and examined the correlation between their expressions and the presence of regional lymph node metastasis. Significantly greater reduction in expression levels of E-cadherin, alpha-catenin and beta-catenin was found in the metastatic group (n=64) compared to the nonmetastatic group (n=95) (P=0.007, 0.001, 0.001, respectively). However, there was no significant correlation between their expressions and the features of the regional metastasis, the number of metastatic lymph nodes or the presence of extracapsular metastasis. These data suggest that evaluation of the immunohistochemical expression of E-cadherin, alpha-catenin and beta-catenin is extremely valuable for the diagnosis of metastatic occurrence.     

Factor inhibiting HIF (FIH-1) promotes renal cancer cell survival by protecting cells from HIF-1α-mediated apoptosis

Background:

Clear cell renal cell carcinoma (CCRCC) is the commonest form of kidney cancer. Up to 91% have biallelic inactivation of VHL, resulting in stabilisation of HIF-α subunits. Factor inhibiting HIF-1 is an enzyme that hydroxylates HIF-α subunits and prevents recruitment of the co-activator CBP/P300. An important question is whether FIH-1 controls HIF activity in CCRCC.

Methods:

Human VHL defective CCRCC lines RCC10, RCC4 and 786–O were used to determine the role of FIH-1 in modulating HIF activity, using small interfering RNA knockdown, retroviral gene expression, quantitative RT–PCR, western blot analysis, Annexin V and propidium iodide labelling.

Results:

Although it was previously suggested that FIH-1 is suppressed in CCRCC, we found that FIH-1 mRNA and protein are actually present at similar levels in CCRCC and normal kidney. The FIH-1 inhibition or knockdown in the VHL defective CCRCC lines RCC10 and RCC4 (which express both HIF-1α and HIF-2α) resulted in increased expression of HIF target genes. In the 786-O CCRCC cell line, which expresses only HIF-2α, FIH-1 attenuation showed no significant effect on expression of these genes; introduction of HIF-1α resulted in sensitivity of HIF targets to FIH-1 knockdown. In RCC4 and RCC10, knockdown of FIH-1 increased apoptosis. Suppressing HIF-1α expression in RCC10 prevented FIH-1 knockdown from increasing apoptosis.

Conclusion:

Our results support a unifying model in which HIF-1α has a tumour suppressor action in CCRCC, held in check by FIH-1. Inhibiting FIH-1 in CCRCC could be used to bias the HIF response towards HIF-1α and decrease tumour cell viability.     

Integrin beta1 is required for the invasive behaviour but not proliferation of squamous cell carcinoma cells in vivo

Integrin beta1 is both overexpressed and in an 'active' conformation in vulval squamous cell carcinomas (VSCCs) compared to matched normal skin. To investigate the significance of integrin beta1 deregulation we stably knocked-down integrin beta1 expression in the VSCC cell line A431. In vitro analysis revealed that integrin beta1 is required for cell adhesion, cell spreading and invasion. However, integrin beta1 is not required for cell growth or activation of FAK and ERK signalling in vitro or in vivo. Strikingly, while control tumours were able to invade the dermis, integrin beta1 knockdown tumours were significantly more encapsulated and less invasive.     

Mutations in BHD and TP53 genes, but not in HNF1beta gene, in a large series of sporadic chromophobe renal cell carcinoma

BHD, TP53, and HNF1beta on chromosome 17 were studied in 92 cases of renal cell carcinoma (46 chromophobe, 19 clear cell, 18 oncocytoma, and nine papillary). Six, thirteen, and zero cases had, respectively BHD, TP53, and HNF1beta mutations, (84% mutations involved chromophobe), suggesting a role for BHD and TP53 in chromophobe subtype.     

In vivo assessment of the antiproliferative properties of interferon-alpha during immunotherapy: Ki-67 (MIB-1) in patients with metastatic renal cell carcinoma

The aim of the present study was to investigate the in vivo antiproliferative effect of interferon alpha (IFN-alpha) in patients with metastatic renal cell carcinoma (mRCC). Core needle biopsies of metastatic and/or the primary kidney cancer were obtained before interleukin-2 (IL-2)- and IFN-alpha-based immunotherapy in 34 patients and repeated after 5 weeks in 25 patients. Tumour proliferation was assessed by use of the anti-Ki-67 antibody MIB-1 and evaluated in multiple, random systematic sampled fields of vision. Ki-67 labelling index (LI) at baseline was median 13.6% (range 1.2-85.0) and median 10.6% (range 1.3-48.6%) at week 5 with a median overall decline of 15.2% (range -95 to +258%) from baseline to week 5. There was no difference between responding and nonresponding patients. Ki-67 LI at week 5 was significantly correlated to survival. Thus, median survival of patients with Ki-67 LI 10.6% (P=0.016). Baseline or change in Ki-67 LI did not correlate to survival. These data suggest that IFN-alpha in vivo has only modest effect on tumour proliferation in patients with mRCC. Tumour Ki-67 (MIB-1) reactivity after 1 month of immunotherapy appears to be a significant predictor of patient survival.     

Overexpression of beta2-microglobulin is associated with poor survival in patients with oral cavity squamous cell carcinoma and contributes to oral cancer cell migration and invasion

beta2-Microglobulin (beta2M), a component of MHC class I molecules, is believed to be associated with tumour status in various cancers. In this study, we examined the expression of beta2M at different malignant stages of oral cavity squamous cell carcinoma (OCSCC). To determine the possible correlation between beta2M expression and various clinical characteristics, 256 samples from patients with OCSCC were evaluated by immunohistochemical staining. Strong beta2M expression was significantly correlated with a relatively advanced tumour stage (P<0.001), positive nodal status (P<0.001), and TNM stage (P<0.001). The cumulative 5-year survival rate was significantly correlated with a relatively advanced tumour stage (P<0.001), positive nodal status (P<0.001), TNM stage (P<0.001), and strong expression of beta2M (P<0.001). Thus, elevated beta2M expression is an indicator of poor survival (P<0.001). In addition, we extended our analysis of beta2M expression to the FaDu and SCC25 oral cancer cell lines. beta2-Microglobulin expression was positively correlated with cell migration and invasion in beta2M-overexpressing transfectants in Transwell chambers. The suppression of beta2M expression using small interfering RNA (siRNA) was sufficient to decrease cell migration and invasion in vitro. Taken together, our results suggest that beta2M expression in the tissues is associated with survival and may be involved in tumour progression and metastasis in OCSCC.     

Immunochemotherapy with interleukin-2, interferon-alpha and 5-fluorouracil for progressive metastatic renal cell carcinoma: a multicenter phase II study. Dutch Immunotherapy Working Party

In patients with metastatic renal cell carcinoma response rates of 7-26% have been achieved with immunotherapy. A high response rate of 48% in 35 patients has been reported for treatment with the combination of interferon-alpha (IFN-alpha), interleukin-2 (IL-2) and 5-fluorouracil (5-FU) (Atzpodien et al (1993a) Eur J Cancer29A: S6-8). We conducted a multicentre phase II study to confirm these results. Metastatic renal cell carcinoma patients were treated as outpatients with an 8-week treatment cycle. Recombinant human IL-2 20 MU m(-2) was administered subcutaneously (s.c.) three times a week (t.i.w) in weeks 1 and 4 and 5 MU m(-2) t.i.w. in weeks 2 and 3. Recombinant human IFN-alpha 2a 6 MU m(-2) was administered s.c. once in weeks 1 and 4 and t.i.w. in weeks 2 and 3, and 9 MU m(-2) t.i.w. in weeks 5-8. 5-FU (750 mg m(-2)) was given as a bolus injection intravenous once a week in weeks 5-8. The treatment cycle was repeated once in case of response or minor response. Fifty-two patients entered the study. All had undergone a nephrectomy and had progressive metastatic disease. The median WHO-performance status was 1, the median number of metastatic sites was 2 (range 1-5) and the median time between the diagnosis of the primary tumour and the start of treatment was 12.9 months (range 1-153). Among the 51 patients, including four patients with early progressive disease, who were evaluable for response, the response rate was 11.8% (95% confidence interval (CI) 2.9-20.7%), with no complete responses. Median duration of response was 8.3 (range 3.8-22.4+) months. Median survival was 16.5 (range 1.8-30.5+) months. Grade 3/4 toxicity (WHO) occurred in 29/52 (55.8%) of the patients in cycle 1 and in 6/16 (37.5%) of the patients in cycle 2. It consisted mainly of anorexia, fatigue, nausea, fever and leucocytopenia. We cannot confirm the high response rate in patients with metastatic renal cell carcinoma treated with the combination of IFN-alpha, IL-2 and 5-FU, as described by Atzpodien et al.     

Expression of interferon-gamma in human adrenal gland and kidney tumours

It is known that interferon-gamma (IFN-gamma) is produced by activated T and NK lymphoid cells, mononuclear cells, and macrophage and dendritic cells. Our previous studies have shown that IFN-gamma-like immunoreactivity also appears in human adrenal cortical tumour and phaeochromocytoma. To investigate whether human tumour cells can produce IFN-gamma, we examined 429 biopsy specimens of 30 kinds of tumour and tumour-surrounding tissues in adrenal glands and in kidneys by using immunohistochemistry and in situ hybridisation. IFN-gamma immunoactivity was shown in 34.3% of the adrenal cortical adenomas, 50% of the adrenal cortical carcinomas, 26.7% of the phaeochromocytomas, 26.7% of the clear cell renal cell carcinomas (RCCs), 22% of the adrenal cortexes and 40% of medullas adjacent to tumours. The positive samples and expression areas were well overlapped between the IFN-gamma mRNA and the immunohistochemistry staining. Western blot analysis has further confirmed the immunohistochemistry results by showing a distinct IFN-gamma band corresponding to 17.4 kDa in tissue extracts from adrenal cortical adenoma, phaeochromocytoma and clear cell RCCs. These results indicate that IFN-gamma is produced by some types of tumour cells, suggesting it may play a dual role in the development of these tumours.     

Clinical implication of expression of cyclooxygenase-2 and peroxisome proliferator activated-receptor gamma in epithelial ovarian tumours

Expression of cyclooxygenase (COX)-2 plays a key role in tumorigenesis and development and peroxisome proliferator-activated receptor gamma (PPARgamma) has been implicated in the control of COX-2 expression in some tissues. The aim of this study is to investigate (1) whether expression of COX-2 and PPARgamma is associated with ovarian carcinogenesis and progression of ovarian tumours and (2) whether COX-2 expression is controlled through ligand-mediated activation of PPARgamma in ovarian carcinoma cells. For this purpose, the presence of COX-2 and PPARgamma was immunohistochemically examined in 71 epithelial ovarian carcinomas, 18 borderline tumours and 23 benign tumours and the levels of COX-2 and PPARgamma proteins were determined by enzyme immunoassay in four benign tumours, three borderline tumours and 12 carcinomas. The frequency of COX-2 and PPARgamma detection was significantly increased and decreased as lesions progressed to carcinoma, respectively. The COX-2 protein was not detected in the three borderline tumours, whereas PPARgamma protein was detected in all of them. COX-2 protein was detected in eight of the 12 carcinomas, whereas PPARgamma protein was detected in only two cases. In addition, PPARgamma protein was not detected in all of the eight carcinomas in which COX-2 protein was detected, suggesting that expression of PPARgamma and COX-2 was in a reciprocal relationship. Furthermore, in cultured ovarian carcinoma cells, Western blot revealed that PPARgamma and COX-2 expression was regulated conversely as a result of stimulation by 15-deoxy-Delta(12, 14) PGJ(2) (15-PGJ(2)), a PPARgamma activator. In addition, 15d-PGJ(2) suppressed tumour necrosis factor-alpha-induced-COX-2 expression, confirming the reciprocal correlation between COX-2 and PPARgamma. From these results, it was suggested that PPARgamma activation might suppress COX-2 expression via the nuclear factor-kappaB pathway in the ovarian carcinoma cells and that low expression of PPARgamma and high expression of COX-2 might be involved in carcinogenesis and progression of ovarian tumours.     

The free beta-subunit of human chorionic gonadotropin as a prognostic factor in renal cell carcinoma

The free beta-subunit of human chorionic gonadotropin beta is expressed in several nontrophoblastic tumours and this is usually associated with aggressive disease. Little is known about human chorionic gonadotropin beta expression in renal cancer. We determined the pretreatment levels of human chorionic gonadotropin beta in serum of patients with renal cell carcinoma, and studied whether elevated levels predicted the clinical outcome. Serum samples were collected before surgery from 177 patients with renal cell carcinoma and from 84 apparently healthy controls. Human chorionic gonadotropin beta in serum was measured by a highly sensitive time-resolved immunofluorometric assay. The prognostic value of human chorionic gonadotropin beta, and of usual clinical and pathological variables was analyzed by the Kaplan-Meier method, the log rank test and Cox multiple hazard regression. The serum concentrations of human chorionic gonadotropin beta were increased in 23% of the renal cell carcinoma patients and they were significantly higher in patients with renal cell carcinoma than in controls (P<0.0001). The concentrations did not correlate with clinical stage and histopathological grade, but patients with increased human chorionic gonadotropin beta levels had significantly shorter survival time than those with levels below the median (cut-off 1.2 pmol l(-1), P=0.0029). In multivariate analysis human chorionic gonadotropin beta, tumour stage and grade were independent prognostic variables. The serum concentration of human chorionic gonadotropin beta is an independent prognostic variable in renal cell carcinoma. The preoperative value of human chorionic gonadotropin beta in serum may be used to identify patents with increased risk of progressive disease.     

Cyclooxygenase-2 in tumor-associated macrophages promotes breast cancer cell survival by triggering a positive-feedback loop between macrophages and cancer cells

Tumor-associated macrophages (TAMs) play an important role in cancer cell survival, however, the mechanism of which remains elusive. In this study, we found that COX-2 was abundantly expressed in breast TAMs, which was correlated to poor prognosis in breast cancer patients. Ectopic over-expression of COX-2 in TAMs enhanced breast cancer cell survival both in vitro and in vivo. COX-2 in TAMs was determined to be essential for the induction and maintenance of M2-phenotype macrophage polarity. COX-2⁺ TAMs promoted breast cancer cell proliferation and survival by increasing Bcl-2 and P-gp and decreasing Bax in cancer cells. Furthermore, COX-2 in TAMs induced the expression of COX-2 in breast cancer cells, which in turn promoted M2 macrophage polarization. Inhibiting PI3K/Akt pathway in cancer cells suppressed COX-2⁺ TAMs-induced cancer cell survival. These findings suggest that COX-2, functions as a key cancer promoting factor by triggering a positive-feedback loop between macrophages and cancer cells, which could be exploited for breast cancer prevention and therapy.     

SLIT2 promoter methylation analysis in neuroblastoma, Wilms' tumour and renal cell carcinoma

The 3p21.3 RASSF1A tumour suppressor gene (TSG) provides a paradigm for TSGs inactivated by promoter methylation rather than somatic mutations. Recently, we identified frequent promoter methylation without somatic mutations of SLIT2 in lung and breast cancers, suggesting similarities between SLIT2 and RASSF1A TSGs. Epigenetic inactivation of RASSF1A was first described in lung and breast cancers and subsequently in a wide range of human cancers including neuroblastoma, Wilms' tumour and renal cell carcinoma (RCC). These findings prompted us to investigate SLIT2 methylation in these three human cancers. We analysed 49 neuroblastomas (NBs), 37 Wilms' tumours and 48 RCC, and detected SLIT2 promoter methylation in 29% of NB, 38% of Wilms' tumours and 25% of RCC. Previously, we had demonstrated frequent RASSF1A methylation in the same tumour series and frequent CASP8 methylation in the NB and Wilms' tumour samples. However, there was no significant association between SLIT2 promoter methylation and RASSF1A or CASP8 methylation in NB and RCC. In Wilms' tumour, there was a trend for a negative association between RASSF1A and SLIT2 methylation, although this did not reach statistical significance. No associations were detected between SLIT2 promoter methylation and specific clinicopathological features in the tumours analysed. These findings implicate SLIT2 promoter methylation in the pathogenesis of both paediatric and adult cancers and suggest that further investigations of SLIT2 in other tumour types should be pursued. However, epigenetic inactivation of SLIT2 is less frequent than RASSF1A in the tumour types analysed.