J. Kilian Mellon

C-erbB-2 in bladder cancer: Molecular biology, correlation with epidermal growth factor receptors and prognostic value

PURPOSE To study the c-erbB-2 oncogene in primary transitional cell bladder cancer. MATERIALS AND METHODS Ninety-five patients with known clinical follow-up and epidermal growth factor receptor (EGFr) status were studied for expression of c-erbB-2 by immunostaining. Possible mechanisms underlying increased staining for c-erbB-2 protein were investigated by analyzing DNA and RNA encoding c-erbB-2. RESULTS Strong positive staining for c-erbB-2 was detected in 20 (21%) tumors, with weaker staining in a further 13 (14%). There was no correlation between increased staining for c-erbB-2 and tumor stage, grade, or EGFr status. There was a low rate of amplification of the c-erbB-2 gene (1 of 24) on Southern blotting with a higher rate of elevated c-erbB-2 mRNA (4 of 44) with dot blot hybridization. For pT1 tumors, the rate of recurrence was higher for those tumors which were positive for c-erbB-2. CONCLUSIONS c-erbB-2 oncoprotein is expressed by a significant proportion of transitional cell tumors of the bladder. In this study, the prognostic significance of c-erbB-2 expression appears limited.

Expression of S100A4 protein is associated with metastasis and reduced survival in human bladder cancer

The calcium‐binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer and its expression correlates strongly with reduced survival in human breast cancer. The expression of S100A4 in normal bladders and 101 bladder tumours has been studied using immunocytochemistry. Moderate or strong expression of S100A4 was found in 28% of the tumours, whilst the remaining tumours and normal urothelium either failed to stain or showed weak staining. S100A4 staining was more frequently observed in invasive bladder tumours than in non‐invasive tumours (p<0.05). In invasive tumours, S100A4 staining was usually strongest in invasive regions and single infiltrating cells. Statistically significant associations were found between S100A4 expression and metastasis (p=0.0003) and reduced survival (p<0.0001). It is concluded that S100A4 expression may play an important role in bladder cancer and may identify a subgroup of patients at increased risk of metastasis who should be considered for adjuvant systemic therapy. Copyright

Transfection of S100A4 Produces Metastatic Variants of an Orthotopic Model of Bladder Cancer

The calcium-binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer, and its expression strongly correlates with reduced survival in human breast and bladder cancer. We have established an orthotopic model of bladder cancer by injecting a cell line derived from a carcinogen-induced rat bladder tumor into the muscular wall of syngeneic rats. MYU-3L cells produce rapidly growing, invasive tumors in the bladder wall but they fail to metastasize. Transfection of MYU-3L cells with a plasmid vector directing overexpression of the S100A4 gene generates variants in which S100A4 expression is elevated by up to sevenfold in comparison with the untransfected cells. Variants overexpressing S100A4 produce primary tumors at similar frequencies and latencies to the parental cell line, a significant number of which metastasize to the para-aortic lymph nodes or lungs. Expression of S100A4 protein in the primary tumors was heterogeneous, but was stronger and more consistent in the metastases, suggesting that transfectants overexpressing S100A4 possess an enhanced ability to form metastatic lesions. We conclude that overexpression of S100A4 can induce the metastatic phenotype in this rodent model of bladder cancer. Taken together with the results from our parallel studies of human bladder cancer, these data suggest a significant role for S100A4 in bladder cancer metastasis and identify a potential new target for systemic therapy in patients with this disease.

Inhibition of STAT Signalling in Bladder Cancer by Diindolylmethane - Relevance to Cell Adhesion, Migration and Proliferation

Effective treatments to prevent recurrence or progression of non-muscle-invasive bladder cancer, or to inhibit metastasis of muscle-invasive forms of the disease, would deliver significant patient benefit. Here the involvement of STAT signalling and the chemopreventive potential of diindolylmethane (DIM) in human bladder cancer were investigated. Muscle-invasive bladder cancer tissues were characterised by nuclear expression of phosphorylated STAT1, 3 and 5. In E-cadherin positive tumour cell lines (RT112, RT4, HT1376), STAT5 was constitutively phosphorylated, while E-cadherin negative lines (J82, T24, UMUC3) contained phosphoSTAT3. Knockdown of STAT3 induced G₀/G₁ arrest and inhibited adhesion in J82 cells. Knockdown of STAT1inhibited migration in J82 and RT112 lines. No significant increase in apoptosis was observed. In response to the Janus kinase inhibitor, AG490, RT112 and J82 cells initially underwent G₀/G₁ arrest, with RT112 cells subsequently exhibiting S phase arrest. Phosphorylation of STAT1(Tyr701), STAT3(Tyr705) and (Ser727) and STAT5(Tyr694) was inhibited by DIM, as was adhesion of J82 cells to collagen, an effect that was enhanced when STAT1 or 3 was reduced by siRNA. However, over-expression of STAT3C partially rescued the DIM inhibitory effect on collagen-mediated adhesion. Migration of both lines was inhibited by DIM, while transfection of constitutively active STAT3C enhanced migration of RT112 cells. DIM induced cell cycle arrest and apoptosis in three cell lines with different degrees of radioresistance. Taken together, these results suggest that inhibition of STAT signalling and/or treatment with DIM may decrease invasiveness of bladder cancer. DIM can induce apoptosis in cell lines which are radioresistant, so in combination with radiotherapy may be useful in overcoming such resistance.