Expression of HER3, HER4 and their ligand heregulin-4 is associated with better survival in bladder cancer patients

Abstract

The epidermal growth factor system has been associated to prognosis in patients with bladder cancer based mainly on the expression of the epidermal growth factor (EGF) receptor 1 (EGFR) and HER2 and their activating ligands. Since limited information exists concerning the expression of other parts of the EGF system, we examined the expression of the receptors HER3 and HER4 and their activating ligands, the heregulins (HRGs), in bladder cancer patients. Biopsies from bladder cancer tumours were obtained from 88 patients followed for a median of 23 months (range, 1 -97 months). The mRNA content of four ligands and their isoforms (HRG1a, HRG1b, HRG2a, HRG2b, HRG3 and HRG4) and two receptors (HER3 and HER4) was quantified by real-time PCR. A significantly lower mRNA expression level of HER3 (P ¼ 0.0003), HRG2a (P ¼ 0.0159), HRG2b (P ¼ 0.0007) and HRG4 (Po0.0001) was observed in muscle-invasive (T2 -T4) tumours as compared to superficial (Ta) tumours. The expression of HER3 mRNA correlated strongly to overall survival (P ¼ 0.0042); increased expression of HER4 (P ¼ 0.0261) and HRG4 (P ¼ 0.0245) was also associated with better prognosis. Interestingly, patients with coexpression of HER3 (P ¼ 0.0034) or HER4 (P ¼ 0.0080) together with their stimulating ligand HRG4 showed even better survival than for HER3 or HER4 alone. Our results together with previous data suggest a dual face for the EGF system. While it is well established that an increased signalling through HER1 and HER2 is related to a poor prognosis, our data suggest that signalling through HER3 and HER4 is related to a favourable outcome in bladder cancer patients.

The relation between survival and expression of HER1 and HER2 depends on the expression of HER3 and HER4: a study in bladder cancer patients

“…However, growing consensus has emerged about the role of HER4, and the few clinical studies carried out so far have suggested that its expression is associated with favourable prognosis (Thybusch-Bernhardt et al, 2001;Suo et al, 2002;Rotterud et al, 2005). We have shown that HER3 and HER4 expression correlated with a better prognosis in bladder cancer patients (Memon et al, 2004). More recent reports also suggest that unlike HER1 and HER2, expression of HER3 and HER4 was not found in more aggressive metastatic squamous cell carcinomas of the oral cavity (Ekberg et al, 2005).…”

“…HER3 and HER4 mRNA expressions have previously been determined on the same samples (Memon et al, 2004). HER1 was expressed in 96% (85 out of 88 Table 2 Correlation between combinations of HER1 and HER3/HER4 and clinicopathological parameters of bladder cancer…”

“…RNA preparation was performed as described before (Memon et al, 2004). Briefly, tumour samples were immediately placed in a denaturing solution (4 mol l À1 guanidine thiocyanate, 25 mmol l À1 sodium citrates (PH7), 0.5% sarkosyl and 0.1 mmol l À1 2-mercaptoethanol) and stored at À801C.…”

“…Real-time reverse transcription -polymerase chain reaction quantification of mRNA Quantification of mRNA was performed by real-time reverse transcription -polymerase chain reaction on the Lightcycler instrument (Roche, Germany) as described before (Memon et al, 2004). Briefly, cDNA was generated in a reverse transcription reaction, where 1 mg RNA was mixed with 2.5 units AMW reverse transcriptase (Applied Biosytems, Foster city, USA) in a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 1 U ml À1 RNase inhibitor, 1 mmol l À1 deoxyribonucleoside triphosphate (dATP, dTTP, dGTP and dCTP), 2.5 mmol l À1 16 mer d(T) 16 primer, 50 mmol l À1 KCl, 6.25 mmol l À1 MgCl 2 in a total volume of 20 ml (all reagents from Applied Biosystem).…”

“…The HER1 primers: 5 0 -AGAGGAGAACTGCCAGAA-3 0 (sense) and 5 0 -GTAGCATTTATG GAGAGTG-3 0 (antisense); HER2 primers: 5 0 -CCAGGACCTGCT GAACTGGT-3 0 (sense) and 5 0 -GTACGAGCCGCACATCC-3 0 (antisense) give rise to a 454 and 272 bp PCR product, respectively. HER3 and HER4 quantifications were previously determined by same method by amplifying a 365 and 265 bp fragment, respectively (Memon et al, 2004). Beta-actin mRNA was used as an endogenous RNA control, which has been used as control gene in various studies on bladder cancer (Vageli et al, 1996;Chiu et al, 2002) as well as on breast cancer (Agudo et al, 2004).…”