Morag Seywright

GRP78 up-regulation is associated with androgen receptor status, Hsp70–Hsp90 client proteins and castrate-resistant prostate cancer†

GRP78/BiP is a key member of the molecular chaperone heat shock protein (Hsp) 70 family. It has a critical role in prostate cancer (PC) including Pten loss‐driven carcinogenesis, but the molecular basis of this remains unclear. We investigated the effect of GRP78 and its putative client proteins, including androgen receptor (AR) in clinical PC. Expression of GRP78 and key Hsp70–hsp90 client proteins (HER2, HER3, AR and AKT) were studied in an incidence tissue microarray (TMA) of prostate cancer. The relationship of GRP78 and AR was further tested in in vitro cell models (LNCaP and its derived LNCaP‐CR subclone) and a matched TMA of hormone‐naïve (HNPC) and castrate‐resistant prostate cancer (CRPC). In vitro and in vivo expression of GRP78 and client proteins were assessed by western blotting and immunohistochemistry, respectively, using the weighted histoscore method. Significant co‐expression of GRP78, pAKT, HER2, HER3 and AR was observed in PC. Abnormal AR, GRP78 and pAKT expression have significant impact on patient survival. GRP78 expression in AR+ tumours was significantly higher than in AR− tumours. In keeping with our clinical data, activation of AR by dihydrotestosterone (DHT) potently activated GRP78 expression in both LNCaP and LNCaP‐CR cells. For the first time, using a matched HNPC and CRPC TMA, enhanced cytoplasmic and membranous GRP78 expression was observed in CRPC. Future prospective studies are therefore warranted to validate GRP78 as prognostic marker and therapeutic target, in the context of the AR and pAKT status. In summary, GRP78 is co‐expressed with Hsp70–hsp90 client proteins. Up‐regulated expression of AR and GRP78 expression in untreated prostate cancer predicts a less favourable outcome. This points to the importance of understanding in the molecular interaction among AR, GRP78 and AKT. Copyright

NFκB signalling is upregulated in a subset of castrate-resistant prostate cancer patients and correlates with disease progression.

Background:Cell line models suggest that activation of NFκB is associated with progression of prostate cancer. This pathway may be a therapeutic target if these observations translate to clinical specimens.Methods:Immunohistochemistry measured NFκBp65 (p65), NFκBp65 nuclear localisation signal (NLS), NFκBp65 phosphorylated at ser 276 (p65ser276), NFκBp65 phosphorylated at ser 536 (p65ser536), IκBα phosphorylated at ser 32/36 (pIκBαser32/36) and MMP-9 protein expression in 61 matched hormone naive prostate cancer (HNPC) and castrate-resistant prostate cancer (CRPC) tumours. Animal and cell models were used to investigate the role of NFκB inhibition in prostate carcinogenesis.Results:In HNPC tumours, NLS expression significantly associated with a shorter time to disease recurrence and disease-specific death. In CRPC tumours p65, pIκBαser32/36 and MMP-9 expression significantly associated with shorter time to death from disease recurrence and shorter disease-specific death. MMP-9 and pIκBαser32/36 expression significantly associated with metastases at recurrence and were independent of Gleason sum and prostate-specific antigen at recurrence. Expression of phosphorylated Akt was associated with increased p65 activation in mouse models and inhibition of NFκB in LNCaP cells significantly reduced cellular proliferation and induced apoptosis.Conclusion:These results provide further evidence that the NFκB pathway could be exploited as a target for CRPC.

Upregulation of MAPK pathway is associated with survival in castrate-resistant prostate cancer

Background:Recent evidence has implicated the MAP kinase (MAPK) pathway with the development of castrate-resistant prostate cancer (CRPC). We have previously reported gene amplification of critical members of this pathway with the development of castrate-resistant disease. In addition, we have shown that rising Raf-1 expression, with the development of CRPC, influences time to biochemical relapse. We therefore sought to further analyse the role of both Raf-1 and its downstream target MAPK in the molecular pathogenesis of CRPC.Methods:Protein expression of Raf-1 and MAPK, including their activation status, was analysed using immunohistochemistry in a database of 65 paired tumour specimens obtained before and after the development of CRPC and correlated with other members of the pathway.Results:Patients whose nuclear expression of MAPK rose with the development of CRPC had a significantly shorter median time to death following biochemical relapse (1.40 vs 3.00 years, P=0.0255) as well as reduced disease-specific survival when compared with those whose expression fell or remained unchanged (1.16 vs 2.62 years, P=0.0005). Significant correlations were observed between protein expression of Raf-1 and MAPK with the type 1 receptor tyrosine kinases, Her2 and epidermal growth factor receptor, as well as the transcription factor AP-1 in CRPC tumours.Conclusion:We conclude that the Her2/Raf-1/MAPK/AP-1 axis may promote the development of CRPC, leading to early relapse, and reduced disease-specific survival. In addition, members of the pathway may act as novel therapeutic and/or diagnostic targets for prostate cancer.

Androgen receptor phosphorylation at serine 515 by Cdk1 predicts biochemical relapse in prostate cancer patients

Background:Prostate cancer cell growth is dependent upon androgen receptor (AR) activation, which is regulated by specific kinases. The aim of the current study is to establish if AR phosphorylation by Cdk1 or ERK1/2 is of prognostic significance.Methods:Scansite 2.0 was utilised to predict which AR sites are phosphorylated by Cdk1 and ERK1/2. Immunohistochemistry for these sites was then performed on 90 hormone-naive prostate cancer specimens. The interaction between Cdk1/ERK1/2 and AR phosphorylation was investigated in vitro using LNCaP cells.Results:Phosphorylation of AR at serine 515 (pARS515) and PSA at diagnosis were independently associated with decreased time to biochemical relapse. Cdk1 and pCdk1161, but not ERK1/2, correlated with pARS515. High expression of pARS515 in patients with a PSA at diagnosis of ⩽20 ng ml−1 was associated with shorter time to biochemical relapse (P=0.019). This translated into a reduction in disease-specific survival (10-year survival, 38.1% vs 100%, P<0.001). In vitro studies demonstrated that treatment with Roscovitine (a Cdk inhibitor) caused a reduction in pCdk1161 expression, pARS515expression and cellular proliferation.Conclusion:In prostate cancer patients with PSA at diagnosis of ⩽20 ng ml−1, phosphorylation of AR at serine 515 by Cdk1 may be an independent prognostic marker.

Prospective study of the role of inflammation in renal cancer

Background: The local and systemic inflammatory responses provide prognostic information in cancer. The modified Glasgow Prognostic Score (mGPS) provides additional prognostic information than C-reactive protein (CRP) alone when assessing the systemic inflammation in cancer. The aim of this study was to determine the role of local and systemic inflammation in renal cancer. Methods: The cohort consisted of 79 patients who had undergone potential curative resection. Systemic inflammation, mGPS, was constructed by measuring preoperative CRP and albumin concentrations and the Klintrup-Makinen score was evaluated histologically for the local inflammatory response. Pathological parameters such as T stage, grade and tumour necrosis were also assessed. The local inflammatory response was assessed by examining all inflammatory cells at the tumour edge on diagnostic haematoxylin and eosin slides. Results: On univariate analysis, T stage (p < 0.001), grade (p = 0.044) and mGPS (p < 0.001) were significant predictors of cancer-specific survival. On multivariate analysis, mGPS (hazard ratio 8.64, 95% confidence interval 3.5–21.29, p < 0.001) was the only significant independent predictor of cancer-specific survival. Conclusion: A preoperative systemic inflammatory response as measured by the mGPS is an independent predictor of poor cancer-specific survival in renal cancer in patients undergoing potential curative resection.

Dermatoses in five related female carriers of X‐linked chronic granulomatous disease

Eight female members of a family with X‐linked chronic granulomatous disease were identified. Five were shown to be carriers of the disease gene. Each of these female carriers of the gene had a history of skin eruptions. The identification of the carrier state is important as genetic counselling should be offered and the prenatal diagnosis of this disorder is possible.

Expression and prognostic significance of Src family members in renal clear cell carcinoma

Background:The aim of this study was to determine whether Src family kinases (SFK) are expressed in renal cell cancer and to assess their prognostic significance.Methods:mRNA expression levels were investigated for the 8 SFK members by quantitative real-time PCR in 19 clear cell cancer tissue samples. Immunohistochemical staining was utilised to assess expression of Src kinase, dephosphorylated Src kinase at Y530 (SrcY530), phosphorylated Src at Y419 (SrcY419) and the downstream focal adhesion kinase (FAK) marker at the Y861 site (FAK Y861) in a cohort of 57 clear cell renal cancer specimens. Expression was assessed using the weighted histoscore method.Results:Src, Lyn, Hck, Fgr and Fyn were the most highly expressed in renal cancer. All members were more highly expressed in T2 disease, and furthermore expression levels between T2 and T3 disease showed a significant decrease for Lck, Lyn, Fyn, Blk and Yes (P=0.032). Assessment of membrane, cytoplasm and nuclear expression of Src kinase, SrcY530 and SrcY419 were not significantly associated with cancer-specific survival. High expression of cytoplasmic FAK Y861 was associated with decreased cancer-specific survival (P=0.001). On multivariate analysis, cytoplasmic FAK Y861 was independently associated with cancer-specific survival (hazard ratio 3.35, 95% CI 1.40–7.98, P=0.006).Conclusion:We have reported that all SFK members are expressed in renal cell carcinoma. The SFK members had their highest levels of expression before the disease no longer being organ confined. We hypothesise that these SFK members are upregulated before the cancer spreading out-with the organ and given that Src itself is not associated with cancer-specific survival but the presence of FAK Y861, a downstream marker for SFK member activity is associated with decreased cancer-specific survival, we hypothesise that another SFK member is associated with decreased cancer-specific survival in renal cell cancer.

The interrelationships between Src, Cav-1 and RhoGD12 in transitional cell carcinoma of the bladder

Background:The aim of this current study was to assess the expression and activity of Src family kinases, focal adhesion kinase (FAK), caveolin (Cav-1) and RhoGD12 in bladder cancer.Methods:Fifty-eight patients with a new diagnosis of bladder cancer undergoing transurethral resection were included. Immunohistochemical staining was utilised to assess expression of c-Src, dephosphorylated (SrcY530), phosphorylated Src (Y419), phosphorylated FAK (FAK Y861), Cav-1 and RhoGD12. Expression was assessed using the weighted histoscore method.Results:High expression of dephosphorylated Y527, phosphorylated Y416 and phosphorylated FAK Y861 in the membrane were associated with increased cancer-specific survival (P=0. 01, P=0.001, P=0.008, respectively) and expression of Y416 in the membrane was an independent factor on multivariate analysis when combined with known clinical parameters (P=0.008, HR 0.288, 95% CI 0.11–0.72).Conclusion:These results demonstrate that in contrast to other solid tumours, activation of the Src family members and downstream signalling proteins are associated with a good prognosis in transitional cell carcinoma of the bladder, and activated Src has a positive relationship with RhoGD12.

Malignant melanoma and systemic mastocytosis a possible association

Lymphoproliferative and myeloproliferative malignancies have been noted in patients with systemic mastocytosis1 and urticaria pigmentosa.2 However, to our knowledge an association between mastocytosis and malignant melanoma has not been reported previously.

The relevance of elevated Borrelia burgdorferi titres in localized scleroderma

A 46-year-old man with a 2-year history of localized scleroderma of his right upper arm and elevated Borrelia burgdorferi titres is described. The association of Borrelia burgdorferi infection and localized scleroderma is discussed.