Pamela McCall

Is PTEN loss associated with clinical outcome measures in human prostate cancer

Inactivating PTEN mutations are commonly found in prostate cancer, resulting in an increased activation of Akt. In this study, we investigate the role of PTEN deletion and protein expression in the development of hormone-refractory prostate cancer using matched hormone-sensitive and hormone-refractory tumours. Fluorescent in situ hybridisation and immunohistochemistry was carried out to investigate PTEN gene deletion and PTEN protein expression in the transition from hormone-sensitive to hormone-refractory prostate cancer utilising 68 matched hormone sensitive and hormone-refractory tumour pairs (one before and one after hormone relapse). Heterogeneous PTEN gene deletion was observed in 23% of hormone sensitive tumours. This increased significantly to 52% in hormone-refractory tumours (P=0.044). PTEN protein expression was observed in the membrane, cytoplasm and the nucleus. In hormone sensitive tumours, low levels of cytoplasmic PTEN was independently associated with shorter time to relapse compared to high levels of PTEN (P=0.028, hazard ratio 0.51 (95%CI 0.27–0.93). Loss of PTEN expression in the nucleus of hormone sensitive tumours was independently associated with disease-specific survival (P=0.031, hazard ratio 0.52, 95%CI 0.29–0.95). The results from this study demonstrate a role for both cytoplasmic and nuclear PTEN in progression of prostate cancer to the hormone-refractory state.

Phosphorylation of the androgen receptor is associated with reduced survival in hormone-refractory prostate cancer patients

Cell line studies demonstrate that the PI3K/Akt pathway is upregulated in hormone-refractory prostate cancer (HRPC) and can result in phosphorylation of the androgen receptor (AR). The current study therefore aims to establish if this has relevance to the development of clinical HRPC. Immunohistochemistry was employed to investigate the expression and phosphorylation status of Akt and AR in matched hormone-sensitive and -refractory prostate cancer tumours from 68 patients. In the hormone-refractory tissue, only phosphorylated AR (pAR) was associated with shorter time to death from relapse (P=0.003). However, when an increase in expression in the transition from hormone-sensitive to -refractory prostate cancer was investigated, an increase in expression of PI3K was associated with decreased time to biochemical relapse (P=0.014), and an increase in expression of pAkt473 and pAR210 were associated with decreased disease-specific survival (P=0.0019 and 0.0015, respectively). Protein expression of pAkt473 and pAR210 also strongly correlated (P<0.001, c.c.=0.711) in the hormone-refractory prostate tumours. These results provide evidence using clinical specimens, that upregulation of the PI3K/Akt pathway is associated with phosphorylation of the AR during development of HRPC, suggesting that this pathway could be a potential therapeutic target.

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.

Abstract 1: Loss of STAT1 predicts outcome in prostate cancer patients at diagnosis by regulating the proliferation of prostate epithelial cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. Dysregulation of STAT factors along with the negative feedback regulators of the Janus-activated kinase (JAK)-STAT pathway, such as members of the SOCS family have been implicated in prostate cancer (PCa) cell growth and survival. Although STAT3 involvement in PCa has been extensively studied, little is known about the role of STAT1. In this study, we aim to determine the clinical significance and functional role of STAT1 expression in PCa. Material and Methods. STAT1 cytoplasmic, nuclear and membrane expression in PCa epithelial cells, was measured by immunohistochemistry (IHC) on 78 patients with hormone naive prostate adenocarcinoma recruited at a referral centre between 1992 -2002. Biochemical relapse, survival from biochemical relapse and disease-specific survival according to high/low protein expression were analysed using Kaplan-Meier methods. Significant findings were included in a cox-regression model. Chi square test was used to assess associations with clinical parameters and Pearsons rank correlation coefficients (c.c) to assess association between protein expression. In in vitro studies we assessed the role of STAT1 in PCa epithelial cell (LNCaP and PC3) proliferation by wst-1 incorporation and real time tracking of growth of cells using XCELLigence (Roche, UK), apoptosis by DNA fragmentation and clonogenic capacity following transient silencing of STAT1 with siRNA (SmartPool, Dharmacon, UK). Results. At diagnosis PCa patients with low membrane expression of STAT1 had significantly shorter time to biochemical relapse (3.8 vs 7.3 years, p=0.021) and overall survival (6.62 vs 9.34 years, p=0.056). To test whether these results stemmed from the functional role of STAT1 in regulating the proliferation of prostate epithelial cells, we silenced STAT1 in PCa cell lines. STAT1 silencing resulted in a moderate increase in LNCaP cells (72h, control: 100 ± 18 %; siRNA STAT1 treated: 114 ± 4.5 %, n=3), however a very pronounced increase was observed in the proliferation of PC3 cells (72h, control: 100 ± 12.1 %; siRNA STAT1 treated: 177.4 ± 38 %, n=4, p<0.001). These results were in accordance with data collected from XCELLigence showing increased PC3 cell numbers in STAT1 silenced cells over 10 days. In clonogenic assay an increased surviving fraction of cells was recorded after 10 days of silecing the cells with STAT1 compared to control. Furthermore, there was no significant change in apoptosis recorded in siRNA STAT1 treated cells. Conclusion. Our results enable us to conclude that loss of STAT1 favors tumor formation in hormone naive PCa cells by leading to increased proliferation of PCa cells. We are currently investigating downstream transcription factors that STAT1 might employ in order to control the proliferation of PCa cells. Citation Format: Sophia Hatziieremia, Pamela McCall, Jennifer Willder, Clare Orange, Morag Seywright, Mark A. Underwood, Joanne Edwards. Loss of STAT1 predicts outcome in prostate cancer patients at diagnosis by regulating the proliferation of prostate epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1. doi:10.1158/1538-7445.AM2013-1

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.

Nuclear factor κB predicts poor outcome in patients with hormone-naive prostate cancer with high nuclear androgen receptor

Despite recent advances in prostate cancer treatments, disease recurrence is common and associated with significant morbidity and mortality. The need for more effective antitumor agents has led researchers to target signaling pathways that drive tumorigenesis by modulating or bypassing androgen receptor signaling--attenuation or blockade of which current treatments aim to effect. The transcription factor nuclear factor κB/p65 has been implicated in prostate cancer progression; however, few studies have examined the involvement of nuclear factor κB in hormone-naive disease. We used immunohistochemistry to investigate expression of p65, androgen receptor, Ki-67, and phosphorylation status of p65 at serine 536, in 154 tumor samples taken from patients before hormone ablation or radical treatment. Nuclear p65 expression was significantly associated with disease-specific mortality: P = .005; hazard ratio, 2.2. When patients were stratified according to androgen receptor status, this relationship was abolished in low androgen receptor-expressing patients and potentiated in high androgen receptor-expressing patients: P = .002; hazard ratio, 3.1. Ki-67 expression was also prognostic of shorter disease-specific mortality: P = .001; hazard ratio, 2.3. When the cohort was stratified according to androgen receptor status, this relationship held for high androgen receptor expressers but not low expressers: P = .0003; hazard ratio, 3.5. Neither androgen receptor nor p65 phosphorylated at S536 were significantly prognostic when considered individually. These data suggest that future prostate cancer treatments that target nuclear factor κB signaling should be assigned primarily to patients with concomitant high nuclear p65 and androgen receptor expression.

Androgen Receptor Phosphorylation at Serine 308 and Serine 791 Predicts Enhanced Survival in Castrate Resistant Prostate Cancer Patients

We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR94), 308 (pAR308), 650(pAR650) and 791 (pAR791). No correlations with clinical parameters were observed for pAR94 or pAR650 in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR308 is significantly associated with a longer time to disease specific death (p = 0.011) and high pAR791 expression significantly associated with a longer time to disease recurrence (p = 0.018) in HNPC tumours and longer time to death from disease recurrence (p = 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p = 0.022) and low proliferating tumours (p = 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer.

Tumoral C-reactive protein and nuclear factor kappa-B expression are associated with clinical outcome in patients with prostate cancer

Prostate cancer (Pca) is the most common form of cancer affecting men, despite recent advances in PCa treatments, one third of patients diagnosed each year succumb to this disease. The inflammatory response has been implicated in prostate cancer progression. The pro-inflammatory transcription factor, NFκB/p65 has been implicated in PCa progression. Few studies have examined the involvement of NFκB and inflammatory signalling in PCa.Immunohistochemistry was employed to investigate the expression of NFκB/p65, C-reactive protein and Ki67 in 61 clinical samples. Tumours expressing high levels of p65 (p=0.004), CRP (p=0.011) and Ki67 (p=0.0003) had a shorter disease specific survival. Upon combining p65 and CRP status it was observed that those tumours with low expression of both proteins had a median survival of 11 years compared to 3.9 years for those with tumours with high expression of both (p=0.005). CRP expression was significantly higher in the 21 patients who died of their disease and on multivariate analysis CRP expression retained independent significance (p=0.005). This study suggests CRP and NFκB may function collectively to drive prostate cancer progression in a subset of patients. Tumoral CRP is a significant independent predictor of cancer specific survival. The relationship between tumour CRP and signalling pathways warrants further investigation in a larger cohort.

High IKKα expression is associated with reduced time to recurrence and cancer specific survival in oestrogen receptor (ER)-positive breast cancer

The aim of our study was to examine the relationship between tumour IKKα expression and breast cancer recurrence and survival. Immunohistochemistry was employed in a discovery and a validation tissue microarray to assess the association of tumour IKKα expression and clinico‐pathological characteristics. After siRNA‐mediated silencing of IKKα, cell viability and apoptosis were assessed in MCF7 and MDA‐MB‐231 breast cancer cells. In both the discovery and validation cohorts, associations observed between IKKα and clinical outcome measures were potentiated in oestrogen receptor (ER) positive Luminal A tumours. In the discovery cohort, cytoplasmic IKKα was associated with disease‐free survival (p = 0.029) and recurrence‐free survival on tamoxifen (p < 0.001) in Luminal A tumours. Nuclear IKKα and a combination of cytoplasmic and nuclear IKKα (total tumour cell IKKα) were associated with cancer‐specific survival (p = 0.012 and p = 0.007, respectively) and recurrence‐free survival on tamoxifen (p = 0.013 and p < 0.001, respectively) in Luminal A tumours. In the validation cohort, cytoplasmic IKKα was associated with cancer‐specific survival (p = 0.023), disease‐free survival (p = 0.002) and recurrence‐free survival on tamoxifen (p = 0.009) in Luminal A tumours. Parallel experiment with breast cancer cells in vitro demonstrated the non‐canonical NF‐κB pathway was inducible by exposure to lymphotoxin in ER‐positive MCF7 cells and not in ER‐negative MDA‐MB‐231 cells. Reduction in IKKα expression by siRNA transfection increased levels of apoptosis and reduced cell viability in MCF7 but not in MDA‐MB‐231 cells. IKKα is an important determinant of poor outcome in patients with ER‐positive invasive ductal breast cancer and thus may represent a potential therapeutic target.