Validation of the prognostic value of NF-κB p65 in prostate cancer: A retrospective study using a large multi-institutional cohort of the Canadian Prostate Cancer Biomarker Network.

BACKGROUND: The identification of patients with high-risk prostate cancer (PC) is a major challenge for clinicians, and the improvement of current prognostic parameters is an unmet clinical need. We and others have identified an association between the nuclear localization of NF-κB p65 and biochemical recurrence (BCR) in PC in small and/or single-centre cohorts of patients. METHODS AND FINDINGS: In this study, we accessed 2 different multi-centre tissue microarrays (TMAs) representing cohorts of patients (Test-TMA and Validation-TMA series) of the Canadian Prostate Cancer Biomarker Network (CPCBN) to validate the association between p65 nuclear frequency and PC outcomes. Immunohistochemical staining of p65 was performed on the Test-TMA and Validation-TMA series, which include PC tissues from patients treated by first-line radical prostatectomy (n = 250 and n = 1,262, respectively). Two independent observers evaluated the p65 nuclear frequency in digital images of cancer tissue and benign adjacent gland tissue. Kaplan-Meier curves coupled with a log-rank test and univariate and multivariate Cox regression models were used for statistical analyses of continuous values and dichotomized data (cutoff of 3%). Multivariate analysis of the Validation-TMA cohort showed that p65 nuclear frequency in cancer cells was an independent predictor of BCR using continuous (hazard ratio [HR] 1.02 [95% CI 1.00-1.03], p = 0.004) and dichotomized data (HR 1.33 [95% CI 1.09-1.62], p = 0.005). Using a cutoff of 3%, we found that this biomarker was also associated with the development of bone metastases (HR 1.82 [95% CI 1.05-3.16], p = 0.033) and PC-specific mortality (HR 2.63 [95% CI 1.30-5.31], p = 0.004), independent of clinical parameters. BCR-free survival, bone-metastasis-free survival, and PC-specific survival were shorter for patients with higher p65 nuclear frequency (p < 0.005). As the small cores on TMAs are a limitation of the study, a backward validation of whole PC tissue section will be necessary for the implementation of p65 nuclear frequency as a PC biomarker in the clinical workflow. CONCLUSIONS: We report the first study using the pan-Canadian multi-centre cohorts of CPCBN and validate the association between increased frequency of nuclear p65 frequency and a risk of disease progression.

Analysis of active surveillance uptake for low-risk localized prostate cancer in Canada: a Canadian multi-institutional study.

PURPOSE: Although the uptake of active surveillance (AS) appears to be increasing in published series, the uptake in most geographic regions remains largely unknown. Our aim was to examine practice patterns around the use of AS in low-risk prostate cancer in Canada. In addition, we examined regional variations in AS uptake, predictors of AS uptake, and persistent use for 12 months. METHODS: This is a retrospective multicentre review of low-risk patients who underwent a prostate biopsy in 2010 in six centres in four provinces (BC, QC, MB and ON). AS was identified based on chart review and required a minimum of 6 months of follow-up after diagnosis without any active treatment. RESULTS: Of 986 patients, 781 patients (mean age 64 years) were incident cases and over three-quarters (77.3 %) chose AS at diagnosis. There were significant differences in uptake of AS by centre (range 65.0-98.0 %, p ≤ 0.05). Key multivariate predictors of pursuing AS included older age (OR 1.34, p = 0.044), centre (p = 0.021), lower number of cores (OR 1.09, p = 0.025), lower number of positive biopsy cores (OR 0.52, p < 0.001), and lower percent core involvement (OR 0.84, p < 0.001). In total, 516 (85.4 %) men remained on AS over 12 months. Maintenance with AS over 12 months differed by centre, ranging from 64.1 to 93.9 % (p = 0.001). Predictors of maintenance with AS over 12 months included older age, centre, and lower number of positive cores. CONCLUSIONS: Active surveillance is widely practiced across Canada, but important regional differences were observed. Further analyses are required to understand the root causes of differences and to determine whether AS uptake is changing over time.

The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo.

BACKGROUND: The involvement of NF-κB signaling in prostate cancer (PCa) has largely been established through the study of the classical p65 subunit. Nuclear localization of p65 in PCa patient tissues has been shown to correlate with biochemical recurrence, while in vitro studies have demonstrated that the classical NF-κB signaling pathway promotes PCa progression and metastatic potential. More recently, the nuclear location of RelB, a member of the alternative NF-κB signaling, has also been shown to correlate with the Gleason score. The current study aims to clarify the role of alternative NF-κB in PCa cells by exploring, in vitro and in vivo, the effects of RelB overexpression on PCa biology. METHODS: Using a lentivirus-expression system, we constitutively overexpressed RelB or control GFP into 22Rv1 cells and monitored alternative transcriptional NF-κB activity. In vivo, tumor growth was assessed after the injection of 22Rv1-derived cells into SCID mice. In vitro, the impact of RelB on 22Rv1 cell proliferation was evaluated in monolayer culture. The anchorage-independent cell growth of derived-22Rv1 cells was assessed by soft agar assay. Apoptosis and autophagy were evaluated by Western blot analysis in 22Rv1-derived cells cultured in suspension using poly-HEMA pre-coated dishes. RESULTS: The overexpression of RelB in 22Rv1 cells induced the constitutive activation of the alternative NF-κB pathway. In vivo, RelB expression caused a lag in the initiation of 22Rv1-induced tumors in SCID mice. In vitro, RelB stimulated the proliferation of 22Rv1 cells and reduced their ability to grow in soft agar. These observations may be reconciled by our findings that, when cultured in suspension on poly-HEMA pre-coated dishes, 22Rv1 cells expressing RelB were more susceptible to cell death, and more specifically to autophagy controlled death. CONCLUSIONS: This study highlights a role of the alternative NF-κB pathway in proliferation and the controlled autophagy. Thus, the interplay of these properties may contribute to tumor survival in stress conditions while promoting PCa cells growth contributing to the overall tumorigenicity of these cells.

ErbB2/Her-2 regulates the expression of Akt2 in prostate cancer cells.

BACKGROUND: We have previously reported that ErbB family members regulate the signaling pathway leading to Akt and NF-kappaB activation in prostate cancer cells. In this study, the regulation of Akt2 expression in LNCaP, DU145, and PC-3 prostate cancer cell lines was investigated. METHODS: Akt-2 expression was analyzed by western-blotting and Q-PCR in cell lines. We also analyzed the Akt-2 protein expression in a tissue microarray from 64 prostate cancer patients. Akt-2 promoter activity was assessed and analyzed by luciferase assay. RESULTS: A concomitant over-expression of Her-2 and Akt2 expression was observed by western-blotting and quantitative-PCR in prostate cancer cell lines. A significant correlation between Her-2 and Akt2 protein expression was also observed by immunohistochemistry assay on prostate cancer tissues. In LNCaP cells, over-expression of Akt2 protein and mRNA was decreased by Her-2 pharmacologic inhibitors as well as small interfering RNA (siRNA) specific to Her-2. Cloning of the AKT2 promoter in a luciferase reporter plasmid further showed that Akt2 over-expression in cell lines is associated with increased AKT2 promoter activity suggesting that Her-2 modulates a signaling pathway involving AKT2 gene regulation. CONCLUSION: This study reveals a novel mechanism of Akt2 regulation in prostate cancer cells.

Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer.

BACKGROUND: Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. METHODS: The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133) were derived from solid tumor (TOV) and ascites (OV), at specific time points at diagnosis and relapse (R). Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133), or cisplatin/topotecan (2295). Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2), the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. RESULTS: All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R) cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R) and OV2295(R2) cell lines. CONCLUSION: The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian cancer.

Macropinocytosis inhibitors and Arf6 regulate ErbB3 nuclear localization in prostate cancer cells.

ErbB3 is a transmembrane tyrosine kinase receptor among the epidermal growth factor receptor (EGFR) family that plays an important role in prostate cancer (PCa) progression. We previously demonstrated that ErbB3 is located in the nucleus of PCa cell lines and PCa tissues. It also was observed that ErbB3 nuclear localization could discriminate between benign and malignant prostate tissues as well as between hormone sensitive and hormone-refractory PCa. Several studies have suggested a role for clathrin-mediated endosomal sorting in the nuclear localization of EGFR and ErbB2 but the mechanisms by which ErbB receptors escape recycling or degradation are not well known. Consequently to determine the role of endocytosis in the nuclear localization of ErbB3, different endocytotic inhibitors and specific si-RNAs were used to discriminate between clathrin-dependent and clathrin-independent pathways. We found that clathrin, caveolin, and membrane domains are not required for endocytosis-mediated nuclear localization of ErbB3 in PCa cells and we provide evidence that amiloride, a macropinocytosis inhibitor, and the ADP-ribosylation factor 6 (Arf6) are implicated in the compartimentalization of ErbB3. In conclusion, evidence for an endocytosis-based mechanism in the nuclear localization of ErbB3 in PCa cells is proposed. These results may help elucidate new therapeutic avenues in PCa that target nuclear ErbB3, which may participate in the progression and aggressivity of the disease.

Identification of Morphologic Criteria Associated with Biochemical Recurrence in Intraductal Carcinoma of the Prostate.

Intraductal carcinoma of the prostate (IDC-P) is an aggressive subtype of prostate cancer strongly associated with an increased risk of biochemical recurrence (BCR). However, approximately 40% of men with IDC-P remain BCR-free five years after radical prostatectomy. In this retrospective multicenter study, we aimed to identify histologic criteria associated with BCR for IDC-P lesions. A total of 108 first-line radical prostatectomy specimens were reviewed. In our test cohort (n = 39), presence of larger duct size (>573 µm in diameter), cells with irregular nuclear contours (CINC) (≥5 CINC in two distinct high-power fields), high mitotic score (>1.81 mitoses/mm2), blood vessels, and comedonecrosis were associated with early BCR (<18 months) (p < 0.05). In our validation cohort (n = 69), the presence of CINC or blood vessels was independently associated with an increased risk of BCR (hazard ratio [HR] 2.32, 95% confidence interval [CI] 1.09-4.96, p = 0.029). When combining the criteria, the presence of any CINC, blood vessels, high mitotic score, or comedonecrosis showed a stronger association with BCR (HR 2.74, 95% CI 1.21-6.19, p = 0.015). Our results suggest that IDC-P can be classified as low versus high-risk of BCR. The defined morphologic criteria can be easily assessed and should be integrated for clinical application following validation in larger cohorts.

Over-expression of IkappaB-kinase-epsilon (IKKepsilon/IKKi) induces secretion of inflammatory cytokines in prostate cancer cell lines.

BACKGROUND: Elevated inflammatory cytokine levels in serum have been associated with advanced stage metastasis-related morbidity in prostate cancer. Several studies have shown that IL-6 and IL-8 can accelerate the growth of human prostate cancer cell lines. Previous studies, in murine embryonic fibroblasts, have shown that Ikappa-B kinase-epsilon (IKKepsilon/IKKi)-deficiency results in the reduction of lipopolysaccharide-mediated expression of IL-6. RESULTS: In this study, we report that over-expression of IKKepsilon in hormone-sensitive 22Rv1 and LNCaP prostate cancer cells induces the secretion of several inflammatory cytokines including IL-6 and IL-8. Both of these cytokines are secreted by hormone-refractory PC-3 prostate cancer cells and IKKepsilon knock-down in these cells correlates with a strong decrease in IL-6 secretion. Furthermore, we demonstrate that IKKepsilon over-expression does not induce the activation of the IKKepsilon classical targets NF-kappaB and IRF-3, two transcription factors involved in the regulation of several cytokines. Finally, we observe that high IKKepsilon expression results in its nuclear translocation, a phenomena that is TBK1-independent. CONCLUSIONS: This study identifies IKKepsilon as a potential prostate cancer gene that may favor chronic inflammation and create a tumor-supporting microenvironment that promotes prostate cancer progression, particularly by the induction of IL-6 secretion that may act as a positive growth factor in prostate cancer.

Regulation of IkappaB kinase epsilon expression by the androgen receptor and the nuclear factor-kappaB transcription factor in prostate cancer.

Although several genes have been associated with prostate cancer progression, it is clear that we are far from understanding all the molecular events implicated in the initiation and progression of the disease to a hormone-refractory state. The androgen receptor is a central player in the initiation and proliferation of prostate cancer and its response to hormone therapy. Nuclear factor-kappaB has important proliferative and antiapoptotic activities that could contribute to the development and progression of cancer cells as well as resistance to therapy. In this study, we report that IkappaB kinase epsilon (IKKepsilon), which is controlled by nuclear factor-kappaB in human chondrocytes, is expressed in human prostate cancer cells. We show that IKKepsilon gene expression is stimulated by tumor necrosis factor-alpha treatment in LNCaP cells and is inhibited by transfection of a dominant-negative form of IkappaBalpha, which prevents the nuclear translocation of p65. Furthermore, we found that tumor necrosis factor-alpha-induced IKKepsilon expression is inhibited by an androgen analogue (R1881) in androgen-sensitive prostate cancer cells and that this inhibition correlates with the modulation of IkappaBalpha expression by R1881. We also noted constitutive IKKepsilon expression in androgen-independent PC-3 and DU145 cells. To our knowledge, this is the first report of an IkappaB kinase family member whose expression is modulated by androgen and deregulated in androgen receptor-negative cells.

NF-kappaB2 processing and p52 nuclear accumulation after androgenic stimulation of LNCaP prostate cancer cells.

Several reports suggest that androgen signalling interferes with canonical RelA-p50 activity in androgen-sensitive cells. Whether this also occurs with non-canonical NF-kappaB subunits has not been studied. Here we report that androgenic stimulation of LNCaP cells with the androgen analogue R1881 appears to positively regulate the non-canonical NF-kappaB pathway as p52 accumulates both in the cytoplasm and nucleus after 48-72 h of stimulation. In contrast to TNF-alpha stimulation, androgen stimulation fails to induce RelB expression and is absent from nucleus of R1881-treated LNCaP cells. Electromobility shift assays reveal a time-dependent change in the nature of NF-kappaB complexes actively bound to DNA after 72 h of androgenic stimulation concomitant with the appearance of p52-containing complexes. Co-immunoprecipitation studies indicate that newly produced p52 can exist as a heterodimer with RelA or p50, but may be mainly present as a homodimer. RNAi experiments targeting IKK-alpha and IKK-beta show that the R1881-induced nuclear accumulation of p52 is IKK-alpha-dependent. These results point to a novel mechanism by which androgens regulate NF-kappaB and provide a rationale for further studies into the biological significance of non-canonical NF-kappaB signalling in prostate cancer.

IκB-Kinase-epsilon (IKKε) over-expression promotes the growth of prostate cancer through the C/EBP-ß dependent activation of IL-6 gene expression.

The inflammatory cytokine IL-6 has been shown to induce the nuclear translocation of androgen receptors in prostate cancer cells and to activate the androgen receptors in a ligand-independent manner, suggesting it may contribute to the development of a castrate-resistant phenotype. Elevated IL-6 serum levels have also been associated with metastasis-related morbidity in prostate cancer patients. We have previously established that over-expression of I-kappa-B-kinase-epsilon (IKKε also named IKKi or IκBKε) in hormone-sensitive prostate cancer cell lines induces IL-6 secretion. We have also reported that prostate cancer cell lines lacking androgen receptor expression exhibit high constitutive IKKε expression and IL-6 secretion. In the present study, we validated the impact of IKKε depletion on the in vitro proliferation of castrate-resistant prostate cancer cells, and characterized how IKKε depletion affects tumor growth and IL-6 tumor secretion in vivo through a mouse xenograft-based approach. We observed a significant growth delay in IKKε-silenced PC-3 cells injected in SCID mice fed with a doxycycline-supplemented diet in comparison with mice fed with a normal diet. We also found a decrease in IL-6 secretion levels that strongly correlated with tumor growth inhibition. Finally, using constructs with various IL-6-mutated promoters, we demonstrated that IKKε over-expression induces a NF-κB-independent stimulation of the IL-6 gene promoter through the activation and nuclear accumulation of the transcription factor C/EBP-ß. Our study demonstrates the pro-proliferative role of the oncogene IKKε in castrate-resistant prostate cancer cell lines, involving the phosphorylation and nuclear translocation of C/EBP-ß that initiates IL-6 gene expression.

BTN3A2 expression in epithelial ovarian cancer is associated with higher tumor infiltrating T cells and a better prognosis.

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR=0.651, p=0.006 and HR=0.642, p=0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR=1.355 p=0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells.

Androgen-regulated expression of arginase 1, arginase 2 and interleukin-8 in human prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens. CONCLUSION/SIGNIFICANCE: Our data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.

Characterization of the intra-prostatic immune cell infiltration in androgen-deprived prostate cancer patients.

INTRODUCTION: Our goal was to study the hormonal regulation of immune cell infiltration in prostate cancer patients treated by androgen deprivation therapy (ADT) using an optimized computer-assistance quantification approach. METHODS: The relative density of immune cell subtypes (CD3(+), CD8(+), CD20(+), CD56(+), CD68(+) and Foxp3(+)) was analyzed by immunohistochemistry in archived prostate specimens from control patients (radical prostatectomy only, n=40) and ADT-treated patients (ADT prior to radical prostatectomy, n=35) using an image analysis software and a whole-slide scanner. RESULTS: ADT-treated patients had significantly increased relative density of CD3(+) (p<0.001) and CD8(+) T lymphocytes (p<0.001) as well as CD68(+) macrophages (p<0.001). Elevated abundance of CD56(+) Natural Killer (NK) cells was associated with a lower risk of prostate cancer progression (p=0.044), while a high density of CD68(+) macrophages was related to an increased risk of biochemical recurrence (p=0.011). CONCLUSIONS: Our results demonstrate that the infiltration of specific immune cell subtypes is modulated by ADT. Furthermore our data confirm that NK cells have a protective role against tumor progression while macrophages seem to favor the development of advanced prostate cancer.

An androgen-independent androgen receptor function protects from inositol hexakisphosphate toxicity in the PC3/PC3(AR) prostate cancer cell lines.

BACKGROUND: Inositol hexakisphosphate (IP6) is a phytochemical exhibiting anticancer activity. Because few prostate cancer (PCa) cell lines have been used to study IP6, we assessed its efficacy in a panel of PCa cell lines. METHODS AND RESULTS: Using WST-1 assays we observed that, although androgens did not modulate its efficacy, IP6 was more active in androgen receptor (AR) negative cells than in AR-positive cells. Stable expression of the AR in PC3 cells (PC3(AR)) decreased the response to IP6, which was reversed by an AR-targeting siRNA. Furthermore, AR expression in PC3 cells resulted in significantly reduced caspase-3 activation (P < 0.001) and DNA fragmentation (P < 0.05) in response to IP6. Similarly, although treatment with IP6 caused the upregulation of NF-kappaB-responsive (IkappaB-alpha, IRF-2) and p53/E2F-responsive genes (Puma, Noxa) in PC3 cells, this increase was reduced in PC3AR cells (P < 0.01). CONCLUSION: We conclude that resistance to IP6 can be linked to a ligand-independent AR function.