Nathalie Delvoye

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.

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.

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

BackgroundCell 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.MethodsThe 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.ResultsAll 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.ConclusionThe 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.

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.

Over-expression of IκB-kinase-ε (IKKε/IKKi) induces secretion of inflammatory cytokines in prostate cancer cell lines

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 Iκ‐B kinase‐epsilon (IKKε/IKKi)‐deficiency results in the reduction of lipopolysaccharide‐mediated expression of IL‐6.

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.

CAAT/Enhancer-binding Proteins Are Involved in β-Globin Gene Expression and Are Differentially Expressed in Murine Erythroleukemia and K562 Cells

Acting in cis with the β-globin locus control region, the CAAT box of the β-globin gene promoter stimulates transcription 10-fold in murine erythroleukemia (MEL) cells but is without effect in K562 cells. Our previous studies suggested that of four proteins from MEL cells that bind to this CAAT box region (CP1, GATA-1, and two factors that were denoted DSFr and DSF1) DSFr is involved in the up-regulation of transcription. In the present report, the DSFr protein in MEL cells was identified as C/EBPγ through expression cloning and antibody studies. C/EBPγ DNA binding activity could not be detected in K562 cells. However, K562 cells, but not MEL cells, were found to express LIP, which is a truncated form of C/EBPβ and is an inhibitor of transcription. Thus, the differential expression of C/EBP members could account for the ability of the β-globin CAAT box to stimulate transcription in MEL cells, but not function in K562 cells. Juxtaposing a specific C/EBP binding sequence next to the β-globin promoter, in constructs in which the CAAT box had been rendered inactive by mutation or deletion, restored full promoter activity in MEL cells only if CP1 still bound to the promoter. In conjunction with previous mutation analyses, these results suggest that C/EBPγ may collaborate with CP1 to enhance transcription through the β-globin CAAT box.

Deletions in the dystrophin gene: analysis of Duchenne and Becker muscular dystrophy patients in Quebec

SummaryWe have analyzed patient DNA samples in 77 unrelated Duchenne (DMD) and Becker (BMD) muscular dystrophy families, 73 of which were of French Canadian origin. We show that the frequency (68%) and distribution of deletions within the dystrophin gene was neither random nor unique in this population. We localized 33% of the deletions to the proximal portion of the dystrophin gene while 63% involved the exons spanning introns 43 through 55 with breakpoint clusters occurring within introns 44 and 50. Whether the dystrophin open reading frame (ORF) is maintained constrains the distribution of DMD/BMD deletions such that BMD deletions tend to be strikingly homogeneous. Finally, the conservation of the dystrophin ORF and the severity of the clinical phenotype were concordant in 95% of the DMD/BMD deletions documented by this work.

Regulation of IκB kinase ε expression by the androgen receptor and the nuclear factor-κB 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-κB 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 IκB kinase ε (IKKε), which is controlled by nuclear factor-κB in human chondrocytes, is expressed in human prostate cancer cells. We show that IKKε gene expression is stimulated by tumor necrosis factor-α treatment in LNCaP cells and is inhibited by transfection of a dominant-negative form of IκBα, which prevents the nuclear translocation of p65. Furthermore, we found that tumor necrosis factor-α–induced IKKε expression is inhibited by an androgen analogue (R1881) in androgen-sensitive prostate cancer cells and that this inhibition correlates with the modulation of IκBα expression by R1881. We also noted constitutive IKKε expression in androgen-independent PC-3 and DU145 cells. To our knowledge, this is the first report of an IκB kinase family member whose expression is modulated by androgen and deregulated in androgen receptor–negative cells. (Mol Cancer Res 2007;5(1):87–94)

ErbB2/Her-2 Regulates the Expression of Akt2 in Prostate Cancer Cells

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.