Ismaël Hervé Koumakpayi

eIF4E phosphorylation promotes tumorigenesis and is associated with prostate cancer progression

Translational regulation plays a critical role in the control of cell growth and proliferation. A key player in translational control is eIF4E, the mRNA 5′ cap-binding protein. Aberrant expression of eIF4E promotes tumorigenesis and has been implicated in cancer development and progression. The activity of eIF4E is dysregulated in cancer. Regulation of eIF4E is partly achieved through phosphorylation. However, the physiological significance of eIF4E phosphorylation in mammals is not clear. Here, we show that knock-in mice expressing a nonphosphorylatable form of eIF4E are resistant to tumorigenesis in a prostate cancer model. By using a genome-wide analysis of translated mRNAs, we show that the phosphorylation of eIF4E is required for translational up-regulation of several proteins implicated in tumorigenesis. Accordingly, increased phospho-eIF4E levels correlate with disease progression in patients with prostate cancer. Our findings establish eIF4E phosphorylation as a critical event in tumorigenesis. These findings raise the possibility that chemical compounds that prevent the phosphorylation of eIF4E could act as anticancer drugs.

PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer

PTEN haploinsufficiency is common in hormone‐sensitive prostate cancer, though the incidence of genomic deletion and its downstream effects have not been elucidated in clinical samples of hormone refractory prostate cancer (HRPC). Progression to androgen independence is pivotal in prostate cancer and mediated largely by the androgen receptor (AR). Since this process is distinct from metastatic progression, we examined alterations of the PTEN gene in locally advanced recurrent, non‐metastatic human HRPC tissues. Retrospective analyses of PTEN deletion status were correlated with activated downstream phospho‐Akt (p‐Akt) pathway proteins and with the androgen receptor. The prevalence of PTEN genomic deletions in transurethral resection samples of 59 HRPC patients with known clinical outcome was assessed by four‐colour FISH analyses. FISH was performed using six BAC clones spanning both flanking PTEN genomic regions and the PTEN gene locus, and a chromosome 10 centromeric probe. PTEN copy number was also evaluated in a subset of cases using single nucleotide polymorphism (SNP) arrays. In addition, the samples were immunostained with antibodies against p‐Akt, p‐mTOR, p‐70S6, and AR. The PTEN gene was deleted in 77% of cases, with 25% showing homozygous deletions, 18% homozygous and hemizygous deletions, and 34% hemizygous deletions only. In a subset of the study group, SNP array analysis confirmed the FISH findings. PTEN genomic deletion was significantly correlated to the expression of downstream p‐Akt (p < 0.0001), AR (p = 0.025), and to cancer‐specific mortality (p = 0.039). PTEN deletion is common in HRPC, with bi‐allelic loss correlating to disease‐specific mortality and associated with Akt and AR deregulation. Copyright

Expression and Nuclear Localization of ErbB3 in Prostate Cancer

Purpose: The ErbB1 and ErbB2 receptors have been implicated in prostate cancer progression, but less is known about the role and biology of other ErbB receptor family members in prostate cancer. The aim of this study was to analyze the expression and localization of ErbB3 in prostate tissues and prostate cancer cell lines. Experimental Design: Immunohistochemistry of ErbB3 was done on prostate cancer tissue sections from 143 patients and on a tissue microarray containing 390 cores of radical prostatectomy-derived specimens representing normal, prostatic intraepithelial neoplasia, and malignant tissues from 81 patients. ErbB3 subcellular localization was studied by Western blot analysis in LNCaP, 22Rv1, PC-3, and DU145 prostate cancer cell lines. Results: Immunohistochemistry analysis of prostate cancer tissues revealed that >90% of prostate cancer tissues displayed cytoplasmic ErbB3 staining. Minimal ErbB3 nuclear staining was observed in normal prostate tissues and benign prostatic hyperplasia tissues; in contrast, ErbB3 was frequently localized in the nucleus of cancerous tissues. This nuclear localization was more frequent (P < 0.001) in hormone-refractory tissues (17 of 17, 100%) compared with hormone-sensitive samples (37 of 92, 40.2%). Additionally, in the tissue microarray, increased nuclear ErbB3 was associated with increasing Gleason grade. Interestingly, Western blot analysis of cytoplasmic and nuclear subcellular fractions showed that ErbB3 nuclear localization was more prevalent in hormone-sensitive prostate cancer cell lines (LNCaP and 22Rv1) compared with hormone-insensitive cell lines (PC-3 and DU145). Conclusions: ErbB3 nuclear localization discriminates normal from malignant prostate tissues and between tumors from hormone-sensitive versus hormone-refractory prostate cancer. ErbB3 nuclear staining seems to be associated with risk of disease progression. The high frequency of ErbB3 nuclear localization in hormone-refractory tissues indicates that ErbB3 warrants further study to understand its association with prostate cancer disease progression.

Expression and localisation of Akt-1, Akt-2 and Akt-3 correlate with clinical outcome of prostate cancer patients.

We investigated the correlation between the expression and localisation of Akt-1, Akt-2, Akt-3, phospho-Akt proteins and the clinicopathological parameters in 63 prostate cancer specimens. More than 60% of cancerous tissues overexpressed Akt-1, Akt-2 or Akt-3. Cytoplasmic Akt-1 expression was correlated with a higher risk of postoperative prostate-specific antigen (PSA) recurrence and shorter PSA recurrence interval. Cytoplasmic Akt-2 did not show any significant correlation with clinicopathological parameters predicting outcomes. Cytoplasmic Akt-3 was associated with hormone-refractory disease progression and extracapsular invasion. Nuclear Akt-1 and Akt-2 expression were correlated with favourable outcome parameters such as absence of lymph node and perineural invasion. Kaplan–Meier analysis and Cox regression model also showed that Akt-1 and Akt-2, but not Akt-3 or phospho-Akt was associated with a significantly higher risk of PSA recurrence. In contrast, nuclear Akt-1 was significantly associated with a lower risk of PSA recurrence. Multivariate analysis revealed that clinical stage, Gleason score and the combined cytoplasmic nuclear Akt-1 marker in cancerous tissues were significant independent prognostic factors of PSA recurrence. This is the first report demonstrating in patients with prostate cancer and the particular role of Akt-1 isoform expression as a prognostic marker depending of its localisation.

The mammalian target of rapamycin pathway is widely activated without PTEN deletion in renal cell carcinoma metastases

Inhibitors of the mammalian target of rapamycin (mTOR) are emerging as promising therapies for metastatic renal cell carcinoma (RCC). Because rational treatment strategies require understanding the activation status of the underlying signaling pathway being targeted at the desired stage of disease, the authors examined the activation status of different components of the mTOR pathway in RCC metastases and matched primary tumors.

Hierarchical clustering of immunohistochemical analysis of the activated ErbB/PI3K/Akt/NF-κB signalling pathway and prognostic significance in prostate cancer

Background:The PI3K/Akt signalling pathway, induced by epidermal growth factor receptor (EGFR) and Her-2, is involved in the constitutive activation of NF-κB in prostate cancer cell lines. In this study, we extended the in vitro observation using an ex vivo model of prostate cancer tissues and assessed the prognostic significance of the PI3K/Ak/NF-κB signalling determinants.Methods:We analysed a prostate cancer tissue microarray of 63 patients for the expression of total and activated EGFR, Her-2 receptors and the signalling molecules PTEN, phospho-PTEN, Akt, phospho-Akt and the NF-κB subunit p65. Data were analysed using Spearmans rho test, Kaplan–Meier curves and multivariate Cox regression analysis. In addition, a non-supervised hierarchical clustering analysis was applied to stratify patients according to prognostic groups in terms of risk of recurrence.Results:The concomitant overexpression of activated EGFR and Her-2 was correlated with the nuclear expression of NF-κB. EGFR, phospho-EGFR, phospho-Her-2, ErbB3 and nuclear NF-κB were associated with the overall biochemical recurrence (BCR) of patients. The non-supervised hierarchical clustering analysis resulted in the separation of patients into five groups according to BCR.Conclusions:These results validate the previous in vitro data on ErbB involvement in NF-κB activation and shows evidence for a significant role of ErbB/PI3K/Akt/NF-κB signalling in the progression of prostate cancer.

Integrative Molecular Profiling Reveals Asparagine Synthetase Is a Target in Castration-Resistant Prostate Cancer

The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level. The overexpression of ASNS was validated by analyzing other public CRPC data sets. ASNS protein expression, as detected by reverse-phase protein lysate array, was tightly correlated with gene copy number. In addition, ASNS protein expression, as determined by IHC analysis, was associated with progression to a therapy-resistant disease state in TMAs that included 77 castration-resistant and 40 untreated prostate cancer patient samples. Knockdown of ASNS by small-interfering RNAs in asparagine-deprived media led to growth inhibition in both androgen-responsive (ie, LNCaP) and castration-resistant (ie, C4-2B) prostate cancer cell lines and in cells isolated from a CRPC xenograft (ie, MDA PCa 180-30). Together, our results suggest that ASNS is up-regulated in cases of CRPC and that depletion of asparagine using ASNS inhibitors will be a novel strategy for targeting CRPC cells.

Low nuclear ErbB3 predicts biochemical recurrence in patients with prostate cancer.

To further evaluate the association between the cytoplasmic or nuclear localization of ErbB3 with biochemical recurrence (BCR) in patients with prostate cancer and positive surgical margins, as there is a greater risk of BCR for such patients after radical prostatectomy (RP).

NOXA and PUMA Expression Add to Clinical Markers in Predicting Biochemical Recurrence of Prostate Cancer Patients in a Survival Tree Model

Purpose: To assess the expression of proapoptotic NOXA and PUMA in prostate tissues and delineate their association with prostate cancer (PCa) recurrence. Experimental Design: Normal, prostatic intraepithelial neoplasia (PIN), hormone-sensitive (HS) PCa, and hormone-refractory (HR) PCa tissues were used to build tissue microarrays encompassing a total of 135 patients. Two observers assessed the intensity of NOXA and PUMA immunohistochemical staining using a composite color scale. One hundred and eighty recursive partitioning and regression tree (RPART) models were generated to predict biochemical recurrence (BCR) within HS cancer patients using NOXA, PUMA, and clinical parameters. Models were then ranked according to the integrated Brier score (IBS). Results: Increasing NOXA expression was associated with PCa progression, reaching the highest levels in HR PCa. Increased NOXA expression was observed in 68% of HS cancer patients and was predictive of BCR (LR = 8.64; P = 0.003). In contrast, PUMA expression was highest in HS cancer, and although 70% of HS cancer patients exhibited increased PUMA expression, PUMA alone could not predict the onset of BCR. Interestingly, the top-ranking RPART model generated [IBS = 0.107; 95% confidence interval (95% CI), 0.065-0.128] included surgical margin status and NOXA and PUMA expression, although recurrent prognostic classification schemes obtained in the top 10 models favored a survival tree model containing margin status, NOXA expression, and preoperative prostate-specific antigen (PSA) (IBS = 0.114; 95% CI, 0.069-0.142). Conclusion: We conclude that NOXA and PUMA expression may be linked to PCa progression and propose further validation of a survival tree model including surgical margin status, NOXA expression, and preoperative PSA for predicting BCR.

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.