Antitumour effect of the mitochondrial complex III inhibitor Atovaquone in combination with anti-PD-L1 therapy in mouse cancer models.

Immune checkpoint blockade (ICB) provides effective and durable responses for several tumour types by unleashing an immune response directed against cancer cells. However, a substantial number of patients treated with ICB develop relapse or do not respond, which has been partly attributed to the immune-suppressive effect of tumour hypoxia. We have previously demonstrated that the mitochondrial complex III inhibitor atovaquone alleviates tumour hypoxia both in human xenografts and in cancer patients by decreasing oxygen consumption and consequently increasing oxygen availability in the tumour. Here, we show that atovaquone alleviates hypoxia and synergises with the ICB antibody anti-PD-L1, significantly improving the rates of tumour eradication in the syngeneic CT26 model of colorectal cancer. The synergistic effect between atovaquone and anti-PD-L1 relied on CD8+ T cells, resulted in the establishment of a tumour-specific memory immune response, and was not associated with any toxicity. We also tested atovaquone in combination with anti-PD-L1 in the LLC (lung) and MC38 (colorectal) cancer syngeneic models but, despite causing a considerable reduction in tumour hypoxia, atovaquone did not add any therapeutic benefit to ICB in these models. These results suggest that atovaquone has the potential to improve the outcomes of patients treated with ICB, but predictive biomarkers are required to identify individuals likely to benefit from this intervention.

Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models.

PURPOSE: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. EXPERIMENTAL DESIGN: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. RESULTS: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. CONCLUSIONS: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

The protease SPRTN and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability.

DNA-protein crosslinks (DPCs) are a specific type of DNA lesion in which proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration, and accelerated aging. DPC proteolysis was recently identified as a specialized pathway for DPC repair. The DNA-dependent protease SPRTN and the 26S proteasome emerged as two independent proteolytic systems. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the cellular response to DPC formation, we identify ubiquitylation and SUMOylation as two major signaling events in DNA replication-coupled DPC repair. DPC ubiquitylation recruits SPRTN to repair sites, promoting DPC removal. DPC SUMOylation prevents DNA double-strand break formation, HR activation, and potentially deleterious genomic rearrangements. In this way, SUMOylation channels DPC repair toward SPRTN proteolysis, which is a safer pathway choice for DPC repair and prevention of genomic instability.

Mitochondrial Inhibitor Atovaquone Increases Tumor Oxygenation and Inhibits Hypoxic Gene Expression in Patients with Non-Small Cell Lung Cancer.

PURPOSE: Tumor hypoxia fuels an aggressive tumor phenotype and confers resistance to anticancer treatments. We conducted a clinical trial to determine whether the antimalarial drug atovaquone, a known mitochondrial inhibitor, reduces hypoxia in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with NSCLC scheduled for surgery were recruited sequentially into two cohorts: cohort 1 received oral atovaquone at the standard clinical dose of 750 mg twice daily, while cohort 2 did not. Primary imaging endpoint was change in tumor hypoxic volume (HV) measured by hypoxia PET-CT. Intercohort comparison of hypoxia gene expression signatures using RNA sequencing from resected tumors was performed. RESULTS: Thirty patients were evaluable for hypoxia PET-CT analysis, 15 per cohort. Median treatment duration was 12 days. Eleven (73.3%) atovaquone-treated patients had meaningful HV reduction, with median change -28% [95% confidence interval (CI), -58.2 to -4.4]. In contrast, median change in untreated patients was +15.5% (95% CI, -6.5 to 35.5). Linear regression estimated the expected mean HV was 55% (95% CI, 24%-74%) lower in cohort 1 compared with cohort 2 (P = 0.004), adjusting for cohort, tumor volume, and baseline HV. A key pharmacodynamics endpoint was reduction in hypoxia-regulated genes, which were significantly downregulated in atovaquone-treated tumors. Data from multiple additional measures of tumor hypoxia and perfusion are presented. No atovaquone-related adverse events were reported. CONCLUSIONS: This is the first clinical evidence that targeting tumor mitochondrial metabolism can reduce hypoxia and produce relevant antitumor effects at the mRNA level. Repurposing atovaquone for this purpose may improve treatment outcomes for NSCLC.

Targeting TOPK sensitises tumour cells to radiation-induced damage by enhancing replication stress.

T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage.

The anti-malarial drug atovaquone potentiates platinum-mediated cancer cell death by increasing oxidative stress.

Platinum chemotherapies are highly effective cytotoxic agents but often induce resistance when used as monotherapies. Combinatorial strategies limit this risk and provide effective treatment options for many cancers. Here, we repurpose atovaquone (ATQ), a well-tolerated & FDA-approved anti-malarial agent by demonstrating that it potentiates cancer cell death of a subset of platinums. We show that ATQ in combination with carboplatin or cisplatin induces striking and repeatable concentration- and time-dependent cell death sensitization in vitro across a variety of cancer cell lines. ATQ induces mitochondrial reactive oxygen species (mROS), depleting intracellular glutathione (GSH) pools in a concentration-dependent manner. The superoxide dismutase mimetic MnTBAP rescues ATQ-induced mROS production and pre-loading cells with the GSH prodrug N-acetyl cysteine (NAC) abrogates the sensitization. Together, these findings implicate ATQ-induced oxidative stress as key mediator of the sensitizing effect. At physiologically achievable concentrations, ATQ and carboplatin furthermore synergistically delay the growth of three-dimensional avascular spheroids. Clinically, ATQ is a safe and specific inhibitor of the electron transport chain (ETC) and is concurrently being repurposed as a candidate tumor hypoxia modifier. Together, these findings suggest that ATQ is deserving of further study as a candidate platinum sensitizing agent.

External Beam Radiation Therapy and Enadenotucirev: Inhibition of the DDR and Mechanisms of Radiation-Mediated Virus Increase.

Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity.

Homeostasis: apoptosis and cell cycle in normal and pathological prostate.

Prostatic diseases such as hyperplasia and cancer are a consequence of glandular aging due to the loss of homeostasis. Glandular homeostasis is guaranteed by the delicate balance between production and cell death. Both cell renewal and apoptosis are part of this delicate balance. We will explore the predictive capacity for biochemical progression, following prostatectomy, of some members of the Bcl-2 family and of proteins involved in cell cycle inhibition in conjunction with established classical markers. The expression of Bcl-2, Bcl-xL, Mcl-1, Bax, Bim, Bad, PUMA, Noxa, p21, p27, Rb and p53 were analyzed by immunochemistry in 86 samples of radical prostatectomy and correlated with each of the markers established clinicopathological tests using statistical tests such as Sperman, Kaplan-Meier curves, unifactorial Cox, and multifactorial. The most relevant results are: (1) Positive correlation between: p27 with clinical T stage; and PUMA with pathological T stage; (2) Negative correlation between: Bcl-2 with clinical T stage, Bcl-xL with survival, Noxa and pRb with Gleason score.Our results suggest that the expression of Bcl-2, Bcl-xL, PUMA, Noxa, p27, and Rb were related to some of the classic markers established to predict biochemical progression after prostatectomy.

CDK1 inhibition sensitizes normal cells to DNA damage in a cell cycle dependent manner.

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner. Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.

Nucleoporin 54 contributes to homologous recombination repair and post-replicative DNA integrity.

The nuclear pore complex (NPC) machinery is emerging as an important determinant in the maintenance of genome integrity and sensitivity to DNA double-strand break (DSB)-inducing agents, such as ionising radiation (IR). In this study, using a high-throughput siRNA screen, we identified the central channel NPC protein Nup54, and concomitantly its molecular partners Nup62 and Nup58, as novel factors implicated in radiosensitivity. Nup54 depletion caused an increase in cell death by mitotic catastrophe after IR, and specifically enhanced both the duration of the G2 arrest and the radiosensitivity of cells that contained replicated DNA at the time of IR exposure. Nup54-depleted cells also exhibited increased formation of chromosome aberrations arisen from replicated DNA. Interestingly, we found that Nup54 is epistatic with the homologous recombination (HR) factor Rad51. Moreover, using specific DNA damage repair reporters, we observed a decreased HR repair activity upon Nup54 knockdown. In agreement with a role in HR repair, we also demonstrated a decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges after IR in cells depleted of Nup54. Our study reveals a novel role for Nup54 in the response to IR and the maintenance of HR-mediated genome integrity.

PI3K pathway and Bcl-2 family. Clinicopathological features in prostate cancer.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathways and Bcl-2 family play a central role in prostate cancer (PC). The aim was to determine influence in the biochemical progression in PC. To evaluate the association between clinic pathological and immunohistochemical variables, Spearman's test was performed. Log-rank test and Kaplan-Meier curves were used for survival comparisons. To explore the correlation of the studied immunohistochemical parameters and the established prognostic variables with biochemical progression, univariate and multivariate Cox proportional Hazard regression analyses were performed. Spearman analysis showed correlation between stroma expression and tumor expression of PI3K with biochemical progression (p = .009, p = .004), respectively, and tumor immunohistochemical score with biochemical progression (p = .051). In the multivariate Cox regression model, only PI3K was retained as independent predictors of biochemical progression. In stroma expression, PI3K is (HR 0.172, 95% CI 0.065-0.452, p = .000); tumor expression, PI3K is (HR 0.087, 95% CI 0.026-0.293, p = .000), and tumor immunohistochemical score (HR 0.382, 95% CI 0.209-0.697 p = .002). Our results suggest a role for prostatic expression of PI3K was prognostic markers for PC. PI3K/AKT/mTOR and Bcl-2 family are becoming an important therapeutic target and predictive biomarkers of onset and progression of PC.

Expression of several cytokines in prostate cancer: Correlation with clinical variables of patients. Relationship with biochemical progression of the malignance.

BACKGROUND: This work is focused on finding new markers that complement or diagnoses currently used towards improving knowledge histological and statistical aspects that allow us to predict the local stage carcinomas and to identify and understand all the factors related to the progression of this disease. MATERIALS AND METHODS: Prostates were obtained from: normal prostates from 20 men, diagnosis of BPH (Benign Prostatic Hyperplasia) from 35 men and prostate cancer from 86 men. We studied the behavior of cytokines that have been implicated in inflammatory processes: TNF-alfa, IL-6, IL-1, EGF and TGF-B. Expression of these cytokines and its receptors was analyzed by immunohistochemistry. Spearman's test, Kaplan-Meier curves, univariate and multivariate Cox proportional hazard regression analyses were performed. RESULTS: Spearman's analysis showed that there was at least one correlation between TGFB-B, IL-6, gp-130, IL-1B, IL-1R, IL-1RII and clinic pathological feature (preoperative serum PSA, clinical t stage, pathological t stage, positive surgical margins, biochemical progression, survival). Immunostaining score was correlated with some of the clinicopathological feature. In Cox multivariate analysis between the prognostic variables (pathological T stage, Gleason score and lymph node) and immunohistochemical parameters (TGF-B, IL-1a, intensity TGFBRI and intensity TGFBRII) only the expression of IL-1a was retained as independent predictors of biochemical progression after radical prostatectomy. CONCLUSIONS: Our results suggest a role for prostatic expression of TGF-B, IL-1a, TGFBRI and TGFBRII as prognostic markers for prostate cancer. The rational combination of novel agents directed toward the inactivation of TGF-B, IL-1a, TGFBRI and TGFBRII could disrupt complementary tumor cell proliferation pathways.

Epidermal growth factor induces p38 MAPK-dependent G0/G1-to-S transition in prostate cancer cells upon androgen deprivation conditions.

Epidermal growth factor (EGF) is thought to contribute to the emergence of castration-resistant (CR) prostate tumors by inducing proliferation of cancer cells despite the low levels of circulating androgens achieved by androgen deprivation therapy. We show that, in LNCaP cells, androgen deprivation induces arrest in the G0/G1 cell cycle phase, and that EGF partially rescues this arrest without affecting cell death. Inhibition of p38 MAPK, but not MEK or IKK-ß, completely abrogates the EGF-induced proliferation of LNCaP cells in androgen-depleted medium, and decreases the fraction of G0/G1-arrested cells. Our results suggest that EGF enables prostate cancer cells to overcome the growth restriction imposed by androgen deprivation by stimulating G0/G1-to-S transition via p38 MAPK. These results suggest the potential of developing therapies for advanced prostate cancer that block the G0/G1 to S transition, such as by targeting p38 MAPK, or that aim to induce apoptosis in G0/G1-arrested cancer cells.

Prognostic value of inhibitors of apoptosis proteins (IAPs) and caspases in prostate cancer: caspase-3 forms and XIAP predict biochemical progression after radical prostatectomy.

BACKGROUND: The expression status of apoptotic regulators, such as caspases and inhibitors of apoptosis proteins (IAPs), could reflect the aggressiveness of tumors and, therefore, could be useful as prognostic markers. We explored the associations between tumor expression of caspases and IAPs and clinicopathological features of prostate cancer--clinical and pathological T stage, Gleason score, preoperative serum PSA levels, perineural invasion, lymph node involvement, surgical margin status and overall survival--and evaluated its capability to predict biochemical progression after radical prostatectomy. METHODS: Protein expression of caspases (procaspase-8, cleaved caspase-8, procaspase-3, cleaved caspase-3, caspase-7 and procaspase-9) and IAPs (cIAP1/2, cIAP2, NAIP, Survivin and XIAP) was analyzed by immunohistochemistry in radical prostatectomy samples from 84 prostate cancer patients. Spearman's test, Kaplan-Meier curves, and univariate and multivariate Cox proportional hazard regression analysis were performed. RESULTS: cIAP1/2, cIAP2, Survivin, procaspase-8, cleaved caspase-8, procaspase-3 and caspase-7 expression correlated with at least one clinicopathological feature of the disease. Patients negative for XIAP, procaspase-3 or cleaved caspase-3 had a significantly worse prognosis. Of note, XIAP, procaspase-3 and cleaved caspase-3 were predictors of biochemical progression independent of Gleason score and pathological T stage. CONCLUSIONS: Our results indicate that alterations in the expression of IAPs and caspases contribute to the malignant behavior of prostate tumors and suggest that tumor expression of XIAP, procaspase-3 and cleaved caspase-3 may help to identify prostate cancer patients at risk of progression.

Clinical significance of both tumor and stromal expression of components of the IL-1 and TNF-α signaling pathways in prostate cancer.

IL-1 and TNF-α, the two major proinflammatory cytokines, have been involved in initiation and progression of several malignancies. They could influence the biological behavior of prostatic tumors and patient outcome, and could be useful as prognostic factors. This study evaluated the prognostic capability for biochemical progression after radical prostatectomy of expression of IL-1, TNF-α and related signaling components, in the tumor and surrounding stroma, as well as its correlation with other clinicopathological features. Expression of IL-1α, IL-1ß, IL-1Ra, IL-1RI, IL-1RII, IRAK-1, TRAF6, TNF-α, TNFRI and TRAF2 was analyzed by immunohistochemistry in radical prostatectomy samples from 93 prostate cancer patients. Spearman's test, Kaplan-Meier curves, and univariate and multivariate Cox proportional hazard regression analyses were performed. Expression of TNF-α, TNFRI, TRAF2, ILRI, IRAK-1 and TRAF6 correlated with at least one clinicopathological feature (clinical T stage, pathological T stage, preoperative serum PSA or Gleason score). Increased tumor expression of TNF-α, TNFRI and IL-1RI, and reduced tumor expression of IRAK-1 were significantly correlated with a poor prognosis in univariate analysis. Reduced stromal expression of IL-1ß and IL-1RII, and increased stromal expression of IRAK-1 were also adverse prognostic factors in univariate analysis. Remarkably, tumor IL-1ß and stromal IL-1RII and IRAK-1 remained as independent prognostic factors after adjustment for preoperative serum PSA, pathological T stage and Gleason score in multivariate Cox models. Our results suggest that prostatic expression of TNF-α, IL-1ß and related signaling proteins (TNFRI, IL-1RI, IL-1RII and IRAK-1) predicts clinical outcome in prostate cancer, and support the involvement of TNF-α and IL-1ß signaling in prostate cancer progression.

Expression of NF-κB-related proteins and their modulation during TNF-α-provoked apoptosis in prostate cancer cells.

BACKGROUND: The involvement of TNF-α in cancer development is controversial, since this cytokine was reported to act either as tumor promoter or suppressor. TNF-α may activate signaling pathways critical for life/death decisions, such as mitogen-activated protein kinases (MAPKs) and the anti-apoptotic NF-κB pathway. In this work, we investigate the activation status of NF-κB-related proteins in human prostate cancerous versus normal epithelium, and the alterations in the NF-κB pathway in relation to cell death in TNF-α-treated LNCaP (androgen-independent cells) and PC3 (androgen-independent) prostate cancer cell lines. METHODS: The expression of phospho-p38-MAPK, phospho-IKK-α/ß and phospho-IκB-α, total IκB-α, and p65- and p50-NF-κB, were analyzed by immunohistochemistry in cancerous and normal prostate samples. The toxicity of TNF-α in LNCaP and PC3 cells, with or without kinase and NF-κB inhibitors, was assessed by changes on viability (MTT assay) and apoptosis (loss of DNA, annexin-V binding, and caspase cleavage/activation). Expression of NF-κB-related proteins in these cell lines was measured by Western blot. RESULTS: Phospho-IκB-α, phospho-IKK-α/ß and phospho-p38 levels, cytoplasmic p50 to IκB-α ratio, and nuclear p50 and p65, levels, were increased in cancerous epithelium, suggesting activation of the NF-κB pathway in prostatic malignance. TNF-α caused apoptosis with higher efficacy in LNCaP cells, and this response was potentiated by p38-MAPK inhibitor (LNCaP cells) and IKK-ß inhibitor (both cell lines). However, the protective action of IKK-ß was mediated by NF-κB only in LNCaP cells. CONCLUSIONS: IKK-ß mediates both NF-κB-dependent and -independent anti-apoptotic functions in prostate cancerous epithelium. IKK-ß and p38-MAPK may represent useful therapeutic targets against prostate cancer.

Immunoreactivity to caspase-3, caspase-7, caspase-8, and caspase-9 forms is frequently lost in human prostate tumors.

Caspases are essential initiators and executioners of apoptosis. Changes in their expression may contribute to the development of proliferative disorders such as cancer, by altering the death-proliferation homeostatic balance. The aim of this work was to analyze the expression of a broad panel of caspases at the epithelial level in human prostate tissues to assess possible prostatic disease-related alterations. We comparatively analyzed by immunohistochemistry the expression of pro-caspase-3, pro-caspase-8, pro-caspase-9, cleaved caspase-3, cleaved caspase-8, and caspase-7, in normal and pathologic (benign hyperplasic, premalignant [high-grade intraepithelial neoplasia], and cancerous [prostate cancer]) human prostate epithelium. Expression of caspases was correlated with clinicopathologic features, including preoperative prostate-specific antigen levels, Gleason scores, and biochemical progression. Percentage of positive samples for all the analyzed caspases decreased in prostate cancer versus normal prostate epithelium. The values obtained for benign prostatic hyperplasia and high-grade intraepithelial neoplasia more qualitatively resembled those of the prostate cancer group. Our results indicate that caspase expression in prostate malignant cells is reduced in a substantial number of patients and that such an alteration occurs in the premalignant stage. Loss of caspase expression could constitute a useful marker for prostate cancer diagnosis. Therapeutic approaches aimed to recover or enhance caspase expression might be effective against prostate cancer.

MAP Kinases and Prostate Cancer.

The three major mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK are signal transducers involved in a broad range of cell functions including survival, apoptosis, and cell differentiation. Whereas JNK and p38 have been generally linked to cell death and tumor suppression, ERK plays a prominent role in cell survival and tumor promotion, in response to a broad range of stimuli such as cytokines, growth factors, ultraviolet radiation, hypoxia, or pharmacological compounds. However, there is a growing body of evidence supporting that JNK and p38 also contribute to the development of a number of malignances. In this paper we focus on the involvement of the MAPK pathways in prostate cancer, including the less-known ERK5 pathway, as pro- or antitumor mediators, through their effects on apoptosis, survival, metastatic potential, and androgen-independent growth.

Relationship between IL-6/ERK and NF-κB: a study in normal and pathological human prostate gland.

BACKGROUND: There is growing evidence that inflammation is a causal factor in cancer, where pro-inflammatory cytokines such as IL-6, IL-1 or TNF-α could induce cellular proliferation by activation of NF-κB. This study focuses on the IL-6/ERK transduction pathway, its relationship with NF-κB, and the consequences of dysregulation in the development of prostate pathologies such as benign prostate hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate cancer (PC). METHODS: Immunohistochemical and Western blot analyses for IL-6, gp-130, Raf-1, MEK-1, ERK-1, p-MEK, ERK-2, p-ERK, NF-κB/p-50 and NF-κB/p-65 were carried out in 20 samples of normal prostate glands, 35 samples of BPH, 27 samples with a diagnosis of PIN (low-grade PIN or high-grade PIN), and 95 samples of PC (23 with low, 51 with medium and 21 with high Gleason scores). RESULTS: Immunoreaction to IL-6, gp-130, ERK-1, ERK-2, p-ERK and NF-κB/p50 was found in the cytoplasm of epithelial cells in normal prostate samples; p-MEK was found in the nucleus of epithelial cells; but not expression to Raf-1, MEK-1 and NF-κB/p65. In BPH, all of these proteins were immunoexpressed, while there was increased immunoexpression of IL-6, gp-130, p-MEK, ERK-1, ERK-2 and NF-κB/p50 (cytoplasm). In PC, immunoexpression of IL-6 and gp-130 were similar to that found in BPH; while immunoexpression of Raf-1, MEK-1, p-MEK, ERK-1, ERK-2, p-ERK, NF-κB/p50 (nucleus and cytoplasm), and NF-κB/p65 (nucleus and cytoplasm) was higher than in BPH. CONCLUSION: Translocation of NF-κB to the nucleus in PC and high-grade PIN could be stimulated by the IL-6/ERK transduction pathway, but might also be stimulated by other transduction pathways, such as TNF-α/NIK, TNF/p38, IL-1/NIK or IL-1/p38. Activation of NF-κB in PC could regulate IL-6 expression. These transduction pathways are also related to activation of other transcription factors such as Elk-1, ATF-2 or c-myc (also involved in cell proliferation and survival). PC is a heterogeneous disease, where multiple transduction pathways might alter the apoptosis/proliferation balance. Significant attention should be give to the combination of novel agents directed towards inactivation of pro-inflammatory cytokines than can disrupt tumour cell growth.

Role of IAPs in prostate cancer progression: immunohistochemical study in normal and pathological (benign hyperplastic, prostatic intraepithelial neoplasia and cancer) human prostate.

BACKGROUND: In this study was investigate IAPs in normal human prostate (NP), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostatic carcinoma (PC), and their involvement in apoptosis/proliferation via NF-kB (TNF-alpha, IL-1) stimulation. METHODS: Immunohistochemical and Western blot analyses were performed in 10 samples of normal prostates, 35 samples of BPH, 27 samples diagnosis of PIN (with low-grade PIN or high-grade PIN) and 95 samples of PC (with low, medium or high Gleason grades). RESULTS: In NP, cytoplasm of epithelial cells were positive to c-IAP1/2 (80% of samples), c-IAP-2 (60%), ILP (20%), XIAP (20%); negative to NAIP and survivin. In BPH, epithelial cells were immunostained to c-IAP1/2 (57.57%), c-IAP-2 (57.57%), ILP (66.6%), NAIP (60.6%), XIAP (27.27%), survivin (9.1%). Whereas low-grade PIN showed intermediate results between NP and BPH; results in high-grade PIN were similar to those found in PC. In PC, epithelial cells were immunostained to c-IAP1/2, c-IAP-2, ILP, NAIP, XIAP (no Gleason variation) and survivin (increasing with Gleason). CONCLUSIONS: IAPs could be involved in prostate disorder (BPH, PIN and PC) development since might be provoke inhibition of apoptosis and subsequently cell proliferation. At the same time, different transduction pathway such as IL-1/NIK/NF-kB or TNF/NF-kB (NIK or p38) also promotes proliferation. Inhibitions of IAPs, IL-1alpha and TNFalpha might be a possible target for PC treatment since IAPs are the proteins that inhibited apoptosis (favour proliferation) and IL-1alpha and TNFalpha would affect all the transduction pathway involucrate in the activation of transcription factors related to survival or proliferation (NF-kB, Elk-1 or ATF-2).