Ilaria Cavarretta

Inhibition of the acetyltransferases p300 and CBP reveals a targetable function for p300 in the survival and invasion pathways of prostate cancer cell lines

Inhibitors of histone deacetylases have been approved for clinical application in cancer treatment. On the other hand, histone acetyltransferase (HAT) inhibitors have been less extensively investigated for their potential use in cancer therapy. In prostate cancer, the HATs and coactivators p300 and CBP are upregulated and may induce transcription of androgen receptor (AR)-responsive genes, even in the absence or presence of low levels of AR. To discover a potential anticancer effect of p300/CBP inhibition, we used two different approaches: (i) downregulation of p300 and CBP by specific short interfering RNA (siRNA) and (ii) chemical inhibition of the acetyltransferase activity by a newly developed small molecule, C646. Knockdown of p300 by specific siRNA, but surprisingly not of CBP, led to an increase of caspase-dependent apoptosis involving both extrinsic and intrinsic cell death pathways in androgen-dependent and castration-resistant prostate cancer cells. Induction of apoptosis was mediated by several pathways including inhibition of AR function and decrease of the nuclear factor kappa B (NF-κB) subunit p65. Furthermore, cell invasion was decreased upon p300, but not CBP, depletion and was accompanied by lower matrix metalloproteinase (MMP)-2 and MMP-9 transcriptions. Thus, p300 and CBP have differential roles in the processes of survival and invasion of prostate cancer cells. Induction of apoptosis in prostate cancer cells was confirmed by the use of C646. This was substantiated by a decrease of AR function and downregulation of p65 impairing several NF-κB target genes. Taken together, these results suggest that p300 inhibition may be a promising approach for the development of new anticancer therapies. Mol Cancer Ther; 10(9); 1644–55. ©2011 AACR.

Interleukin-6 stimulation of growth of prostate cancer in vitro and in vivo through activation of the androgen receptor

It is hypothesized that ligand-independent activation of the androgen receptor is one of the mechanisms implicated in tumour progression. However, supportive evidence is limited to the effect of HER-2/neu that stimulates prostate cancer progression through activation of the androgen receptor. In the present study, we have asked whether the proinflammatory cytokine interleukin-6 (IL-6), which is known to stimulate androgen receptor activity and expression of its downstream target genes, may also induce growth of androgen-sensitive cells. We have found that IL-6 differentially regulates proliferation of LAPC-4 and MDA PCa 2b cells. In MDA PCa 2b cells, growth stimulation by IL-6 was reversed by administration of either the non-steroidal anti-androgen bicalutamide or the inhibitor of the mitogen-activated protein kinase pathway PD98059. Neither cell line was found to express endogenous IL-6. Interestingly, the treatment of those prostate cancer cells did not increase phosphorylation of STAT3. The effect of IL-6 on stimulation of androgen receptor activity in MDA PCa 2b cells was lower than that of androgen, comparable with findings reported by other researchers. However, growth of MDA PCa 2b xenografts in castrated animals treated with IL-6 was similar to that in non-castrated animals. In addition, bicalutamide showed an inhibitory effect on IL-6-regulated growth in vivo. Taken together, data in the present study demonstrate that IL-6 may cause growth of androgen receptor-positive tumours in vitro and in vivo through activation of the androgen receptor.

The antiapoptotic effect of IL-6 autocrine loop in a cellular model of advanced prostate cancer is mediated by Mcl-1

Levels of the proinflammatory cytokine interleukin-6 (IL-6) are increased in therapy-resistant prostate cancer. IL-6 has been considered a positive growth factor in late-stage prostate cancer cells and a potential target for therapeutic interference. Effects of inhibition of IL-6 on cell survival were studied in LNCaP-IL6+ cells, a model system for advanced prostate cancer, which produce IL-6. We show that the autocrine IL-6 loop is responsible for resistance to apoptosis and increased cellular levels of myeloid cell leukemia-1 (Mcl-1) protein, an antiapoptotic member of the Bcl-2 family. Treatment of cells with a chimeric anti-IL-6 antibody (CNTO 328) led to the induction of apoptosis and downregulation of Mcl-1 protein levels. Specific knockdown of Mcl-1 gene expression by small interfering RNA also yielded an increase in apoptosis of LNCaP-IL-6+ cells. Vice versa, inactivation of IL-6 autocrine loop had no influence on apoptosis levels in the absence of Mcl-1, thus suggesting this molecule as a mediator of the survival action of IL-6. Mcl-1 protein regulation by the endogenous cytokine directly involved the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway. Our data support the concept of anti-IL-6 targeted therapy in therapy-resistant prostate cancer.

Interleukin-6 trans-signalling differentially regulates proliferation, migration, adhesion and maspin expression in human prostate cancer cells.

Interleukin-6 (IL-6) is suggested to have a pathogenic role in the progression of prostate cancer (PC), therefore representing an attractive target for new therapies. However, due to the pleiotropy of this cytokine, targeting IL-6 results in different and unpredictable responses. In order to better understand the mechanisms underlying the different responses to the cytokine, we focused our attention on IL-6 receptors (IL-6Rs) that represent the first element in the cascade of cytokine-activated signalling pathways. IL-6 signal transduction may indeed occur through the membrane IL-6R (classical signalling) and/or through the less studied soluble IL-6R (sIL-6R; IL-6 trans-signalling (IL-6TS)). We provide the first evidence how responses to IL-6 may depend on the different content of IL-6Rs in PC. In particular, the studies of 3H-thymidine incorporation and exploitation of different approaches (i.e. activation or inhibition of IL-6TS in sIL-6R-negative and -positive cell lines and transfection of IL-6R siRNA) allowed us to demonstrate that IL-6TS specifically accounts for an anti-proliferative effect of the cytokine in three PC cell lines that are known to respond differently to IL-6. Additionally, by applying migration-, scratch- and adhesion assays, we show that IL-6TS increases motility and migration and decreases adhesion of prostate cells facilitating thereby processes that determine metastasis initiation and spread. Finally, by western analyses, we uncovered an IL-6- and sIL-6R-dependent downregulation of the tumour suppressor maspin. Collectively, these data suggest that selective targeting of IL-6TS might allow to refine the currently available experimental anti-IL-6 therapies against PC.

Suppressor of cytokine signalling-3 is up-regulated by androgen in prostate cancer cell lines and inhibits androgen-mediated proliferation and secretion

Suppressors of cytokine signalling (SOCS) are induced by interleukins (ILs) and various peptide hormones and may prevent sustained activation of signalling pathways. We have previously shown that SOCS-3 antagonizes regulation of cellular events by cAMP and is expressed in human prostate cancer. To investigate possible effects of androgen on SOCS-3 protein expression, two prostate cancer cell lines (PC3-AR and LAPC4) were treated with different concentrations of R1881. Western blot analyses revealed induction of SOCS-3 protein expression in both cell lines by androgen, an effect which can be blocked by the anti-androgen bicalutamide. To further characterize the effects of R1881 on the SOCS-3 gene, promoter-reporter assay and real-time PCR were performed. We found no influence of androgen on promoter activity or SOCS-3 mRNA levels, thus suggesting a post-transcriptional effect of androgen. Concordant with our previous findings, we show a significant increase of SOCS-3 protein after androgen treatment in cells in which transcription was blocked, but not in those with impaired translation. In order to understand implications of SOCS-3 regulation by androgen, we used SOCS-3-negative LNCaP-IL-6 cells and stably transfected them with a tetracycline-responsive SOCS-3 Tet-On plasmid. We report that androgenic effects on cell proliferation and prostate-specific antigen secretion are significantly diminished following up-regulation of SOCS-3. In conclusion, androgen up-regulates SOCS-3 protein via post-transcriptional effects. SOCS-3 inhibits androgen-stimulated proliferation by influencing cell cycle regulation. Taken together with previous findings showing androgen receptor activation by IL-6, our results imply that androgen and cytokine signalling pathways interact at multiple levels in prostate cancer.

Corticosteroids protect oligodendrocytes from cytokine-induced cell death

The present data show that the simultaneous exposure to tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) induces cell death with characteristics of apoptosis in cultured rat oligodendrocytes; TNFα alone was ineffective. We have also demonstrated that different corticosteroids (aldosterone, deoxycorticosterone, dexamethasone and corticosterone) protect rat oligodendrocytes in culture from apoptosis induced by TNFα plus IFNγ. This effect seems to be exerted via the interaction with both type I and type II corticosteroid receptors since all steroids considered are effective. Since oligodendrocyte apoptosis represents an important event in multiple sclerosis and in several demyelinating diseases, the present observations might be considered an interesting background for further researches directed to the possibility of controlling in vivo the death of these cells.

The Microbiome of the Prostate Tumor Microenvironment

BACKGROUND The advent of molecular-based methods of identification and characterization of complex microbial populations has led to a new era of microbial discovery. A detailed and comprehensive analysis of the microbial ecosystem of the pathologic and healthy prostate tissues has not been yet reported. OBJECTIVES To characterize the microbiome possibly associated to the pathologic prostate microenvironment. DESIGN, SETTING, AND PARTICIPANTS The microbiome profile of tumor, peri-tumor, and nontumor tissues was assessed on 16 radical prostatectomy-specimens. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Microbiome analysis was assessed by massive ultradeep pyrosequencing. Bacteria load was expressed as a percentage of the total number of bacteria. The statistical significance of differences among specimen-groups was tested with Friedmans test (Dunn posthoc test) and Wilcoxon rank-sum test. RESULTS AND LIMITATIONS Three phyla, six classes, nine orders, 14 families, and 11 genera were above the set threshold value of 1%, respectively. Significant differences in specific microbial populations among tumor/peri-tumor and nontumor prostate specimens were observed at certain taxonomic levels. Among genera, Propionibacterium spp. were the most abundant. Staphylococcus spp. were more represented in the tumor/peri-tumor tissues (p<0.05). The restricted number of specimens represents a potential limitation. CONCLUSIONS The prostate contains a plethora of bacteria, which set themselves within the gland with a distribution dependent on the nature of the tissue, thus suggesting a possible pathophysiological correlation between the composition of the local microbial niche and the presence of the tumor itself. Future studies will help to clarify the role of these specific bacteria and their potential to be exploited as new biomarkers. PATIENT SUMMARY The pathological prostate is populated by specific microbial populations, whose distribution varies according to the nature of the tissue. This finding opens interesting perspectives for the identification of novel therapeutic approaches and biomarkers.

Mesenchymal stem cells expressing therapeutic genes induce autochthonous prostate tumour regression

Mesenchymal stem cells (MSC) as vehicles of therapeutic genes represent a unique tool to activate drugs within a neoplastic mass due to their property to home and engraft into tumours. In particular, MSC expressing the cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) have been previously demonstrated to inhibit growth of subcutaneous prostate cancer xenografts thanks to their ability to convert the non-toxic 5-fluorocytosine into the antineoplastic 5-fluorouracil. Since both the immune system and the tumour microenvironment play a crucial role in directing cancer progression, in order to advance towards clinical applications, we tested the therapeutic potential of this approach on animal models that develop autochthonous prostate cancer and preserve an intact immune system. As cell vectors, we employed adipose-tissue and bone-marrow MSC. CD-MSC toxicity on murine prostate cancer cells and tumour tropism were verified in vitro and ex-vivo before starting the preclinical studies. Magnetic Resonance Imaging was utilised to follow orthotopic tumour progression. We demonstrated that intravenous injections of CD-MSC cells, followed by intraperitoneal administration of 5-fluorocytosine, caused tumour regression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which develops aggressive and spontaneous prostate cancer. These results add new insights to the therapeutic potential of specifically engineered MSC in prostate cancer disease.

Human Prostate Tissue-derived Extracellular Matrix as a Model of Prostate Microenvironment

BACKGROUND Clinical experience highlights the wide heterogeneity of primary prostate cancer (PPCa), even when potentially related to the same grade and stage. Currently available prediction tools and biomarkers do not always allow for early recognition of PPCa aggressive phenotype, sometimes making it impossible to distinguish among men harbouring indolent tumours or life-threatening disease. OBJECTIVE To establish a novel ex vivo/in vitro model suitable to estimate the invasive phenotype of PPCa cells (PPCaC). DESIGN, SETTING, AND PARTICIPANTS The ability of PPCaC to infiltrate the prostate extracellular matrix (ECM) was used as an index of invasion. ECM was obtained by decellularising 24 NT-prostate specimens from radical prostatectomy. PPCaC were obtained from six tumours with different Gleason patterns and pathological stages. Invasion ability was estimated in direct-cocolture experiments. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The extent of ECM invasion by PPCaC was quantified by counting the number of infiltrated cells. Mann-Whitney test was utilised for statistical comparisons. RESULTS AND LIMITATIONS Samples of ECM resulted to be free of cells and DNA and with a preserved three-dimensional structure and stromal protein content. The system resulted to be reliable since well characterised normal-, benign-, and malignant-prostate cell lines either re-epitheliased or invaded the matrices, according to their specific nature. Similarly, PPCaC invaded the ECMs consistently with their stage and biochemical recurrence. Of notice, this model was able to identify a different invasive phenotype even among tumours with equal Gleason patterns and pathological stages. The small sample size represents a limitation. CONCLUSIONS We developed an ex vivo/in vitro model able to reproduce the original PPCa-microenvironment and suitable to recognise the inherent invasive behaviour of PPCaC. PATIENT SUMMARY We developed a novel ex vivo/in vitro system which enables us to uncover which prostate tumours host potentially aggressive cancer cells. The identification of cancer cells with different invasive abilities will likely lead to the identification of new biomarkers to safely predict disease progression.

Analysis of the Enteric Microbiome: First Tentative Steps Towards a Comprehensive Work-up of Prostate Cancer?

The introduction of new instruments and novel technologies has often been followed by relevant advances in scientific understanding of biological systems, the discovery of new etiological mechanisms, and the expansion of innovative therapeutic options. In particular, the advent of high-throughput nextgeneration sequencing techniques has started a new era of microbial discovery that could allow a more comprehensive understanding of the complex microbial communities that persistently or transiently colonise our body areas [1–3]. In this context, a significant example is the prostate gland. Both the prostate and the prostate tumor–associated microflora have recently been characterised. Interestingly, a number of microbiome differences have been identified, even among paired prostate tumour, peritumour, and nontumour samples, showing that the distribution of bacterial microbes depends on the nature of tissue within the same gland [2]. These observations are intriguing and may point the way to novel prognostic, diagnostic, prophylactic, and therapeutic strategies. However, for integration into everyday clinical routine, less invasive approaches are certainly needed. It is now accepted that an altered enteric microbiome and increased intestinal permeability may lead to a systemic inflammatory status, which in turn is associated with (or is possibly the cause of) different pathological conditions, including cancer risk factors such as obesity [4–8]. These observations are of great importance from a clinical standpoint, since the enteric, or more precisely the faecal, microbiome is much easier to access. However, this approach has been only partly followed for prostate cancer (PCa).