Hypoxia and Inflammation in Prostate Cancer Progression. Cross-talk with Androgen and Estrogen Receptors and Cancer Stem Cells.

Tumors are complex tissues in which transformed cells communicate with the surrounding microenvironment and evolve traits promoting their own survival and malignancy. Hypoxia and inflammation are constant characteristics of prostate tumor microenvironment influencing both cancer stem cells and differentiated tumor cells. HIFs and NF-kB are the key regulators of the transcriptional response to hypoxic and inflammatory stresses, respectively, and a crosstalk between HIFs and NF-kB pathways has been widely documented. Similarly, androgen and estrogen signaling, that play important roles in the growth and function of normal prostate gland, when deregulated, have a significant part in the acquisition of hallmarks of malignant diseases. Moreover, androgen and estrogen receptors have been shown to intersect with the HIF/NF-kB signaling in prostate cancer. Aim of this review is to present the current knowledge regarding the crucial role, in prostate cancer progression, of a molecular network linking hypoxia, pro-inflammatory response and steroid receptors.

HIF3α: the little we know.

Hypoxia-inducible factors (HIFs) are key regulators of the transcriptional response to hypoxic stress. Three inducible isoforms of HIF are present in mammals. HIF1α and HIF2α are the best characterized and structurally similar isoforms, while HIF3α is the most distantly related and is less studied. The HIF3α gene undergoes complex regulation and produces a large number of long and short mRNA splice variants, which are translated into different polypeptides. These molecules primarily act as negative regulators of HIF1α and HIF2α activity and transcriptional activators of target genes, according to the variant and the biological context. The present review provides an overview of the available, fragmented and sometimes contradictory information concerning the structure, expression and distinct roles of the HIF3α variants, in both hypoxic adaptation and in hypoxia-unrelated activities. The pathological consequences of HIF3α deregulation are also illustrated.

Distinct phenotypes of human prostate cancer cells associate with different adaptation to hypoxia and pro-inflammatory gene expression.

Hypoxia and inflammation are strictly interconnected both concurring to prostate cancer progression. Numerous reports highlight the role of tumor cells in the synthesis of pro-inflammatory molecules and show that hypoxia can modulate a number of these genes contributing substantially to the increase of cancer aggressiveness. However, little is known about the importance of the tumor phenotype in this process. The present study explores how different features, including differentiation and aggressiveness, of prostate tumor cell lines impact on the hypoxic remodeling of pro-inflammatory gene expression and malignancy. We performed our studies on three cell lines with increasing metastatic potential: the well differentiated androgen-dependent LNCaP and the less differentiated and androgen-independent DU145 and PC3. We analyzed the effect that hypoxic treatment has on modulating pro-inflammatory gene expression and evaluated the role HIF isoforms and NF-kB play in sustaining this process. DU145 and PC3 cells evidenced a higher normoxic expression and a more complete hypoxic induction of pro-inflammatory molecules compared to the well differentiated LNCaP cell line. The role of HIF1α and NF-kB, the master regulators of hypoxia and inflammation respectively, in sustaining the hypoxic pro-inflammatory phenotype was different according to cell type. NF-kB was observed to play a main role in DU145 and PC3 cells in which treatment with the NF-kB inhibitor parthenolide was able to counteract both the hypoxic pro-inflammatory shift and HIF1α activation but not in LNCaP cells. Our data highlight that tumor prostate cell phenotype contributes at a different degree and with different mechanisms to the hypoxic pro-inflammatory gene expression related to tumor progression.

Modulators of HIF1α and NFkB in Cancer Treatment: Is it a Rational Approach for Controlling Malignant Progression?

HIF1α and NFkB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression, and resistance to chemotherapy. In fact, HIF1α and NFkB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic reprograming, inflammatory reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7, and some TLRs, are activated by HIF1α; and that, in turn, alarmin receptors strongly activate NFkB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these molecules are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors with reduction of metastasis formation and, more importantly, with a net increase in survival. This review highlights the central role of HIF1α activated in hypoxic regions of the tumor, of NFkB activation and proinflammatory gene expression in transformed cells to understand their progression toward malignancy. Different molecules and strategies to inhibit these transcription factors will be reviewed. Finally, the central role of a new class of deacetylases called Sirtuins in regulating HIF1α and NFkB activity will be outlined.

Action of retinoic acid receptor on EGFR gene transactivation and breast cancer cell proliferation: Interplay with the estrogen receptor.

In the present report, we investigated the action of retinoic acid (RA) on the transactivation of the epidermal growth factor receptor (EGFR) gene promoter. In a previous study, we showed that the estrogen receptor (ER) α activated by 17ß-estradiol (E2) increased EGFR expression by enhancing the binding of the transcription factor Sp1 to the EGFR minimal promoter in HeLa cells. Here, we demonstrate that ligand-activated RA receptor (RAR) α inhibited EGFR transactivation by competing with Sp1 for binding to the same promoter fragment in the same cell model. When RARα and ERα were coexpressed, the inhibitory effect of RA on transactivation of the EGFR promoter counteracted the enhancement induced by E2-activated ERα and became more pronounced in the presence of ligand-free ERα. In the MCF7 breast cancer cell line, which endogenously expresses RARα and ERα, RA exerted anti-proliferative effects in the presence of ligand-free ERα. Moreover, interplay between the pathways mediated by the two receptors was observed, as RA counteracted E2-induced cell proliferation. Our results suggest that the interference with the activity of Sp1 on the EGFR promoter could be related to the observed RA-mediated growth suppression of breast cancer cells.

Up-regulation of the inflammatory-reparative phenotype in human prostate carcinoma.

BACKGROUND: Recent studies have underlined the role of tumor cells in the endogenous synthesis of pro-inflammatory molecules. We tested whether malignant progression in prostate cancer was associated with the activation of a phenotype typical of the innate immune system. METHODS: The expression of a set of molecules involved in tissue inflammation and repair was measured by real-time PCR and Western blot analysis in prostate samples in the absence or slight presence of a detectable leukocyte infiltrate. Whole tumor and non-tumor samples were analyzed in addition to laser-capture microdissected tumor and host epithelium. Receptor for advanced glycation end products, purine receptor, inducible enzymes cyclooxygenase-2 and nitric oxide synthase-2, pentraxin-3 and growth-survival factor receptors such as epithelial growth factor and estrogen alpha and beta receptors were all studied. RESULTS: A global survey approach showed an up-regulation in tumor samples of all of the studied genes, with the exception of ERbeta. A laser-capture microdissection approach highlighted over-expression of pro-inflammatory molecules in each tumor sample examined. Nuclear translocation of nuclear factor-kB subunit p65 was observed in tumor tissues. CONCLUSIONS: These data support the evidence that molecules typical of the innate immune system, similar to that of activated leukocytes, are produced by prostate epithelial cells and that their expression is up-regulated in malignant cells. We suggest that the observed pro-inflammatory and repair process activation may represent an important molecular mechanism in the progression of prostate cancer.

Oestrogens and selective oestrogen receptor (ER) modulators regulate EGF receptor gene expression through human ER alpha and beta subtypes via an Sp1 site.

Through the analysis of the transient expression of the luciferase reporter gene in HeLa cells, an evaluation has been made of the transcriptional activity of oestrogens and of selective oestrogen receptor (ER) modulators (SERMs), mediated by the alpha and beta isoforms of the ER, on the epidermal growth factor receptor gene promoter. Oestrogen-activated ERbeta presents a lower transcriptional activity compared with ERalpha, probably due to structural differences in the AF-1 regions of the receptors. Also SERMs induce different responses depending on the receptor isoform bound. Indeed, the phyto-oestrogens, genistein and daidzein, act as weak agonists of the oestrogenic activity via ERalpha, but as full agonists when bound to ERbeta. The synthetic SERM 4OH-tamoxifen, on the other hand, displays an opposite behaviour since it exerts a full agonist action through ERalpha, but acts as a full antagonist via ERbeta. As we have previously shown for ERalpha, an ERbeta/Sp1 functional synergism has also been highlighted, by means of gel mobility shift assays. Moreover, our results show that the sensitivity of target tissues to oestrogens and SERMs can be affected by coexpression of ERs, depending on the formation of appropriate levels of homo- and heterodimers, thus providing a useful approach to predict the effects of hormonal treatment.