Role of AHR in gliomas carcinogenesis

There have been a limited number of investigations on the potential role of AHR signalling in gliomas. However, the role of AHR in glioma carcinogenesis in the absence of exogenous ligand remains to be determined.

1.13.9.1. AHR expression in normal brain and gliomas

In rats, AHR and ARNT mRNAs were found widely distributed throughout the brain, especially in hypothalamic and brainstem regions involved in the regulation of appetite and circadian rhythms (Petersen et al., 2000). In other study, and also in murine cells, both astrocytes and endothelial cells express abundant levels of AHR protein in brain (Filbrandt et al., 2004).

In 2009, Gramatzki and coworkers, in order to predict the possible value of targeting AHR in human glioma patients in vivo, have studied the AHR expression in human normal brain and in a large series of human gliomas of WHO grade II-IV, using immunohistochemistry (Gramatzki et al., 2009).

The AHR gene was expressed in normal brain regions including cortex and white matter of frontal, parietal, temporal and occipital lobes, basal ganglia, thalamus, hippocampus, midbrain and cerebellum. On the other hand, no AHR expression was found in pyramidal hippocampal neurons, nigral neurons of the midbrain, pyramidal cortical neurons or granular cerebellar cells (Gramatzki et al., 2009).

A strong AHR expression was seen especially in astrocytes. Expression was observed in the cytoplasm and strongly in the nuclei of glial cells but not in endothelial cells of proliferating vessels. Both in normal brain and in gliomas the pattern of expression was heterogeneous (Gramatzki et al., 2009).

The mean AHR expression levels showed a decrease with WHO grade, showing the lowest levels in grade IV glioma, being the difference in the expression levels, between grade II gliomas when compared with grade IV gliomas, statistically significant. The authors have also found that the nuclear staining intensity was higher in WHO grade IV gliomas when compared with the grade II and III gliomas (Gramatzki et al., 2009).

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1.13.9.2. Epigenetic regulation of AHR expression in human cancer

Surprisingly few studies have been focused on the mechanisms that control expression of the human AHR gene. There are numerous examples in which alterations of the level of AHR expression determines the responsiveness of downstream pathways, however, the underlying cellular and molecular mechanisms responsible for the observed changes in AHR expression remain for the most part elusive (Harper et al., 2006).

It was demonstrated that expression of the AHR gene may be down regulated by epigenetic mechanisms, namely by DNA hypermethylation. Mulero – Navarro and co-workers analysed the promoter methylation status as an epigenetic mechanism regulating AHR expression in 19 human tumor cell lines, representing 16 human tumor types (Mulero-Navarro et al., 2006).

They found that only cell lines from acute lymphoblastic leukaemia and chronic myeloid leukaemia had significant AHR promoter hypermethylation. Twenty-one human primary acute lymphoblastic leukaemia tumors were also analysed and 33% of the patients presented AHR promoter hypermethylation (Mulero-Navarro et al., 2006).

In non-lympohoid human tumors, namely, from breast, colon, lung, melanoma, osteosarcoma, testis carcinoma, neuroblastoma, rhabdomyosarcoma and choriocarcinoma minimal AHR promoter hypermethylation was found (Mulero-Navarro et al., 2006).

The results from this study suggest that promoter hypermethylation could represent a mechanism regulating AHR expression in human tumor cells in a cell-specific manner, points AHR as a negative regulator of cell growth and proliferation in acute lymphoblastic leukemia in the absence of xenobiotics and supports the putative role of tumor suppressor of AHR gene (Mulero-Navarro et al., 2006).

AHR expression was also found to be regulated through CpG promoter hypermethylation in mantle cell lymphoma (MCL), in 32% of samples, and is correlated with higher proliferation, increased number of chromosomal abnormalities and shorter survival of the patients. Five genes, including AHR, (SOX9, HOXA9, AHR, NR2F2, and ROBO1) were frequently methylated in these tumors suggesting that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumors (Enjuanes et al., 2011).

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1.14. The Transforming growth factor beta

Transforming growth factor-beta (TGF-β) is a multifunctional regulatory polypeptide that is the prototypical member of a large family of cytokines which is comprised of over thirty members including activins, nodals, bone morphogenetic proteins, growth and differentiation factors. Is secreted by several cell types and controls many aspects of cellular function, including cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance, and survival (Jakowlew, 2006).

Three different isoforms have been identified in mammals, namely TGF-β1, TGF-β2 and TGF-β3, which share about 80% amino acid sequence identity (Roberts, 1998).

The active form of the TGF-β is a 25kDa dimer of two polypeptides stabilized by hydrophobic interactions strengthened by a bisulfite bond. Before binding to its receptors, TGF-β is activated from a large latent complex comprised of Latent TGF-β binding protein (LTBP) and Latency Associated Peptide (LAP).

Once activated, TGF-β signals through two classes of receptors, the TGF-β type I receptor (TβRI) and TGF-β type I receptor (TβRII). Type I and II receptors are serine/threonine kinase receptors that form a heterodimeric complex upon TGF-β binding. TGF-β interacts with TβRII which catalyses the phosphorylation of the TβRI and allows the subsequent incorporation of the TβRI generating a ligand-receptor complex formed by a ligand dimer, two TβRI and two TβRII. The only known function of the TβRII is to activate the TβRI. Once activated by the TβRII, TβRI phosphorylates Smads through its catalytic domain.

Smad proteins are transcription factor and only known TGF-β receptor substrates capable of signal transduction. Activated Smads regulate diverse biological effects by partnering with transcription factors resulting in cell-state specific modulation of transcription. The activity of the Smad pathway is terminated through a number of mechanisms including ubiquitinylation and proteosomic destruction of the Smads (Weiss and Attisano, 2013; Seoane, 2006; Rich, 2003).

In addition, TGF-β can signal through Smad-independent pathways (Moustakas and Heldin, 2005).

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1.14.1. TGF-β role in human carcinogenesis

The role of TGF-β in cancer is complex once it has a dual role acting as both a tumor suppressor in early tumorigenesis and tumor promoter in later cancer stages (Figure 24) (Derynck et al., 2001; Akhurst et al., 2001).

In normal epithelial cells and early well-differentiated epithelial tumor cells TGF-β acts as a suppressor of cell proliferation and apoptosis promoter (Massagué, 1998).

Studies shown that pancreatic cancers have non-functional TGF-β pathways that suggest that inactivation of TGF-β signalling pathways plays an important role in human pancreatic tumorigenesis and support the tumor suppressor role of TGF-β in those tumors (Goggins et al., 1998; Villanueva et al., 1998).

In contrast, in human colorectal cancer TGF-β is expressed in high levels and is associated with disease progression (Tsushima et al., 1996). In prostate cancer, TGF-β1 upregulation is associated with angiogenesis, metastasis and poor patient prognosis (Wikström et al., 1998).

Furthermore, a significantly greater number of invasive breast carcinomas expressed higher levels of TGF-β than in situ breast carcinomas and the strongest expression was observed in invasive carcinomas with associated lymph node metastasis, supporting a relationship between TGF-β production and tumor progression (Walker and Dearing, 1992; Gorsch et al., 1992).

The oncogenic role of TGF-β has prompted the development of therapeutic strategies based on the inhibition of the TGF-β pathway but a better understanding of the mechanisms that mediate the malignant transformation of TGF-β will improve the development of rational and successful therapeutic strategies.