*1.1. The Central Role of WNT Pathway Overactivation in the Tumorigenesis of ACP*

The one consistent genomic mutation that appears to be present in the majority, if not all, of ACPs is an activation mutation in the *CTNNB1* gene of the WNT/wingless pathway [9,11,16]. Most commonly this involves a point mutation in exon 3 of the *CTNNB1* gene. A number of studies have demonstrated various different mutations, most commonly involving serine or threonine phosphorylation sites encoded by exon 3 [13,17]. Ordinarily, and in the absence of WNT activation, beta-catenin is marked for destruction by a destruction complex consisting of AXIN, glycogen synthase kinase-3β (GSK3β), and APC, among other proteins. This complex binds to and phosphorylates specific residues encoded by exon 3 of *CTNNB1* and results in degradation of the protein [13,18]. In the presence of WNT activation, WNT ligands bind to Frizzled and its co-receptor LRP (Low-density lipoprotein receptor-related protein) at the cell membrane. This in turn leads to the activation of Disheveled (DVL) and the binding of AXIN at the cell membrane. Consequently, the normal destruction complex is broken up and beta-catenin is released. Eventually this stabilized beta-catenin will accumulate in first the cytoplasm, and subsequently the nucleus resulting in the expression of WNT pathway target genes [18]. In the pathological state present in ACP, the various point mutations prevent the binding of GSK3β to beta-catenin, and the subsequent phosphorylation of the serine and threonine residues. This results in a degradation-resistant form of beta-catenin, resulting in aberrant nuclear accumulation of the protein in certain cells within the tumor. In the nucleus, beta-catenin acts as a transcription factor, leading to overactivation of the WNT/beta-catenin pathway [16,18,19]. Although this aberrant overactivation of the WNT pathway is thought to be crucial in the pathogenesis of ACPs, the resulting nuclear accumulation of beta-catenin is only observed in a minority of cells, specifically in whorl like epithelial cell clusters (Figure 1D). These cells are thought to be crucial in the tumorigenesis of ACP and various mechanisms have been proposed as to how they may drive tumor growth [16,20,21] (Figure 2).

One such theory involves a paracrine mechanism whereby these cell clusters induce tumor growth by expressing a large array of growth factors, chemokines, and cytokines and act as a kind of signaling center that promotes tumor progression [21]. It has also been hypothesized that the nuclear accumulation of beta-catenin and overactivation of the WNT pathway in these cell clusters might also play a crucial role in the invasion of adjacent structures (e.g. hypothalamus and pituitary) in ACP [20]. Microscopically, a digitate invasion/growth pattern into structures such as the hypothalamus can be seen and is thought to be an important factor in the neuro-endocrine disorders frequently seen in children with ACP [3,22]. In addition, this invasive nature can preclude the neurosurgeon from obtaining a gross total resection at the time of surgery leading to tumor recurrence and a more aggressive clinical course. Hölsken et al. [20] noted that beta-catenin accumulating whorls/clusters are found at the tips of these invading projections of tumor and hypothesized that this may suggest a role for these clusters in the promotion of tumor invasion [20]. In addition, Apps et al. [23] used micro-CT to produce 3-D models of ACP tumor samples. Using this novel technique, they visualized cell clusters in tumor protrusions into surrounding tissue. In a separate paper, the same group used laser capture microdissection to separate out these cell clusters and analyze their transcriptomic profiles [10]. They found that these cell clusters express high levels of the FGF, BMP and WNT families of secreted factors and were able to demonstrate downstream activation of the MAPK/ERK that was particularly prominent at the tips of the invading tumor epithelium. These facts lend further credence to the theory that these clusters drive tumor invasion in a paracrine manner. Hölsken et al. [20] cultured a total of 6 ACP samples and measured their invasion capacity via two methods, namely Boyden chamber assays, and wound-healing assays. They then suppressed beta-catenin expression in the samples by introducing small interfering RNA (siRNA) directed against the *CTNNB1* gene and repeated the assays. They found that after treatment with the siRNA the accumulation of beta-catenin was significantly reduced and resulted in a significant decrease in tumor cell migration and invasion capacity [20]. They also demonstrated that the treatment with siRNA resulted in the reduced expression of the Fascin protein. Fascin is a member of the actin cross-linking family of proteins and plays a crucial role in cell-matrix adhesion, cell migration, and remodeling of the cell cytoskeleton/architecture [24,25]. In addition, the aberrant overexpression of the Fascin protein has been demonstrated in a number of cancers, including oral squamous cell carcinoma, and prostate cancer [24,26]. Hölsken et al. [20] demonstrated that the beta-catenin accumulating cells in ACP also over expressed Fascin. They then showed that treatment with the siRNA lead to a decrease in not only beta-catenin accumulation, but also Fascin levels. They proposed that this increase in Fascin expression may represent the mechanism by which WNT overactivation in these ACP cells may increase tumor cell migration and invasion into adjacent structures [20].

Given the seemingly crucial role of WNT overactivation in ACPs, targeting the WNT pathway would appear to represent an attractive strategy for tackling these tumors. The WNT pathway has been shown to play a crucial role in a number of cancers such as colorectal cancers, non-small cell lung cancer, and chronic myeloid leukemia [27,28]. This has resulted in significant efforts to better understand the pathway and to develop therapies that target it [27,28]. Despite all these efforts, no drug targeting the WNT pathway has been approved. The reasons for the difficulty in targeting the WNT pathway are legion and complex but one major area of concern is the important role the pathway plays in the maintenance of normal stem cells for tissue regeneration [27,29]. The potential issues that may arise with WNT pathway targeting was illustrated by Zhong et al. [30] who demonstrated significant intestinal toxicity associated with tankyrase inhibitors in mice.

Due to the difficulties that have been encountered in targeting the WNT pathway in more aggressive cancers, it seems likely that such therapies with acceptable efficacy and toxicity will remain elusive for some time to come [28]. It is unlikely such a therapy will become a viable option in the treatment of ACP in the near future and as a result, the need to discover other effective therapies has become imperative. ACP is a very rare disease and developing novel therapies specifically for this tumor type is currently not practical or realistic. As a result, much work has focused on identifying alternative targets with extant treatments, which may offer better results in the treatment of ACP. These efforts have resulted in the identification of multiple molecular pathways involved in the pathogenesis of ACP [6]. A number of these pathways result in the upregulation of pro-inflammatory/immune genes that may be amenable to targeted therapies [10,11,31–33]. The immune/inflammatory cells seen in ACP samples are varied and can include CD4-T-Lymphocytes, CD20-B-Lymphocytes, CD-68-Macrophages, and CD-56-NK cells. The presence of all these cells is not consistent among all ACP samples and this fact is reflective of the histologically heterogeneous nature of these tumors [34]. Work is ongoing to

investigate whether these pathways may present potential therapeutic targets and ultimately leads to better outcomes and reduced morbidity for patients. The following is a review of the evidence that highlights the potential importance of the inflammatory/immune response in the generation of these tumors and the potential in targeting these pathways in the treatment of this often-devastating disease.
