IMPORTANCIA DE LA COMUNICACIÓN EFECTIVA EN LA FAMILIA

Advanced-stage prostate cancer is often associated with skeletal complications related to the spread of this disease to its preferred metastatic site, the bone. In fact, approximately 90% of patients that die as a result of prostate cancer have bone metastases (Bubendorf, Schöpfer et al. 2000). Typically, when the disease reaches this stage, a mixed osteolytic/osteoblastic bone response can be seen in the same patient at different metastatic sites (Roudier, Morrissey et al. 2008), although the interactions between the prostate cancer cells and the bone matrix predominantly yield an osteoblastic response. While the pathological events that take place during prostate cancer metastasis are well established, there is only a limited understanding of the exact molecular events involved. So far, studies have implicated a few protein families in this process (Coleman 2006), the BMP family being one of them.

As previously discussed, there are over 20 members of the BMP family which exert their effects through a heteromeric complex of two types of serine threonine kinase transmembrane receptors, termed type-I and type-II (Bragdon, Moseychuk et al. 2011). The extracellular BMP homo- or hetero-dimers utilise three type-I receptors, BMPRIA, BMPRIB and ActRIA, and three type-II receptors, BMPRII, ActRIIA and ActRIIB. When the BMP ligands bind to the BMPRs, they initiate the Smad-dependent or the Smad-independent pathway to subsequently modulate the transcription of target genes affecting key cellular processes like cell survival, apoptosis, migration and differentiation (see figure 1.7 for schematic of signalling pathways). The Smad- dependent pathway is initiated when the BMP dimer binds to a preformed complex of homodimeric type-I and type-II BMPRs, resulting in the phosphorylation of a regulatory domain within the type-I receptor, known as the GS box. The catalytically activate type-I receptor then recruits and phosphorylates BMP-specific intracellular members of the Smad family, R-Smads 1, 5 or 8/9, on their conserved SSXS motif. Once activated, the R-Smads dissociate from the type-I

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receptor, presumably due to a change in conformation, and form an oligomeric complex with another R-Smads and a Co-Smad (Smad4), which, with the help of nuclear import and export factors, transits into the nucleus to regulate the transcription of target genes. In addition to this type of pathway, BMP signalling may also exert its effects by completely bypassing the use of Smads via a pathway known as the Smad-independent pathway. For this pathway to be triggered, the BMP dimer needs to interact with just one of the BMPR dimers, triggering the recruitment of a second BMPR dimer for the formation of the heteromeric BMPR complex. This pathway then typically proceeds through one or more MAPK signalling pathways (ERK, p38 or JNK) to indirectly bring about changes in target gene transcription.

Since BMPs are most commonly known for their inherent osteoinductive capacities, many have hypothesised their involvement in osteoblastic lesion formation arising in advanced prostate cancer. This was a theory that was first brought forward by Bentley and colleagues in the early 90s, who, by screening for the mRNA expression of BMPs, demonstrated the presence of BMPs 1-6 in prostatic adenocarcinoma (Bentley, Hamdy et al. 1992). As part of this study, they also compared the expression levels of these BMPs between patients with skeletal metastases and those without, reporting BMP-6 to be selectively expressed in bone-scan positive metastatic disease. Henceforward, the expression of BMPs has been examined in the different stages of prostate cancer to assess their roles in this disease. For example, Bobinac et al (2005) reported the expression of BMP-2, -4, -5, -6 and -7 in normal prostate tissue, while prostate carcinoma samples predominantly expressed BMP-2 and -4, with significantly decreased levels of BMP-7. Spanjol et al (2010) reported mostly similar findings, demonstrating an increased expression of BMP-6 and decreased levels of BMP-7 in localised prostate cancers, and bone metastases expressing high levels of BMP-2, -4, -6 and -7. Contrary to Bentley et al however, Spanjol and colleagues reported decreased expression levels of BMP-2/4, -6 and -7 in metastatic prostate cancers. Thus, despite the slightly conflicting data from these expression studies, the evidence

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gathered has indicated BMP-2, -4, -6, and -7 to be potential players in the pathophysiology behind skeletal metastases produced by prostate cancer.

Physiologically, BMPs constitute a pivotal group of morphogenetic signals that orchestrate nearly all of tissue architecture throughout the vertebrate body (Hogan 1996). As such, BMP activity is tightly regulated at different levels of signalling, from the intracellular phosphatases and I-Smads, to the extracellular pseudoreceptor BAMBI, and BMP antagonists. Interestingly, BMP antagonists have been shown to be particularly integral to BMP function, not only due to their canonical inhibitory capacities but also as agonists when present in low concentrations (Walsh, Godson et al. 2010). This dual feedback between BMPs and their antagonists is mainly demonstrated in developmental studies. For instance, while it is well established that BMPs, BMP-2, BMP-4 and BMP-7 more specifically, are critical in limb-bud development, their activity is carefully modulated by their antagonists, with disruptions or alterations in this ligand- antagonist balance leading to congenital malformations (Walsh, Godson et al. 2010, Pignatti, Zeller et al. 2014). Furthermore, BMP antagonist action has also been shown to be crucial in bone formation, with BMP exposure inducing the production of such antagonists as Noggin, Follistatin and Gremlin by osteoblasts, thus binding and sequestering BMP action ultimately enabling proper skeletal development (Gazzerro, Gangji et al. 1998, Pereira, Economides et al. 2000, Abe, Abe et al. 2004). For example, studies on Noggin null mice have reported a failure to initiate joint formation in test mice, as well as excessive cartilage amongst other skeletal anomalies, which were likely brought on due to excessive BMP action (Tylzanowski, Mebis et al. 2006).

Given the information gathered above, we hypothesise that since the feedback between BMPs and BMP antagonists is so crucial in physiological conditions that perturbations in this feedback could also participate in the progression of prostate cancer and more particularly, the spread of

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this disease to the bone. Indeed, due to the impact BMPs and their antagonists have on skeletal phenotype, it is possible that alterations in their relationship may have a role in the type of lesions produced during prostate cancer related bone metastasis. The study presented in this chapter aims to investigate this hypothesis by assessing the expression profiles BMP and their antagonists in prostate cancer cell lines associated with different bone lesion phenotypes. By also evaluating the expression profiles of related receptors and some of the intermediate signalling molecules, we also hope to elucidate parts of BMP signalling that could be in play.