REFLEXIÓN SOBRE LA LENGUA Y SU APRENDIZAJE 1. Conocimiento de la lengua

The data presented in this thesis have shown that FASN affects the stability of p65 protein, and that the specific phosphorylation site Thr254 is involved in p65 protein stability, thereby indicating that FASN may affect

phosphorylation of p65 at this site. Though it appears palmitate is likely involved in regulating p65 protein stability downstream of FASN according to the data in

Figure 8 of this thesis, it remains largely unclear how FASN is able to impact

phosphorylation of p65 at Thr254 to affect p65 protein stability. As discussed earlier in the introduction, palmitate is known to play a significant role in cancers through a variety of processes. Specifically, it has been shown that protein modification through palmitoylation of cysteine residues can have a direct effect on the activity of signaling molecules such as Wnt proteins and Ras GTPases

(Rohrig and Schulze 2016; Eisenberg et al. 2013). As a result, a logical hypothesis for the role of FASN in affecting phosphorylation of p65 at Thr254 would likely begin with investigation into a potential role for protein palmitoylation, whether that be directly on p65 to perhaps affect the conformation of the protein and disrupt phosphorylation, or on a different protein to induce a subsequent effect on p65 phosphorylation.

As shown in Appendix A, blocking palmitoylation with an inhibitor resulted in a decrease in total p65 protein level in M3K doxorubicin resistant breast

cancer cells. This finding suggests that high FASN expression does not lead to direct palmitoylation of p65 to suppress p65 expression. Further supporting this finding, many proteomics studies have been carried out to determine

palmitoylated proteins in various mammalian genomes. Compilation studies of these protein palmitoylomes, though finding many proteins appearing to be palmitoylated that have significant roles in cancer, did not find p65 or any other NF-kB family proteins to be palmitoylated (Sanders et al. 2015; Ko and Dixon 2018).

Though it does not appear likely FASN leads to the palmitoylation of p65, it is possible FASN impacts p65 expression through an indirect mechanism that involves palmitoylation, seeing as blocking palmitoylation activity resulted in a decrease in p65 protein level. In such a mechanism, when FASN expression is high, FASN production of palmitate could lead to the palmitoylation of a kinase, subsequently leading to the suppression of the activity of that kinase to prevent phosphorylation of p65 at Thr254 and disrupt p65 protein stability. Another

possible mechanism could involve FASN-mediated palmitoylation of a

phosphatase, wherein palmitoylation results in increased phosphatase activity. Neither the canonical kinase responsible for the phosphorylation of p65 at Thr254, nor any phosphatases responsible for removing phosphoryl groups from this site are known. Based upon the uncovered relationship between FASN and this p65 phosphorylation site, a logical place to start investigating a potential kinase or phosphatase in the context of breast cancer cells would be to first investigate those known to have a mechanistic relationship with FASN.

One particular kinase, PKC, has been linked to both FASN and p65. Specifically, one study has shown that, in response to FASN inhibition with the cerulenin derivative C93, the classical PKC isoform PKCa was activated and able to phosphorylate IkBa to result in its degradation and the subsequent activation of NF-kB (Lemmon et al. 2011). Further, multiple PKC isoforms have been found to be pamitoylated in proteomics studies, while the palmitoylation of PKCe has been verified in in vitro studies (Sanders et al. 2015; Dasgupta et al. 2011). Interestingly, PKC is commonly known to translocate to the nucleus upon its activation. Given that it is believed the phosphorylated form of p65 is likely present in the nucleus, this suggests that activated PKC and p65 in this context would both be localized in the nucleus. As a result, it is possible FASN may affect the palmitoylation of a specific PKC isoform, thereby affecting its ability to

phosphorylate p65 at Thr254.

Multiple studies have also linked FASN inhibition to the activation of AMP- activated protein kinase (AMPK) (Landree et al. 2004; Zhou et al. 2007). Though

it has not been found in palmitoylome studies that AMPK can be palmitoylated, it is possible AMPK is involved in this process, as it has been shown that AMPK activation can increase NF-kB nuclear translocation and DNA binding activity (Liu et al. 2010). As a result, examining the potential relationship between FASN, NF- kB/p65, and these kinases may lead to the determination of a more detailed mechanistic understanding of the ability of FASN to regulate NF-kB/p65.

In further consideration of the effects on total p65 level with the inhibition of palmitoylation activity shown in Appendix A, though 2-bromopalmitate is known to block palmitoylation of proteins, there is also evidence to suggest that it can behave similarly to palmitate in cells with respect to signaling processes (Resh 2006). For example, it has been shown that 2-bromopalmitate treatment can lead to gene expression changes through activation of peroxisome

proliferator-activated receptors (PPARs), which are receptors for which palmitate is a common ligand (Brandes et al. 1995; Bastie et al. 2000). As a result, it must be considered that 2-bromopalmitate treatment in breast cancer cells could, in fact, be acting as a palmitate analog and could be affecting total p65 protein level not through a mechanism involving inhibition of palmitoylation, but rather through a cell signaling mechanism that is independent of palmitoylation activity and mirrors the effects of exogenous palmitate treatment. If this is the case, it is possible palmitate affects total p65 protein stability by a mechanism involving its other roles in cell signaling, such as modulation of membrane composition and lipid raft formation.