Análisis de Varianza

Given the important roles that the AKT signalling pathway plays in EOC spheroids both in mediating responses to BMP signalling but also in contributing to spheroid formation-induced dormancy, I wanted to identify other candidate pathways known to interact with the PI3K/AKT pathway. Taking into account the dormant state of the cells within spheroids and their decreased metabolism an obvious candidate for investigation was the AMPK signalling cascade given its unique ability to respond to changes in extracellular energy and nutrient supply. The AMPK and PI3K/AKT signalling pathways converge on mTORC1 and have opposing regulatory effects on this complex20. We have demonstrated that in ovarian cancer spheroids decreased AKT activity and induction of cellular dormancy is associated with enhanced and sustained AMPK activity (Figure 4.1).

Other studies in our laboratory have linked cellular dormancy and quiescence within multicellular spheroids with induction of autophagy (Correa, DiMattia, and Shepherd, unpublished data). AMPK has the ability to directly induce autophagy by phosphorylating and positively regulating ULK1, a critical protein for autophagy initiation21-23. AMPK can also indirectly induce autophagy through its ability to inhibit mTORC1 by phosphorylation of TSC2 and Raptor. A study by Avivar-Valderas and colleagues in 2012 highlighted the potential for interaction between mTORC1, AMPK and a member of the unfolded protein response (UPR) pathway, protein kinase (PKR)- like endoplasmic reticulum kinase (PERK) in autophagy induction and anoikis-resistance

in mammary epithelial cells24. It was noted that in response to loss of ECM attachment

PERK was able to activate AMPK through its upstream kinase, LKB1 although the precise mechanism through which this occurs remains unclear. This suspension-induced AMPK activity was required for inhibition of mTORC1 and induction of autophagy. This was the first study to implicate the LKB1/AMPK/mTORC1 signalling cascade as a key regulator of anoikis in epithelial cells.

Figure 4.1: Contribution of BMP and LKB1/AMPK signalling pathways to ovarian cancer metastasis.

A number of pathways and processes are uniquely altered in ovarian cancer spheroids, aiding in cellular aggregation and induction of dormancy. Specifically, the BMP signalling pathway is down-regulated during ovarian cancer spheroid formation and aids in cellular aggregation and cohesion. Alternatively, the LKB1/AMPK signalling cascade is up-regulated during spheroid formation and contributes to spheroid formation-induced dormancy. Many of these signalling aberrations are reversed during spheroid reattachment. We propose that upon spheroid reattachment to serosal surfaces of various peritoneal organs (omentum, appendix, intestine), carcinoma-associated fibroblasts (CAFs) secrete a number of cytokines, such as BMPs and TGF-βs, to enhance proliferation, motility and invasion.

Applying these findings to our system, I chose to focus on LKB1 and mTORC1 as critical upstream and downstream mediators of AMPK signalling in EOC spheroids. As discussed in this thesis, suspension-induced AMPK activation in ovarian cancer cells is in fact associated with enhanced phosphorylation of its upstream kinase LKB1 and decreased activity of mTORC1. Surprisingly, targeted knockdown of AMPK did not have any effect on viability of cells within ovarian cancer spheroids. This was unexpected given the important role AMPK is known to play in cell survival under nutrient replete conditions. However, targeted knockdown of LKB1 results in significant reduction in viability of cells within multicellular aggregates, indicating an important role for this kinase in anoikis-resistance of ovarian cancer cells independent of AMPK.

As described in the introduction to this thesis, LKB1 phosphorylates 12 other kinases in addition to AMPK, termed the AMPK-related kinases (ARKs). Interestingly, one of these kinases, microtubule affinity-regulating kinase 4 (MARK4) has very recently been shown to phosphorylate Raptor on the same residue as AMPK, resulting in

inhibition of mTORC1 signalling25 (Figure 4.2). Another of these kinases, NUAK1 may

also be an interesting target to investigate in our system as it has been shown to be a

target of AKT in addition to LKB126. Similar to AMPK, NUAK1 is able to promote cell

survival during times of nutrient deprivation27. Although their functions are not as well characterized as AMPK, it is likely that one or more of the ARKs may be important mediators of the pro-survival functions of LKB1 in our system. Targeted knockdown of each of these ARKs (MARK1-4, NUAK1, NUAK2, SIK 1-3, SNRK, BRSK1, BRSK2) and assessment of cell viability in suspension would be the most effective method through which to determine our target(s) of interest.

These studies are the first to identify a pro-survival function for the tumour suppressor LKB1 in a metastatic cancer setting. Further studies will focus on examining the effect that LKB1 loss has on cellular quiescence and autophagy induction in ovarian cancer spheroids, as well as determining downstream mediators of LKB1 in our system. Perhaps we have uncovered a unique LKB1 signalling axis crucial in mediating anoikis- resistance in ovarian cancer cells.

Figure 4.2: Proposed mechanisms of dormancy induction in ovarian cancer spheroids.

AMPK and AKT have opposing regulatory effects on mTORC1 via phosphorylation of TSC2. mTORC1 activity is decreased in multicellular spheroids and this corresponds with decreased AKT activity and enhanced AMPK activation. LKB1 not only functions as an upstream kinase for AMPK but also for a number of AMPK-related kinases (ARKs). One of these ARKs, MARK4, phosphorylates Raptor leading to inhibition of mTORC1. This is another potential kinase with elevated activity in dormant ovarian cancer spheroids. We propose that LKB1 is an important mediator of spheroid-formation induced dormancy independent of AMPK.