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A negative energy balance, resulting from both reduced production of ATP and increased energy expenditure is commonplace in cancer patients (Jager et al., 2007; Kemp et al., 2007; Kjobsted et al., 2018; Koh et al., 2008). The increase of energy expenditure in cachexia is prevalent in human and pre-clinical cachexia (Brown, Rosa- Caldwell, Lee, Blackwell, et al., 2017; Sandri, 2016; Tsoli et al., 2014; White, Baynes, et al., 2011b). The energy sensing molecule adenosine monophosphate protein kinase AMPK regulates energy metabolism and homeostasis in response to metabolic stressors such as fasting and exercise (Bujak et al., 2015; Friedrichsen et al., 2013; Kjobsted et al., 2018). AMPK is known for facilitating a host of metabolic processes and couples cellular energy status to protein synthesis. AMPK activity is disrupted during the progression of cancer cachexia (Hardee et al., 2017; Sanchez, Csibi, et al., 2012; Sandri, 2016). Multiple pre-clinical models of cachexia demonstrate an elevation of AMPK activity during the later stages of cachexia (Brown, Rosa-Caldwell, Lee, Blackwell, et al., 2017; Puppa et al., 2012; Rohm et al., 2016; White, Baynes, et al., 2011a). Often coinciding with the elevation of AMPK in cachectic muscle is the suppression of protein synthesis (mTORC1) and acceleration of degradation (E3 ligases and autophagy)

pathways (Brown, Rosa-Caldwell, Lee, Blackwell, et al., 2017; Puppa et al., 2012; Rohm et al., 2016; White, Baynes, et al., 2011a). While this induction of AMPK activity has been established, very little has been done to mechanistically examine the role of AMPK in the regulation of protein turnover. Our laboratory has previously suggested that AMPK inhibition in cultured myotubes incubated with IL-6 and LLC media is capable of restoring mTORC1 and protein synthesis (White, Baynes, et al., 2011b; White, Puppa, Gao, et al., 2013). However, recent evidence has suggested that the timing of AMPK activation may contribute to cachexia induced muscle mass loss (Rohm et al., 2016). Activation of AMPK via AICAR immediately following C26 tumor implantation attenuated tumor growth and cachexia induce muscle mass loss (Rohm et al., 2016). Taken together these results although ambivalent, definitively demonstrate a role for AMPK in the regulation of muscle mass during cancer cachexia. The current dissertation is the first study to mechanistically examine the cellular processes AMPK regulates in response to fasting, feeding, and activity during cancer cachexia.

AMPK Regulation of TSC2 and Raptor: Fasting, Feeding, and Activity in Cachexia

An intriguing question among researchers is whether cachectic muscle retains anabolic plasticity (Hardee et al., 2017). This could prove beneficial for clinicians looking to provide treatment and improve muscle mass and function in cachectic cancer patients. While circadian fluctuations in protein turnover are the established norm in muscle mass regulation, it has become evident that protein synthesis is highly responsive to environmental stimuli (Chang et al., 2017; Lefta, Wolff, & Esser, 2011). Fasting, feeding, and activity are extremely potent regulators of skeletal muscle protein synthesis

perturbed sensitivity to these anabolic signals (Argiles, Lopez-Soriano, et al., 2015; Bodine, 2013; Bowen et al., 2015). Short term fasting is a normal physiological process that often occurs during sleep and contributes to the diurnal variation of protein turnover (Lefta et al., 2011). Research has demonstrated that short term fasting in normal healthy skeletal muscle does not activate the AMPK signaling pathway is well tolerated (Bujak et al., 2015; Canto et al., 2010; Galic et al., 2018). However, our laboratory was one of the first to suggest that the chronic activation of AMPK may be responsible for the cachexia disruption of protein synthesis after an acute 5 hour fast (White, Baynes, et al., 2011a). The current studies demonstrate that the fasting induction TSC2 and Raptor require AMPK in cachectic skeletal muscle. Furthermore, we demonstrate that cancer cachexia accelerates the skeletal muscle physiological response to fasting by robustly inducing AMPK after a mere 12-hour fast during the light cycle. Min mice that were fasted for 12-hours during the light cycle exhibited a robust activation of both TSC2 and Raptor phosphorylation by AMPK which were not present in their fed counterparts. The AMPK phosphorylation of TSC2 and Raptor are believed to serve as a metabolic checkpoint that shuts off mTORC1 activity (Gwinn et al., 2008). However, recent evidence has suggested that the phosphorylation of Raptor and not TSC2 is critical in this process (Gwinn et al., 2008).

Since cancer cachexia cannot be reversed by conventional nutritional supplementation, it is also necessary to further our understanding of anabolic resistance (Bruggeman et al., 2016; Dillon et al., 2012; Hardee et al., 2017). Growing evidence is supporting the disruption of protein synthesis during cancer cachexia; however, very few studies have examined if nutrients or feeding can stimulate anabolic signaling (Evans et

al., 2008; A. M. H. Horstman et al., 2016; Kitagawa, Haji, & Amagai, 2017). We provide evidence in the current studies that 1-hour of refeeding following a 12-hour short term fast suppresses AMPK and its phosphorylation of downstream targets TSC2 and Raptor. Interestingly, this suppression of AMPK signaling was not reflected in downstream puromycin incorporation which is indicative of protein synthesis. The disparity between the two may be an issue of timing as the refeeding paradigm was only 1 hour. However, it is interesting to speculate how long the suppression of AMPK occurs for post feeding, the decrease in phosphorylation may only be an acute transient suppression. Furthermore, the composition of the food may contribute to protein synthesis as well, cancer patients are still capable of inducing protein synthesis in response to protein ingestion however, the degree of activation appears to be severely impaired (Dillon et al., 2012). A newer approach using specially formulated medical food that is high in protein and leucine has shown promise in cancer patients with involuntary weight loss and may be applicable to the current data presented (Deutz et al., 2011).

Physical activity and exercise have been deemed critical regulators of skeletal muscle AMPK (Canto & Auwerx, 2009; Z. P. Chen et al., 2000; Friedrichsen et al., 2013; Laker et al., 2017). Acute exercise has been extensively investigated to induce skeletal muscle AMPK to compensate for the energy stress (Friedrichsen et al., 2013; Jorgensen et al., 2005). Increased physical activity as a treatment modality has become an important area of interest in cancer cachexia (Alves et al., 2015; Argiles et al., 2012; Grande et al., 2014). Numerous studies have investigated the association between physical activity, developing cancer, and skeletal muscle protein turnover (Ali & Garcia,

2014; Alves et al., 2015; Argiles et al., 2012; Aversa et al., 2017; Grande et al., 2015; Hardee et al., 2016; Hardee et al., 2017; Khamoui et al., 2016). Studies and clinical trials have also suggested that prognosis is improved in physically active cancer patients due to exercise performed after the cancer diagnosis (Gould et al., 2013; Grande et al., 2015). Additionally, similar results have been reported in pre-clinical models of cancer cachexia as well, wheel running activity in C26 cancer mice blocked the induction of AMPK mediated degradation pathways in gastrocnemius muscle (Pigna et al., 2016b). In the current study we provide evidence that 4 weeks of voluntary wheel activity prior to fasting is sufficient to inhibit AMPK phosphorylation and activation of Raptor. These results also demonstrated that wheel activity blocked the fasting suppression of protein synthesis. Our laboratory has previously demonstrated that two models of contraction are capable of suppressing AMPK and inducing protein synthesis in cachectic skeletal muscle (Puppa, Gao, et al., 2014; White, Baynes, et al., 2011a). Seven bouts of high frequency electrical stimulation as well as an acute bout of low frequency stimulation were able to suppress AMPK, restore mitochondrial biogenesis, and induce protein synthesis in Min and LLC mice (Hardee et al., 2016; Puppa, Murphy, et al., 2014).

AMPK Mediated Protein Degradation in Cachexia

It remains mostly unclear how tumors initiate the signal to stimulate muscle protein degradation and cachexia. The often anatomically distant location between tumors and the wasting muscles are suggestive of tumor derived factors such as cytokines into circulation to initiate signaling processes that cause muscle wasting (Baracos, 2006a; Bowen et al., 2015; Johns et al., 2012). A hallmark feature of cancer cachexia is the net loss of skeletal muscle protein through selective degradation pathways (Penna et al.,

2014; Pettersen et al., 2017; Tisdale, 2009). A consistent finding that translates from pre- clinical models to the clinic is the elevation of muscle protein degradation while synthesis is suppressed (Penna, Ballaro, Beltra, De Lucia, & Costelli, 2018). While cancer cachexia may be a multifactorial condition, clear evidence suggest that the ubiquitin proteasome system and autophagy lysosomal system are major contributors to muscle mass regulation (Penna, Costamagna, et al., 2013; Sandri, 2016; Yuan et al., 2015).

AMPK is a critical regulator of both the ubiquitin proteasome system through FOXO3a and the autophagy lysosomal system through ULK-1 (Ju et al., 2016; K. Nakashima & Yakabe, 2007; Sandri, 2016). Phosphorylation of these two downstream targets induces both E3 ligases and autophagy indices (Ju et al., 2016; K. Nakashima & Yakabe, 2007; Sanchez, Csibi, et al., 2012; Sandri, 2010, 2016). Activation of these two degradation pathways has been studied extensively in catabolic conditions such as fasting and cachexia and are believed to be key mediators of muscle mass loss (Ju et al., 2016; K. Nakashima & Yakabe, 2007; Pettersen et al., 2017; Pigna et al., 2016b; Sandri, 2016). AMPK regulation of FOXO3a and ULK-1 has been demonstrated through loss and gain of function studies (Sandri et al., 2004). Treatment of mice with AICAR infusion has demonstrated a robust activation of FOXO3a and E3 ligases Atrogin-1 and MuRF-1 (Sanchez, Csibi, et al., 2012). Multiple pre-clinical models and human cancer patients have reported elevated E3 ligase expression (Bodine & Baehr, 2014; Bowen et al., 2017; Mueller et al., 2016b). In addition to E3 ligase expression, the process of autophagy has recently become a target of interest in cachexia (Penna et al., 2014). Both human cancer patients and pre-clinical models of cachexia have demonstrated inductions of the autophagy lysosomal system (Penna, Costamagna, et al., 2013). In the current

dissertation we demonstrate that a short term 12-hour fast induces FOXO3a and ULK-1 phosphorylation in Min mice and this induction requires AMPK. The phosphorylation of these proteins leads to the induction of Atrogin-1, MuRF-1, and LC3B (II/I). Interestingly, in line with our protein synthesis data described above, re-feeding following a 12-hour fast suppressed the induction of these proteolytic systems. The suppression of these signaling processes by re-feeding suggest that despite the failure to induce protein synthesis, lowering AMPK via re-feeding may alleviate the elevation of muscle protein degradation acutely. While the fasting induction of these proteins is not a new concept the current dissertation is the first to report these findings in cachexia and that acute re-feeding is capable of suppressing that fasting activation. AMPK mediated protein degradation during a fast in healthy skeletal muscle is typically not activated until 24-48 hours of fasting is achieved (Galic et al., 2018). We demonstrate that cachectic skeletal muscle induces these pathways after only 12-hours.

Many cancer patients suffer from chronic fatigue, either from the disease itself or its treatment, which is often a confounder that limits regular exercise practice (Grande et al., 2015). This lack of physical activity or exercise has been demonstrated to occur in both the clinic and pre-clinical models (Grande et al., 2015; Roberts et al., 2013). While the targeting of aberrant protein degradation processes has been suggested as a potential treatment for cachexia, it is entirely plausible that inhibiting protein degradation could prove detrimental to systemic metabolism and muscle function (Cohen, Nathan, & Goldberg, 2015). Furthermore, damaged proteins induced by exercise must be removed for successful regeneration and muscle function. Muscle protein degradation is stimulated by both acute endurance and resistance exercise (Vainshtein, Tryon, Pauly, & Hood,

2015; Wolfe, 2006). In contrast, exercise training can decrease indices of protein breakdown in muscle, which may be related to improved protein quality in previously cachectic muscle (Vainshtein et al., 2015). Indeed, voluntary wheel running decreased muscle E3 ligase mRNA expression and autophagy protein expression in tumor bearing mice (Pigna et al., 2016b) . In the current studies we demonstrate that 4 weeks of voluntary wheel activity suppressed the fasting induction of Atrogin-1,MuRF-1, P62 and LC3B. These results are in line with previous reports demonstrating treadmill exercise prior to tumor inoculation decreased muscle protein breakdown in rats (Salomao, Toneto, Silva, & Gomes-Marcondes, 2010). While exercise-induced improvements have not been observed in all preclinical studies, this could be related to variables such as tumor type or exercise intensity (Khamoui et al., 2016).