THE ROLE OF FoxO1 DURING CANCER CACHEXIA

By

BRANDON ROBERTS

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2016

© 2016 Brandon Roberts

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ACKNOWLEDGMENTS

The process of earning a doctorate is long, demanding, and is certainly not done

alone. I would like to express the deepest appreciation to my advisor, Dr. Andrew

Judge, for his mentorship. He has taught me how to think like a true academic. His wit

and lightheartedness have kept my spirits high when experiments fail. While his intellect

and persistence has encouraged me to grow. I would also like to thank past and present

members of the Judge lab for their friendship and help - specifically, Dr. Sarah Judge

and Dr. Adam Beharry, who have provided critical support and have truly helped me to

develop as a scientist. In addition, I would also like to thank, Dr. Leo Ferreira, for his

guidance and collaboration throughout my graduate studies. Furthermore, I am indebted

to my committee members, Dr. Krista Vandenborne, and Dr. Scott Powers, for their

guidance and for being exceptional role models in academia. Importantly, I would like to

thank my friends and family for their support throughout my studies. And finally, I would

like to thank Lisa for all of her support and understanding.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................................................................................................. 3

LIST OF FIGURES .......................................................................................................... 6

LIST OF ABBREVIATIONS ............................................................................................. 7

ABSTRACT ..................................................................................................................... 8

CHAPTER

1 LITERATURE REVIEW ............................................................................................. 10

Cachexia ................................................................................................................. 10 Cancer Cachexia .................................................................................................... 11 C26 Cancer Cachexia ............................................................................................. 12 Upstream Mediators of Cancer Cachexia ............................................................... 16 Anabolic Signaling During Cancer Cachexia .......................................................... 19 Catabolic Signaling During Cancer Cachexia ......................................................... 22 FoxO in Cancer Cachexia ....................................................................................... 24

2 EXPERIMENTAL OUTLINE ...................................................................................... 30

Experimental Outline............................................................................................... 30 Specific Aim ............................................................................................................ 30 Hypothesis .............................................................................................................. 30 Experiments ............................................................................................................ 30

3 METHODS ................................................................................................................ 31

Animals ................................................................................................................... 31 Cancer Cachexia .................................................................................................... 31 RNA Isolation, cDNA Synthesis and RT-PCR ......................................................... 31 Histochemistry ........................................................................................................ 32 Electron Microscopy................................................................................................ 32 AAV Vector & Delivery ............................................................................................ 33 Microarray ............................................................................................................... 33 Statistical Analysis .................................................................................................. 33

4 RESULTS AND DISCUSSION .................................................................................. 35

FoxO1 in Cancer Cachexia ..................................................................................... 35 C26 Mice Characteristics ........................................................................................ 35 Muscle Wasting in Cancer Cachexia ...................................................................... 36

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FoxO1 Signaling Increases in Cancer Cachexia ..................................................... 37 FoxO1 is Necessary for Muscle Wasting in C26 ..................................................... 38 Knockdown of FoxO1 Rescues Ultrastructural Changes in C26 Skeletal Muscle ... 40 Microarray Analysis to Identify Direct or Indirect FoxO1 Target Genes during

C26 Cancer Cachexia.......................................................................................... 42 FoxO1 is Necessary for Cancer-induced Downregulation of Genes Encoding

Collagen and Sarcomeric Proteins ...................................................................... 43 Validation of Transcripts Regulated by FoxO1 ........................................................ 45 Ubiquitin Proteasome Pathway Enriched Among FoxO1 Targets Upregulated in

Cachectic Muscle ................................................................................................ 45 FoxO1 Mediates Part of the Autophagy and Apoptosis Systems ............................ 46 FoxO1 May Alter Cytokine and Other Upstream Signaling ..................................... 48 FoxO1 Regulation of Transcription Factors ............................................................ 51

5 CONCLUSION AND FUTURE DIRECTIONS ........................................................... 53

LIST OF REFERENCES ............................................................................................... 71

BIOGRAPHICAL SKETCH ............................................................................................ 79

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LIST OF FIGURES

Figure page 4-1 Whole body wasting during cancer cachexia ...................................................... 57

4-2 Body characteristics of cancer cachexia ............................................................. 58

4-3 Muscle wasting during cancer cachexia ............................................................. 59

4-4 FoxO family of transcription factors are increased in cancer-induced muscle wasting. .............................................................................................................. 60

4-5 FoxO1 is required for cancer-induced muscle wasting. ...................................... 61

4-6 Muscle fiber cross sectional area in the TA. ....................................................... 62

4-7 Muscle fiber representative images of the TA..................................................... 63

4-8 Gene expression of TA.. ..................................................................................... 64

4-9 Identification of differentially regulated genes. .................................................... 65

4-10 DAVID analysis of the most highly enriched processes from upregulated and downregulated FoxO1 target genes. .................................................................. 66

4-11 MSigDB analysis of the most highly enriched processes from upregulated and downregulated FoxO1 target genes (FC≥±1.4fold). ..................................... 67

4-12 Heatmap of gene expression changes.. ............................................................. 68

4-13 Transcription factor binding. ............................................................................... 69

4-14 Enriched gene networks downregulated via FoxO1 during cancer cachexia. ..... 70

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LIST OF ABBREVIATIONS

ActRIIb

CSA

DMSO

FoxO

activin type-2 receptor

cross-sectional area

dimethyl sulfoxide

Forkhead boxO

FoxO1 Forkhead box O1

FoxO3 Forkhead box O3

H&E

HAT

HDAC

hematoxylin and eosin

histone acetyltransferase

histone deacetylase

IFN- γ interferon γ

IGF-1 insulin-like Growth Factor 1

IL-1 interlukin-1

IL-6 interlukin-6

MuRF1 muscle RING finger 1

NF-B nuclear factor B

PBS phosphate buffered saline

PCR polymerase chain reaction

PI3K phosphatidylinositol 3-kinase

TA tibialis anterior

TNF-α

DAVID

tumor necrosis factor-α

database for annotation, visualization and integrated discovery

RT-PCR reverse transcription polymerase chain reaction

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

THE ROLE OF FoxO1 DURING CANCER CACHEXIA

By

Brandon Roberts

August 2016

Chair: Andrew R. Judge Major: Rehabilitation Science

Cancer cachexia is a devastating condition that affects up to 80% of patients with

advanced stage cancer. This metabolic syndrome results in significant skeletal muscle

wasting and weakness. Importantly, the muscle wasting translates to a reduced physical

function and independence. Therefore, developing treatments to prevent cancer

cachexia is important to enhancing the quality of life and survival of cancer patients.

However, a better understanding of the mechanisms which drive muscle wasting during

cancer is needed. Recent data published from our lab demonstrates Forkhead box O

(FoxO)-dependent transcription is required for muscle fiber atrophy in a C26 model of

cancer cachexia. Using a dominant negative (d.n.) FoxO AAV to repress FoxO

transcriptional activity we have shown attenuation of atrophy and force deficits in the

muscles of cachectic mice. However, this d.n. FoxO construct was highly homologous

with FoxO3a, FoxO1 and FoxO4 which could have prevented the activation of multiple

FoxO family members. Consequently teasing out the role of each individual FoxO factor

is challenging. New data from our lab has shown elevated levels of FoxO1 mRNA, but

not FoxO3a in muscle biopsies from cachectic pancreatic cancer patients. In

agreement, others show a 5-fold increase in FoxO1 in the abdominal muscle of

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cachectic patients. Therefore, in the current study we aimed to determine the role of

FoxO1 in skeletal muscle wasting during cancer cachexia. We injected a short hairpin

(sh) FoxO1 adeno-associated virus to knock down FoxO1 expression in vivo. Our data

shows shFoxO1 AAV can attenuate the muscle weight and muscle fiber size loss during

cancer cachexia. Next, we used an Affymetrix Gene 2.0 ST array to identify differentially

regulated genes. We analyzed muscle taken from control and C26 mice injected with a

scrambled shorthairpin AAV or shFoxO1 AAV (-1.4 ≥ fold change ≥ 1.4). We identified

5,823 genes that were differentially regulated in C26 compared to control. Furthermore,

of those genes we identified 2,007 which were direct or indirect FoxO1 target genes.

Subsequent analysis identified 960 of these genes were upregulated ≥1.4 fold change

and 1047 had fold change ≤-1.4 in shFoxO1 C26 compared to C26. Based on these

findings we have identified several novel FoxO1 target genes in skeletal muscle.

Cumulatively, these findings identify FoxO1 as an important regulator of cachexia-

induced muscle wasting.

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CHAPTER 1 LITERATURE REVIEW

Cachexia

Until 2008, there was no widely agreed upon definition of cachexia. However,

that year a group clinicians and scientists convened for the cachexia consensus

conference. They defined cachexia as a complex metabolic syndrome characterized by

whole body wasting that is associated with an underlying illness (Evans et al, 2008).

The prominent clinical feature of cachexia is weight loss. Cachexia is also commonly

associated with anorexia, inflammation, insulin resistance and increased muscle protein

breakdown. It is important to note that cachexia is distinct from malnutrition, sarcopenia,

and hyperthyroidism and is associated with increased morbidity.

The clinical criterion for a diagnosis of cachexia was also established at this

consensus group. The main component is a 5% loss of edema-free body weight during

12 months or less. Patients must also present with three of five criterions which include

decreased muscle strength, fatigue, anorexia, low muscle mass, and biochemical

abnormalities. There is also a classification system in respect to body weight loss with

mild cachexia being >5%, moderate >10% and severe cachexia >15%. This standard

definition has allowed the unification of cachexia research and diagnostic evaluation

(Evans et al, 2008).

Muscle wasting is an important part of the pathology of cachexia. The increased

loss of skeletal muscle mass separates cachexia from weight loss that occurs from a

reduction in energy intake. Numerous independent investigators have suggested that

actin and myosin are selectively targeted for degradation in conditions associated with

cachexia (DeJong et al, 2005). Indeed, one research group concluded that a common

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transcriptional program is associated with multiple conditions with skeletal muscle

atrophy including: uremia, fasting, cancer and diabetes. (Lecker et al, 2004). Since

skeletal muscle wasting is a common feature of cachexia, therapies targeting

degradation pathways may be effective in reducing the loss of muscle mass.

Cancer Cachexia

Cachexia occurs in up to half of all patients diagnosed with cancer and

represents a significant physical and psychological burden (Dewys et al, 1980). For

example, the classic patient with advanced cancer cachexia demonstrates severe

weight loss, anorexia, loss of appetite, weakness and anemia (Fearon & Preston, 1990).

These patients also present with reduced: muscle mass, muscle glycogen and

mitochondrial number. All of these effects cumulate in a decrease in quality of life

(Fearon et al, 2006), decreased physical performance, increase risk of failed treatment,

and increased mortality rate. Therefore, cachexia plays an integral role in morbidity of

cancer patients. Moreover, cancer cachexia accounts for over a quarter of cancer-

related deaths, which is thought to be due, in part, to the wasting of skeletal muscle.

Cancer results from genetic changes and the identification of genes involved in

tumor development. Progress in the area of genetics has been a prominent goal of

cancer research for decades. Although it is important to catalogue genome variants,

there are obvious difficulties in bridging the gaps between high-throughput sequencing

information and the molecular mechanisms behind cancer. In this regard, there is an

unprecedented need for animal models of cancer. Interestingly, mice with naturally

occurring oncogenic mutations have provided important information along with

genetically engineered mice. These two have emerged as important tools for both drug

discovery and mechanistic research in the cancer field.

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C26 Cancer Cachexia

In the 1970s there was an effort to establish an animal derived colon tumor

model for biological and therapeutic studies. One of the first tumors to survive the initial

transplant experiments was the colon tumor 26. The C26 line was deemed highly

tumorigenic and had a low tendency to metastasize in addition to having a high mortality

rate (Sato et al, 1981). This animal model was shown to suffer from extensive weight

loss (~40%) with no change in food intake (Tanaka et al, 1990). Importantly, this has

become the standard model for cancer cachexia research.

The C26 model of experimental cancer cachexia has recapitulated limb muscle

weakness, with several groups independently showing a decrease in maximal tetanic

force in the extensor digitorum longus (EDL) and tibialis anterior (TA) (Gorselink et al,

2006). These muscles are commonly studied during cancer cachexia because they are

mainly glycolytic muscles. Notably, cancer cachexia is known to affect glycolytic

muscles to a greater extent than oxidative muscles (Reed et al, 2012).

Until recently there were no studies showed a significant difference when

comparing specific force in control and C26 mice. Specific force is defined as force

normalized to cross sectional area, or mass. Our lab was among the first to

demonstrate specific force deficits in the C26 model. This indicates that the muscle is

intrinsically weaker and that the weakness is not entirely due to a decrease in muscle

mass (Roberts et al, 2013b). In this study we found a significant decrease in maximal

tetanic force in the solei and EDL, which agrees with previous research. However, we

also found a significant 17% decrease in specific force of the solei from C26 mice

compared to controls. This is considerably less than the 30% decrease in maximal

tetanic force. Furthermore, the soleus of C26 mice also showed an increase in

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fatigability, and prolonged contraction and relaxation times demonstrating contractile

deficits in this muscle. The soleus is a postural muscle therefore its weakness and

increased fatigability during cancer cachexia could play a role in compromising physical

function.

We also determined the contractile properties of the EDL to identify whether our

model of severe cancer cachexia also plays a role in affecting the contractile function of

a glycolytic muscle, which was not found to show statistically significant decreases in

other experiments. Yet, we found that the EDL was significantly decreased by 13%

when measuring maximal specific force, compared to control.

The decreases in both the oxidative and glycolytic muscles suggest that, in our

model of severe cancer cachexia, the muscle weakness is a result of additional factors

other than muscle atrophy. Indeed, electron micrographs have shown disorganization of

myofibrils in mice during cancer cachexia which could contractile dysfunction and

altered molecular signaling.

In addition to limb muscle, it has also been reported diaphragm function is

compromised in cachectic mice. In the early 1990s it was shown that there was no

change in diaphragm muscle mass during cancer cachexia in Yoshida ascites

hepatoma AH-130 rats (Tessitore et al, 1993). Likewise, a study done in 2011 by

Murphy et al., showed no change in the muscle fiber cross sectional area of the

diaphragm in Lewis lung carcinoma (LLC) mice (Murphy et al, 2011). However, this

study only had a moderate (9%) decrease in body weight compared to controls and also

used a different line of cancer cells. We have recently found a significant decrease in

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CSA of the diaphragm during severe cancer cachexia. Furthermore, when we measured

specific force we also found a significant decrease compared to control.

Although it is unclear to know what these force deficits in the diaphragm were

caused by, there is potential for it to be due to alteration in membrane excitability, Ca2+

handling or the contractile apparatus as seen in limb muscle. Some patients with cancer

demonstrate respiratory muscle dysfunction, shown by an inability to generate

inspiratory pressure and volume. This results in decreased airflow and inability to cough

effectively. If airway clearance is impaired, it could cause the development of

pneumonia or other respiratory related problems. It is presumed that respiratory

muscles play an integral role in morbidity and mortality in cancer patients.

Interestingly, a study completed by Murphy et al., reported a 10% lower specific

force and absolute force using strips of diaphragm from C26 mice that lost ~22% body

weight, which would be classified as severe cachexia (Murphy et al, 2012). Our lab has

also found that mice bearing C26 tumors have decreased isometric diaphragm specific

force compared to control (Roberts et al, 2013a). These previous two studies are

consistent, showing diaphragm weakness could explain the possible impairment of

generation of airway pressure and lung volume. Indeed, during baseline conditions,

using the rapid shallow breathing index, it was found that C26 mice are significantly

elevated compared to controls. During brief hypoxic exposure control mice are able to

increase breathing frequency and minute ventilation, but mice bearing C26 tumors are

not able to. This clearly demonstrates that severe experimental cancer cachexia causes

ventilatory deficits due to muscle weakness. The lack of cancer mice to increase minute

ventilation during a challenge suggests that human patients may also have a

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compromised ability to increase ventilation, such as from walking up a flight of stairs. It

is also possible that parts of this ventilatory deficit are due to neural control of breathing.

There are several explanations for the impairments in contractile properties in

the diaphragm of cachectic mice. First, the sarcolemma is disrupted, causing membrane

excitability or intracellular signaling events to occur. We have found both sarcolemma

morphology and permeability to be altered in cachectic mice. Another possibility is

disruption of calcium homeostasis, which could be affected by disorganization of the

sarcoplasmic reticulum and has previously been shown (de Oliveira et al, 2011). We

also found significant targeted degradation of myosin heavy chain, which could explain

the contractile deficits.

Our studies of permeabilized single-fibers in the diaphragm lend further insight

into potential mechanisms of contractile dysfunction in cancer. We found a significant

decrease in calcium-activated specific force in the diaphragm (18%) of C26 mice

compared to controls. This could indicate a reduced number in force generating cross

bridges. Other experiments have shown that force kinetics were 30% slower in

cachectic mice compared to controls. These measurements reflect the rate of cross-

bridge binding from weakly bound, to strongly bound, which causes force generation. A

reduced rate of strong binding could contribute to decreases in maximal force. C26

cancer cachexia also decreases calcium sensitivity and cooperativity of the contractile

apparatus.

Breathing requires diaphragm shortening, which induces changes in contractile

properties. Accordingly, we found that cancer impaired Vmax and peak power in the

diaphragm. However, since the force-velocity relationship was unchanged, the decrease

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in peak power is caused by the decrease in Vmax and Po. The decrease in Vmax can

reflect reductions in the level of activation and cross-bridge cycling. Our experiments

from single fiber studies suggest both mechanisms explain the decrease in Vmax, Po and

peak power. Hence, the decreased level of activation and cross bridge slowing play an

important role in ventilation.

One other variable that can alter muscle function is fiber-type distribution, which

has previously been shown to shift toward more glycolytic fibers during C26 cancer

cachexia in the soleus. However, this shift did not reach significance in the plantaris,

gastrocnemius, or TA. Moreover, it is unlikely a small shift in fiber type in skeletal

muscle during cancer cachexia could cause the contractile changes that we find.

Cumulatively, all of these findings suggest that skeletal muscle has a heterogenic

response to cancer cachexia, with a decrease in maximal force in multiple muscles

during severe cancer cachexia. Understanding the contributing factors to skeletal

muscle weakness for clinically relevant outcomes is vital. The literature has established

a healthy standard for use of the C26 model. This recapitulates the diagnostic criteria

for cachexia including: decreased muscle strength, weight loss >5%, fatigue, and

abnormal biochemistries.

Upstream Mediators of Cancer Cachexia

The muscle wasting that occurs during cancer cachexia is driven by factors that

affect the metabolic activity of the whole body. These metabolic changes are thought to

be due to factors secreted by the tumor. Tumor burden can grow up to 15% of total

body mass in animals, but is generally less than 1% in humans. Therefore, this

indicates factors secreted from the tumor effect mechanisms on a metabolic level. This

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is demonstrated by the heterogeneity of muscle atrophy and dysfunction throughout the

entire body in cancer patients suffering from cachexia.

The syndrome caused by cancer cachexia is multifactorial and cannot be

reversed by dietary intervention. For several decades, investigators have searched for

mediators of cancer cachexia in hope of developing novel therapeutics to protect

against tumor-induced adipose and skeletal muscle loss. One of the most researched

mediators is Tumor Necrosis factor α (TNF α), a proinflammatory cytokine. Initially

called “cachectin”, it was associated with wasting in rabbits with leishmaniasis(Beutler &

Cerami, 1986). Later, this cytokine was shown to induce cachexia in mice (Oliff et al,

1987) as well as inhibiting both adipocyte and skeletal myocyte differentiation (Guttridge

et al, 2000). Additionally, TNFα is sufficient to promote atrophy in cultured myotubes by

inducing E3 ligases that mediate protein breakdown (Li et al, 2005). It is important to

note that the origin and relevance of TNFα to cancer cachexia is unclear, since there is

much controversy in the literature to whether these cytokine levels increase in human

patients with cancer. Furthermore, recent trials of anti-TNFα antibodies in patients with

cancer cachexia have shown no benefit (Jatoi et al, 2010). It is possible that TNFα is a

facilitator but is not sufficient to promote muscle atrophy in humans and that additional

signaling activities are needed.

Another candidate that may interact with TNFα as a driver of systemic

inflammation in cancer cachexia is Interleukin 6 (IL-6). The pathway of IL-6 signaling is

through its membrane bound receptor, gp130, which leads to activation of the

JAK/STAT pathway with activated STAT proteins then translocated to the nucleus.

Numerous cancer types secrete IL-6 and this can be amplified by host-derived

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proinflammatory cytokines. Unlike TNFα, circulating levels of IL-6 have been shown to

correlate to weight loss in cancer patients and reduced survival (Scott et al, 1996).

Studies also show the requirement of IL-6 in regulating experiments in cachectic mice

(Strassmann et al, 1993). However, only supraphysiological doses of IL-6 are able to

induce muscle atrophy in the absence of a tumor (Baltgalvis et al, 2008). An IL-6

dependent mouse model of cachexia showed suppression of muscle protein synthesis

occurred early and increased with progression of body weight loss. This could explain

the increase in muscle wasting with the progression of the cancer phenotype. Recent

trials of anti-IL-6 antibody in lung cancer patients have shown reversal of fatigue,

anorexia, and anemia but have not shown any effects on loss of muscle mass (Bayliss

et al, 2011). This may implicate IL-6 as only a factor in the cachectic phenotype, not the

source driving muscle wasting.

One of the recent factors associated with cancer cachexia is myostatin, a

Transforming Growth factor Beta (TGF-β) superfamily of cytokine member. It has a

clear role in muscle hypertrophy since animals and human that are genetic nulls show

significant increases in muscle hypertrophy (Mosher et al, 2007). Myostatin is created

and secreted from muscle cells, then activates signaling through the activin type II

receptor, which recruits the Activin Receptor-like (ALK) family kinase, resulting in

activation of Mothers Against Decapentaplegic, Drosophila, Homolog Of, 2 (Smad2/3)

transcription complex (Trendelenburg et al, 2009). Indeed, overexpression of myostatin

in mice leads to significant skeletal muscle atrophy (Zimmers et al, 2002). However,

these mice are only hypertrophic, showing no increases in muscle strength. The

mechanism by which myostatin causes muscle loss has not been fully discovered, but

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experiments suggest that there might be multiple pathways that inhibit Protein Kinase B

(Akt) and downstream Mammalian Target of Rapamycin (MTOR) pathways that control

protein synthesis. Recent studies have used an Activin Receptor 2B (ActRIIB) receptor

trap to treat cancer cachexia in the C26 model. This trap blocked cachexia in the C26

model without effecting tumor growth. Importantly, these mice experienced a 30%

increase in survival rates (Zhou et al, 2010). The myostatin pathway may not be the

main cause of muscle wasting in cancer cachexia, but these studies show that it plays

an important role.

One novel factor found to be increased in cachectic patients is an increase in IL-

8, which has been ignored as a mediator of cancer cachexia (Krzystek-Korpacka et al,

2007). IL-8 was also found to be predictive of survival (Reitter et al, 2014) and is

frequently found in the serum of cachectic patients. More work is needed to determine

its role during cachexia.

Anabolic Signaling During Cancer Cachexia

Cancer cachexia is a multi-factorial disease. While anorexia has been associated

with cancer cachexia and may play a role in patient weight loss, it is not the major cause

of skeletal muscle wasting, since it can be reversed through nutritional intervention.

Indeed, patients on total parenteral nutrition, with a perfectly controlled diet, still lose

weight and show cachexia symptoms (Evans et al, 1985).

Cachectic patients may have a shift in metabolism. For example, energy

expenditure has been found to vary between 50-150% of predicted values in resting

cancer patients (Lelbach et al, 2007). Moreover, patients with types of cancer that are

frequently associated with cachexia may have elevated resting energy expenditure.

Inefficient energy metabolism can include unnecessary heat production and

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unnecessary metabolic cycles such as the Cori Cycle to meet the demands of the tumor

(Tisdale, 2002).

Skeletal muscle is metabolically active and accounts for ~40% of total body

mass. Therefore, a change in metabolism could also be caused by increased protein

turnover. Muscle plays an important role by regulating the metabolism of substrates

such as glucose. It has also been recently reported to function as an endocrine organ

since it has the ability to secrete myokines and IL-6 to communicate with other tissues

(Pedersen & Febbraio, 2008).

Currently, the only validated treatment for cancer cachexia is exercise, which has

the ability to reduce muscle atrophy (Lira et al, 2014). However, exercise is not a viable

option for frail, bed-ridden, or older individuals with other chronic diseases. Thus, there

is an urgent need to develop therapeutic interventions that can increase muscle mass.

Moreover, the need for a drug therapy that can increase muscle function, quality of life,

and independence is crucial to increase survival.

A range of mediators are associated with the cancer cachexia, including multiple

pro-inflammatory cytokines such as TNFα, IL-6 and interferon-gamma (IFNγ).

Hormones such as insulin-like growth factor 1 (IGF-1) and glucocorticoids have also

been implicated. IL-6 and TNFα antibodies have previously been mentioned, so now

we’ll focus on IGF-1.

IGF-1 is an anabolic hormone which binds to the insulin receptor and IGF-1

receptor which is regulated by specific binding proteins. IGF-1 promotes protein

synthesis and inhibits protein catabolism by activation of the Phosphoinosidide 3-kinase

(PI3K) and Akt pathway. Circulating IGF-1 has been found to correlate with weight loss

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in patients with lung cancer. Specifically, it was found to be significantly lower in patients

with more than 10% weight loss compared to those with less than 10% weight loss

(Simons et al, 1999). However, treatment with IGF-1 failed to reverse cachexia in tumor

bearing murine models. This suggests that alternative pathways may have a dominant

role in muscle wasting.

Myostatin, a major inhibitor of muscle growth, binds to the activin A receptor type

II B (ActRIIB) in skeletal muscle, activating SMAD2 and SMAD3 transcription factors.

This activation leads to the activation of the atrophy pathways and the inhibition of

satellite cell activation (McCroskery et al, 2003). Interestingly, C-26 mice injected with

soluble activin receptor IIB to antagonize the ActRIIB pathway show no loss of muscle

mass or decreased function. Importantly, they show an extended life span compared to

C-26 mice with the same tumor burden and cytokine levels (Zhou et al, 2010).

Furthermore, this decoy receptor has prevented cachexia development in multiple

cancer models, while increasing muscle function. Moreover, when this treatment was

started after the development of cachexia, it completely reversed not only muscle loss,

but also the loss of cardiac mass, even though multiple pro-inflammatory cytokines were

high(Zhou et al, 2010). Thus, myostatin has the potential to be a therapeutic

intervention during cachexia. There is currently one clinical trial in process using a

myostatin-specific antibody in humans with pancreatic cancer (NCT0105530).

However, another clinical trial (NCT01099761) that was using myostatin decoy

receptors to treat muscular dystrophy was terminated due to bleeding. This side effect

was probably caused the binding of other TGF family members to the circulating

receptors, and could presumably be avoided by a more selective antibody-based

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approach. Novartis has also begun clinical trains with an antibody (BYM338) that binds

to ActRIIb and therefore specifically prevents myostatin. These trials were considered

by the FDA for accelerated (Lach-Trifilieff et al, 2014) approval.

Significant progress has been made recently in our understanding of the

molecular mechanisms that mediate the loss of muscle mass during cancer cachexia.

Several novel mechanisms have been discovered and used as drug targets, with

multiple clinical trials currently ongoing. These therapeutic trials involve major

challenges for the pharmaceutical industry. Specifically, evaluating strength, nitrogen

balance or aerobic capacity is challenging, especially in older patients or those who are

bed-ridden. Perhaps the most dramatic affect that could occur is an increase in quality

of life and extended lifespan, whether this is through manipulating muscle mass or other

avenues. Although these are considerable challenges, the medical benefits of such

therapies will be monumental.

Catabolic Signaling During Cancer Cachexia

The loss of skeletal muscle mass is due to the imbalance of protein synthesis

and protein degradation. This holds true in cancer cachexia, with the previously

mentioned mediators controlling the upstream signaling of atrophy. There are multiple

signaling mediators that are required to increase the expression of key E3 ligases. E3

ligases include muscle RING finger-containing protein 1 (MuRF1) and muscle atrophy F

box protein (Atrogin-1/MAFbx). These two E3 ligases mediate sarcomeric breakdown

and inhibition of protein synthesis (Glass, 2010). MuRF1 is upregulated in multiple

models of muscle atrophy and is responsible for mediating the ubiquitination of myosin

heavy chain (MyHC) and other thick filament components (Clarke et al, 2007). Atrogin-1

is also a marker of muscle atrophy and is upregulated in multiple models of cachexia

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(Gomes et al, 2001). The literature suggests that both MurF1 and Atrogin-1 contribute to

skeletal muscle atrophy. Indeed, in the absence of either MuRF1 or Atrogin-1, rates of

atrophy are diminished (Bodine et al, 2001). Therefore, it is thought that cytokines and

other mediators secreted by tumors induce downstream signaling pathways that

upregulate E3 ligases to induce skeletal muscle degradation.

One signaling pathway upstream of these E3 ligases that is commonly increased

during cancer cachexia is the FoxO signaling pathway. There are three Forkhead box O

(FoxO) family members in skeletal muscle (FoxO1, FoxO3a, and FoxO4). The

importance of this family of transcription factors has been demonstrated in a number of

genetic studies. Overexpression of FoxO1 or FoxO3a is sufficient to cause skeletal

muscle atrophy in murine models (Senf et al, 2010). Furthermore, inhibition of FoxO

transcriptional activity by a dominant negative (DN) FoxO partially attenuates muscle

atrophy during immobilization (Reed et al, 2012). The regulation of muscle mass by

FoxO transcription factors is due to its regulation of atrophy-related genes. For example,

four genes whose expression levels are commonly upregulated during models of

skeletal muscle atrophy are: Atrogin-1, MuRF1, cathepsin L, and Bnip3 (Milan et al,

2015). There is evidence to support each of these as FoxO target genes. Indeed,

overexpression of FoxO3a is sufficient to cause an increase in both Atrogin-1 and

MuRF1 promoter reporter (Senf et al, 2010) and Atrogin-1 mRNA (Sandri et al, 2004).

Expression of cathepsin L, a lysosomal protease, is increased in muscles

overexpressing FoxO1 (Kamei et al, 2004). Kamei et al., also showed a FoxO1

transgenic mouse has significant muscle atrophy and a fiber type switch compared to

control.

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It was first shown that a cachectic condition increases FoxO transcriptional

activity in skeletal muscle by our lab in 2012. This study found that FoxO transcriptional

activity is increased in two metabolically distinct skeletal muscles during cancer and

sepsis. Furthermore, inhibition of FoxO transcriptional activity in control muscles

increased satellite cell proliferation and fusion of myofibers along with inhibiting

myostatin transcription.

There is only a small amount of data in humans on FoxO signaling during cancer

cachexia. One group measured the ratio of phosphorylated FoxO3a to total FoxO3a and

found that it is significantly lower in the abdominal muscles of humans with pancreatic

carcinoma (Schmitt et al, 2007). Moreover, FoxO1 was also upregulated in the muscle

of human cancer patients, and was recently identified as a cachexia-associated gene

(Skorokhod et al, 2012). Cumulatively, these studies show that both human cancer

patients and animal models of cancer cachexia support that FoxO transcription factors

play a role in skeletal muscle atrophy.

FoxO in Cancer Cachexia

As previously mentioned, findings from both human cancer patients and multiple

animal models have implicated the role of FoxO in cancer cachexia. Indeed, our lab has

used an adeno-associated virus (AAV) to knockdown FoxO1/FoxO3/FoxO4 during a C-

26 model of cancer cachexia (Judge et al, 2014b). This study found that blocking FoxO

expression prevented C26-induced muscle fiber atrophy of both locomotor muscles and

the diaphragm. Furthermore, it significantly attenuated muscle force deficits. This

sparing of muscle mass and function was associated with the differential regulation of

over 500 transcripts which increase or decrease during C26. Dominant negative (d.n.)

FoxO was injected with an AAV9-d.n.FoxO construct which also expressed GFP as a

25

non-fusion protein to help visualize it. Importantly, the d.n. FoxO sequence consists of

only that which codes for the FoxO3a DNA binding domain, but it shares 85% homology

with the binding domain of FoxO1 and 75% sequence homology with that of FoxO4, all

of which share greater than 90% sequence conservation within this region. The d.n.

FoxO thereby acts by outcompeting endogenous FoxOs for the FoxO binding elements

(FBE). Since it lacks a transactivation domain, this blocks FoxO dependent

transcription.

Using AAV, we were able to achieve >95% transduction efficiency in muscle

fibers in the TA and diaphragm, but only ~75% transduction of the fibers in the EDL.

The average cross-sectional area of muscle fibers in the TA, EDL and diaphragm were

all significantly attenuated in mice bearing C26 tumor bearing mice with AAV9-dnFoxO

compared to C26 tumor bearing mice. To determine whether this attenuation of muscle

mass was carried over to muscle function, the EDL was harvested for in vitro contractile

measurements. We found that EDL muscles from C26 mice showed a 40% decrease in

maximum absolute force and an 11% decrease in specific force when compared to

muscles in C26 mice transduced with an empty vector. However, muscles from C26

mice transduced with AAV9-dnFoxO showed a 28% decrease in absolute force and a

6% decrease in specific muscle force, when compared to muscle from C26 mice

transduced with an empty vector. It is important to note that although the reduction in

force deficits by dnFoxO was not complete, these data are comparable with the effect

on fiber size in EDL muscles of C26 mice, where we only found a partial sparing of

CSA. Furthermore, contractile properties of the EDL demonstrated no differences in

time to peak tension. Conversely, half-relaxation time was significantly elevated in

26

response to cancer cachexia, which was abolished in muscles transduced with AAV9-

dnFoxO. Cumulatively, this data show that FoxO dependent transcription is necessary

for muscle wasting induced by cancer cachexia in the limb and diaphragm (Judge et al,

2014b).

FoxO factors are well known to regulate genes involved in protein degradation

and this was confirmed by the previous study. Importantly, it provided novel evidence

that during cancer FoxO is necessary for the regulation of various atrophy-related

transcription factors, including Signal transducer and activator of transcription 3 (Stat3),

Myocyte enhancer factor 2c (Mef2c), and CCAAT/enhancer binding protein beta

(Cebpb). This demonstrates FoxO is a critical factor controlling diverse transcriptional

networks in skeletal muscle during cancer cachexia, and that it could play a role in the

muscle atrophy program through other transcription factors.

To systematically identify gene networks that were changed in response to C26

cancer cachexia which require FoxO-dependent transcription, TA muscles with AAV9-

dnFoxO or empty vector were taken for microarray analysis. After identifying over 2,000

genes that were differentially expressed between C26 injected with AAV9-dnFoxO and

control mice plus empty vector (fold change ±1.5, q

27

related to protein degradation. An additional seven pathways were associated with the

inflammatory processes.

The identification of Stat3 and Cebpb as targets of the FoxO family during cancer

cachexia was novel since both of these has been shown to regulate muscle atrophy.

Indeed, Cepbp is necessary for LLC-induced muscle wasting (Bonetto et al, 2011) and

Stat3 is necessary for C26 induced muscle wasting (Zhang et al, 2011). To further

establish FoxO factors were regulators of these atrophy-related transcription factors,

mRNA levels of Stat3 and Cebpb were measured after muscles were injected and

electroporated with constitutively active FoxO1 (FoxO1 TM) or constitutively active

FoxO3a (FoxO3a TM). FoxO1 TM significantly increased the mRNA levels of Cebpb (3-

fold) and Ubr2 (3.5.-fold). Ubr2 is a bona fide Stat3 target gene.

This manuscript was the first to perform a genome-wide microarray analysis of

transcripts regulated (directly or indirectly) by FoxO factors in skeletal muscle during

cancer cachexia. It was previously well known that FoxO factors regulated genes

involved in protein degradation (which our study confirmed), but it also provided new

evidence that FoxO is necessary for the increased expression of multiple atrophy-

related transcription factors. In conclusion, this study found FoxO to be a critical

transcription factor for skeletal muscle wasting in C26 cancer cachexia. Furthermore, it

puts FoxO in the category of controlling various gene networks which make up the

response to cachexia (Judge et al, 2014b).

Another recently published manuscript shows specific deletion of FoxOs in

skeletal muscle can attenuate the muscle loss and dysfunction in response to multiple

muscle wasting conditions (Milan et al, 2015). This group used FoxO1/3/4-floxed mice

28

crossed with a transgenic line expressing Cre recombinase under the control of a

muscle-specific MLC1f promoter. Interestingly, these triple knockouts were similar in

appearing to control mice and histological analysis revealed normal muscle

characteristics. Under conditions of muscle wasting FoxO1/3/4 knockout mice were

spared from muscle loss and dysfunction. Furthermore, contractile proteins were

decreased (myosin) in controls mice compared to the FoxO1,3,4 knockout mice. These

findings suggest that FoxO factors play an important role in muscle atrophy and

function.

Further experiments revealed 29 of the 63 atrophy related genes require FoxO

for normal induction during muscle wasting. Indeed, the induction of genes involved in

protein breakdown was fully blunted in knockout mice. To substantiate their findings,

somatic deletion of FoxO in adult mice via tamoxifen-inducible muscle-specific FoxO

knockout model was used. Similar to previous findings, somatic deletion of FoxO1,3,4

attenuated muscle loss and dysfunction during a muscle wasting condition. To

determine if these atrogenes requires direct binding of FoxO factors, a ChIP was done.

They found FoxO3a was recruited to the promoter region of the following atrogenes:

atrogin-1, MuRF1, Bnip3, LC3, Cathepsin L and 4EBP1. Furthermore, they found

FoxO1 was recruited to the promoter regions of: MuRF1, p62, Cathepsin L and TGIF.

This indicates that some genes are regulated by different FoxO family members. These

findings strongly support the FoxO family as a central regulator of the muscle atrophy

program in multiple wasting conditions (Milan et al, 2015). It is important to note that all

of this work with the FoxO1,3,4 null mice was done in muscle wasting induced by

starvation or denervation. While this is may be a useful tool to discover the molecular

29

signaling pathways, it may not be the most physiological. Therefore, there is an unmet

need to discover the role of independent FoxO family members play during a

physiological condition.

As proven by multiple experiments from independent labs, the FoxO family of

transcription factors plays a diverse role in muscle wasting conditions. However, to date

no experiments have attempted to tease out the importance of the separate FoxO family

members.

We believe FoxO1 may be the central regulator of muscle wasting during cancer

cachexia. Recent transcriptome-wide characterization of skeletal muscle biopsies

utilizing the Real-Imaging cDNA-AFLP approach from pancreatic cancer patients shows

FoxO1 is five-fold increase in the abdominal muscle, but FoxO3a was not increased

(Skorokhod et al, 2012). Furthermore, unpublished data from our lab shows FoxO1

mRNA is increased 6 fold in the muscle of pancreatic cancer patients with >15% weight

loss. Therefore, it is the aim of this study to determine the extent to which FoxO1

regulates skeletal muscle mass, ultrastructure and identify FoxO1 dependent gene

networks in C26 cancer cachexia.

30

CHAPTER 2 EXPERIMENTAL OUTLINE

Experimental Outline

The overall goal of this study is to isolate the role of FoxO1 in the regulation of

skeletal muscle mass during C26 cancer cachexia.

Specific Aim

To determine the extent to which FoxO1 regulates skeletal muscle mass,

ultrastructure and identify FoxO1 target genes during cancer cachexia.

Hypothesis

Short hairpin FoxO1 will attenuate muscle atrophy, rescue muscle morphology

and regulates numerous target genes that play a role in the extracellular matrix,

metabolism, and contractile apparatus.

Experiments

1) Validate knockdown of FoxO1 in vivo during cancer cachexia. We will also

measure other FoxO isoforms to determine if there is any compensatory upregulation.

2) Determine muscle fiber size, morphology and ultrastructure during FoxO1

knockdown in cancer cachexia.

3) Identify novel FoxO1 target genes during cancer cachexia using an Affymetrix

Gene 2.0 ST array. Use Bioinformatics to functionally categorize target genes for

analysis.

31

CHAPTER 3 METHODS

Animals

Male CD2F1 mice were purchased from Charles River Laboratories (Wilmington,

Massachusetts) weighing 20g at 8 weeks old and were used for C26 cancer cachexia

experiments. All animals were maintained in a temperature controlled facility with 12-h

light/dark cycle. Food and standard diet were provided ad libitum. The University of

Florida Institutional Animal Care and Use Committee approved all animal procedures.

Cancer Cachexia

Colon-26 (C26) cells were received from the National Cancer Institute Tumor

Repository (Frederick, MD, USA). These cells were passaged in RPMI1640 media

(Mediatech, Herndon, VA,USA) supplemented with 10% Fetal Bovine Serum, 100

micrograms per milliliter streptomycin, 100 U/ml penicillin at 37◦C in a 5% CO2

humidified chamber. Cancer cachexia was induced by subcutaneous injection of 1

x10^6 cells into each flank, and muscles were removed for analysis when mice reached

endpoint (24-28 days).

RNA Isolation, cDNA Synthesis and RT-PCR

RNA was extracted from the TA using TRIzol as previously described (Senf et al,

2008). RNA (1 microgram) was reverse transcribed to create cDNA using Ambion’s

RETROscript first-strand synthesis kit (Ambion, Austin, TX, USA). cDNA was used as a

template for real-time PCR using the primers listed below and a 7300 real-time PCR

system (Applied Biosystems, Foster City, CA, USA). TaqMan probe based chemistry

was used to detect PCR products and quantification was performed using a relative

standard curve. Primers used were FoxO1, FoxO3a, FoxO4, Atrogin-1, Bnip3, and

32

MuRF1. All primers were purchased from Applied Biosystems. 18S expression was

used for normalization, if necessary.

Histochemistry

Transduction of AAV was determined in 10um cross-sections via direct

visualization of Green Fluorescent Protein (GFP) using a Leica DM500B microscope

(Leica Microsystems, Wetzlar, Germany) following incubation with alexa flour-

conjugated wheat germ agglutinin (Invitrogen) for ~20 minutes. Leica application suite,

version 3.5.0 software was used to trace and measure muscle fiber cross-sectional area

as previously described (Roberts et al, 2013a). For H& E staining, slides were brought

to room temperature prior to sequential submersions in the following solutions: 100%

ethanol for 1 min, 70% ethanol for 1 min, dH20 for 2 min and Gill's Hematoxylin for 2

min. Sections were then washed thoroughly in dH20 followed by sequential submersions

in the following solutions: Scott's Solution for 15 seconds, dH20 for 2 seconds, 70%

ethanol for 1 minute, Eosin for 2 minutes, 95% ethanol with gentle shaking for 1 minute,

100% ethanol for 30 seconds and Xylene for 3 minutes. Slides were allowed to dry for

30 minutes and then mounted with glass cover-slips using Permount.

Electron Microscopy

Mice were anesthetized using isoflurane and the TA was removed by cutting a

0.5 x 0.2 x 0.2cm strip of muscle from the sample, with the longitudinal axis of the

muscle parallel to the in vivo direction of the muscle. The strip of muscle was then

immediately placed in a buffer solution. The buffer solution consisted of 2.5%

glutaraldehyde and 2% paraformaldehyde in 0.1 M PBS, pH 7.4, and was allowed to

incubate for 24 h at 4°C. The samples were then rinsed with PBS, post-fixed with 1%

OsO4 for 2 h at room temperature, dehydrated with acetone and embedded in Epon

33

812. Muscle was analyzed using a Hitachi 7600 with both digital camera and film

camera.

AAV Vector & Delivery

The shorthairpin FoxO1 adeno-associated virus used to attenuate the increase in

FoxO1 due to cancer cachexia was from Vector Biolabs (SKU# ADV-209271). Mice

were anesthetized with isoflurane gas delivered through a nose cone. The vector was

diluted in lactated Ringer solution so that 1x10^11 vector genomes were injected in 25ul

into the TA. For AAV delivery small incisions were made the lateral leg and the TA was

exposed. This minimally invasive technique causes widespread transduction of the TA.

AAV was injected into the TA seven days prior to C26 inoculation to allow for maximal

AAV expression at endpoint.

Microarray

An Affymetrix GeneChip Gene 2.0 ST array system for mouse was used. Samples were

pooled from three muscles in each group and sent to the Boston University Medical

Campus Microarray and Sequencing Resource.

Statistical Analysis

All data were analyzed using either a 2-way ANOVA followed by a Bonferroni

post hoc test, a 1-way ANOVA followed by a Bonferroni post hoc test, or a Student’s t

test (GraphPad Software, Sand Diego, CA, USA). All data are expressed as means ±

standard deviation. Significant was established at P≤ .05. The website

http://www.cs.uiowa.edu/~rlenth/Power/ was used to conduct a power analysis. Using a

Multivariate ANOVA design, an alpha level of .05, and a power value of .8, the program

provided a sample size of 4 per sample, to determine a 15% difference in key outcomes

between animals. However, based on previous experience in similar studies using the

34

same biochemical, histological, and other measures proposed in this project, we have

found that n=3 mice per group is sufficient. Thus, in order to make an attempt to reduce

the number of mice, we will use n=3 muscles per group for CSA, n =4 muscles for RT-

PCR, and n = 2 muscles for EM.

35

CHAPTER 4 RESULTS AND DISCUSSION

FoxO1 in Cancer Cachexia

The current study demonstrates FoxO1 is required for skeletal muscle wasting

during cancer cachexia. We show a significant attenuation of muscle fiber cross

sectional area, as well a reduction in muscle fiber ultrastructure damage when FoxO1

mRNA is knocked down by a shFoxO1 AAV in cachectic mice. This data indicates that

FoxO1 is an important regulator of muscle mass and the muscle atrophy program. In

addition, we identified FoxO1 target genes using a microarray with subsequent

bioinformatics analysis. Examination revealed there were 2,007 FoxO1 target genes

differentially regulated during cancer cachexia. Of the differentially regulated genes

there were 960 increased and 1,047 decreased by ±≥1.4 fold. Further analysis revealed

upregulated pathways included those of the olfactory receptors and antigen processing.

FoxO1 was also necessary for the downregulation of several genes involved in skeletal

muscle development and myoblast formation.

C26 Mice Characteristics

The C26 model was first established in 1975 in an effort to create a tumor model

to study biological and chemotherapy treatments (Corbett et al, 1975). In 1981, the C26

model was characterized in vivo by inoculation of suspended cells into a BALB/c

mouse. Multiple labs have contributed to confirming the C26 model as the current

standard in studying cancer cachexia. This model has been the foundation of studying

multiple pathways in cancer cachexia.

We found C26 tumor-bearing mice show a progressive weight loss (Figure 1A).

Indeed, we find a 25% tumor-free bodyweight loss after ~26 days of cancer cachexia in

36

our murine model. During this period tumors grow to 2-3cm in diameter, weighing 1.5g

and 3.3g cumulatively at mid and endpoint, respectively (Figure 2B). Mice also lose

significant fat mass and at endpoint have little to no fat remaining (Figure 2A). We have

also found, much like the literature suggest, anorexia plays no role in this model

because food intake is not changed (unpublished data). Therefore, we believe this

model recapitulates cancer cachexia in human patients. One limitation of this model is

that cachectic mice die within ~30 days of cancer inoculation, while humans typically

suffer from cachexia for much longer.

Muscle Wasting in Cancer Cachexia

The loss of skeletal muscle during cancer cachexia leads to muscle weakness

and functional loss of both locomotor and respiratory muscles which is responsible for

over a third of cancer related deaths. We found a progressive decline in tumor-free

bodyweight during the progression of cancer cachexia (Figure 1A). There was

significant muscle weight loss at endpoint in all muscles (Figure 3B). Furthermore, we

found a trend of decreasing muscle weights as the disease progressed.

The earliest measurements were taken at day 14, which is meant to mimic a

pre-cachexia stage in humans. During this period the tumor is ~.5cm in diameter. At this

time point the muscle weight of the TA is decreased 6%. There is also a 12%, 25%, 9%

and 6% decrease in the gastrocnemius, soleus, plantaris, and quad, respectively.

Our endpoint for these experiments was day 26 and is similar to that of severe

cachexia in humans with weight loss of >15%. At endpoint we found significant

decreases in all muscle weights compared to control. Specifically we found a 32%,

55%, and 26% decrease in the TA, EDL and soleus, respectively at endpoint (Figure

3A).

37

In addition to muscle weight loss there is also a decrease in muscle function.

Previous work form our lab has shown absolute force is decreased ~32% and ~30%, in

the EDL and soleus, respectively. We have also found when force is normalized to

muscle size (specific force) there is a decrease in muscle function in cachectic mice.

Another important finding from this study was the significant decrease in MyHC protein

levels. Interestingly the protein levels of sarcomeric actin were unchanged along with

troponin and tropomyosin levels. These findings suggest MyHC is specifically targeted

for degradation during cancer cachexia which could help explain the impaired

contractile function.

Cumulatively, this data indicates there may be a deeper underlying pathology

associated with the extracellular matrix or contractile apparatus of the muscle to

account for the fact that the muscle is not only smaller, but also weaker when

normalized to size.

FoxO1 Signaling Increases in Cancer Cachexia

We found a significant increase in both FoxO1 and FoxO3a in skeletal muscle of

cachectic mice (Figure 4A). Specifically, we found a ~6-fold increase and ~4-fold

increase in FoxO1 and FoxO3a mRNA, respectively. FoxO1 and FoxO3a play a role in

upregulating E3 ubiquitin ligases during muscle atrophy conditions. Indeed, in our model

we find MuRF-1 is upregulated ~25 fold and Atrogin-1 is upregulated 13-fold (Figure

4B). It has been well established MuRF1 and Atrogin-1 are markers of protein

degradation (Bodine et al, 2001). Furthermore, the activation of these two E3 ligases is

required for muscle wasting in multiple animal models of muscle wasting.

Transgenic mice overexpressing FoxO1 in skeletal muscle have increased

expression of Atrogin-1 levels. The upregulation of these canonical pathways drives

38

ubiquitin proteasome proteolysis resulting in muscle atrophy. The translocation of FoxO

family members to the nucleus is required to induce muscle wasting. This translocation

can be inhibited by Akt phosphorylation, or by acetylation. Data from our lab indicates

HDAC proteins can contribute to activation of FoxO and induction of the muscle atrophy

program (Beharry et al, 2014). HDAC1 is sufficient to activate FoxO and cause muscle

atrophy and is necessary for the atrophy of muscle fibers in disuses atrophy. Our

unpublished data shows that HDAC6 may be an upstream regulator of FoxO1 via

acetylation status. Indeed, we have found acetylated-FoxO1 decreases during C26

cancer cachexia. Furthermore we have found WT HDAC6 can increase FoxO reporter

activity. We also find FoxO1 and HDAC6 complex via immunoprecipitation experiments.

We did not include any experiments to further identify upstream regulators of FoxO1 in

this manuscript, but it does merit future consideration.

FoxO1 is Necessary for Muscle Wasting in C26

The FoxO family of transcription factors plays an important role in cachexia

induced atrophy as well as many other wasting conditions. We recently completed an

unbiased microarray using a dominant negative form of FoxO in cancer cachexia. We

found that not only did inhibition of FoxO transcriptional activity prevent increases in

canonical genes such as Atrogin-1 and MuRF1, but it also repressed genes related to

the structural integrity of the muscle.

A recent study by Skorhold et al., identified gene expression in skeletal muscle

biopsies from cancer patients with or without cachexia using genome-wide expression

analysis. Patients with cachexia had a median of 14% weight loss, which fits within the

weight loss we see in our C26 model. This study yielded ~183 cachexia-associated

genes which were further classified using Gene Ontology (GO). GO analysis revealed

39

changes in genes related to muscle contraction, cytoskeletal arrangement, protein

degradation, and acute-phase response proteins. Importantly, one gene identified as a

cachexia-associated gene was FoxO1, which had a 5-fold increase in cachectic cancer

patients. Other groups have found no change in p-FoxO3 to total FoxO3 at the protein

level (Pretto et al, 2015). This data, in combination with our own showing FoxO1 is more

highly elevated in cachectic mice, leads us to believe that FoxO1 may be the main

factor causing muscle atrophy during cancer cachexia.

Muscle loss is an important factor in the fatigue and weakness of cancer patients.

However, studies aimed at elucidating the mechanisms of muscle wasting have not

distinguished the specific role of each FoxO family member during cancer cachexia.

Thus, in this study we focused on FoxO1 in cancer-induced wasting of limb muscle in

response to C26. To inhibit endogenous FoxO1, we transduced muscles with shFoxO1-

AAV (or a control AAV) this expressed a green fluorescent protein, GFP, to visualize

transduction. To transduce the TA we performed a single intramuscular injection of AAV

into the midbelly of the muscle. Immediately following AAV injection, mice were

assigned to groups, and either injected with PBS (control) or C26 cells. Muscles were

then harvested from all groups ~26 days post C26 inoculation.

Tibialis Anterior muscle was isolated from cachectic and control mice then

weighed to confirm muscle atrophy. As shown in figure 4A, we found a 23% decrease in

wet weight in C26 compared to control muscle. Furthermore, we found 74% attenuation

in muscle weight in the shFoxO1-AAV C26 group compared to control. Similar to

muscle weight, we also found a significant decrease in muscle fiber cross sectional area

of the TA in cachectic mice. Conversely, TAs injected with a shFoxO1 had attenuated

40

atrophy by ~50% (Figure 6a). This data therefore demonstrates that blocking FoxO1 is

sufficient to prevent C26-induced muscle wasting in limb muscle, which adds to the

previous literature that FoxO is necessary for C26, LLC, and S-180 induced cancer

cachexia.

Knockdown of FoxO1 Rescues Ultrastructural Changes in C26 Skeletal Muscle

The muscles of cachectic cancer patients and mice show evidence of significant

muscle damage with the lack of injury (He et al, 2013). For example, several

components of the dystrophin associated protein complex (DAPC) are altered in

muscles of cachectic cancer patients. Furthermore, several cachectic cancer patients

show a dramatic loss of dystrophin, which is correlated with decreased survival time

(Acharyya et al, 2005). This disruption of the Z-disc and M-line could result in the

perturbance of cell integrity as well as changing cell signaling. Since the Z-disc serves

as the structural anchor of the membrane, it provides a direct link to the sarcomere. In

fact, patients with Duchenne Muscular Dystrophy (DMD) have the inability to regenerate

muscle fibers which is known to be due to the lack of the dystrophin protein. We believe

that this disruption could be a key initiating mechanism for the regulation of transcription

of critical proteins for cell integrity. Others agree, showing dystrophin downregulation

causes cachectic muscle to become fragile which may contribute to muscle wasting.

Indeed, several unbiased microarray studies in preclinical cancer cachexia

models have shown that a significant number of muscle structural genes are

downregulated in response to a tumor burden (Judge et al, 2014a). Moreover, a

transcriptome analysis of muscle from patients losing weight with upper GI cancer

showed substantially more genes are downregulated than upregulated. These genes

41

were identified to be important to maintaining the structural integrity of the muscle cell

(Gallagher et al, 2012).

We used electron microscopy to determine if there were alterations in muscle

membrane structure in cachectic muscle compared to control. The processing and

visualization of skeletal muscle using EM is technically challenging due to the

accumulation of artifact if using an inappropriate protocol. This is due to water in the

tissue being increased by submersion in fluid. However, EM is necessary to determine

changes at a nanometer level. Therefore we used the EM imaging core at the University

of Florida to process our tissue after removal from animals.

Cachectic muscle fibers show abnormal membrane morphology. Our EM images

(Figure 7B) from cachectic mice appear wrinkled and irregular with fraying at the

sarcolemma. However, control muscle membranes are smooth and have a well-defined

sarcolemma. Other studies have shown a myofilament protein content reduction and

sarcomeric alignment alteration in longitudinal sections of cachectic muscle. Aulino et

al., found the sarcomeres in cachectic muscle were disrupted in cross-sections with a

poorly defined perimeter of the sarcomere. Morphological changes at the membrane

level may indicate a possibility that membrane damage is associated with the

pathogenesis of cancer cachexia. We and others have found that laminin staining is

blurred in muscle from cachectic mice, which could mean there is disorganization of the

basement membrane. Indeed, data collected from our lab (unpublished) using skeletal

muscle biopsies from cachectic pancreatic cancer patients show a disruption to normal

architecture and substantial ultrastructural pathology.

42

We believe that the transcriptional repression of muscle structural genes may be

mediated by FoxO and could be the cause of the ultrastructural pathology in cachectic

muscle. In support of this, when we previously inhibited FoxO transcriptional activity we

found the most highly enriched functional categories of genes repressed in cachectic

muscle were related to the extracellular matrix and sarcomere. In the current study we

also found highly enriched processes downregulated belonging to the ECM and proteins

associated with this complex were identified as FoxO1 targets (Figure 9).

Microarray Analysis to Identify Direct or Indirect FoxO1 Target Genes during C26 Cancer Cachexia

We performed an unbiased microarray to comprehensively identify gene

networks that were differentially regulated in response to C26 cancer cachexia which

require FoxO1-dependent transcription. We analyzed TA muscle taken from control and

C26 mice injected with a scrambled shorthairpin AAV or shFoxO1 AAV (-1.4 ≥ fold

change ≥ 1.4). We identified 5,823 genes that were differentially regulated in C26

compared to control. Furthermore, of those genes we identified 2,007 which were direct

or indirect FoxO1 target genes. Subsequent analysis identified 960 of these genes were

upregulated ≥1.4 fold change and 1047 had fold change ≤-1.4 in shFoxO1 C26

compared to C26 (Figure 9). A heatmap of genes of interest in skeletal muscle wasting

is available in Figure 4-12.

In order to identify broad gene networks, molecular pathways, and canonical

pathways regulated in response to our conditions, gene profiles were analyzed using

DAVID Bioinformatics database and the Broad Institute Molecular Signatures Database

(MSigDB). Then we analyzed transcripts that bind 2kb upstream or downstream of the

start codon. Pathway analysis has become one of the best methods to gain insight into

43

the mechanisms of differentially expressed genes. Using bioinformatics reduces the

complexity of the microarray data, allowing us to identify complicated networks of

interactions to fully understand the context of the biological processes. It gives a global

perspective on the data and underlying question. The value of pathway analysis lies in

the ability to explore the overall picture of a complex system.

Among the indirect or direct Foxo1 target genes (960) that were upregulated in

muscle of C26 mice, the most highly enriched biological clusters identified by DAVID

analysis were related to the proteasome, sarcomere, and transcription regulation.

Ranked in order of significance based on taking the negative log of the p-value provided

by DAVID the most highly enriched annotation terms from each of the top 10 annotation

clusters are shown (Figure 8). For comparison, the top 10 canonical pathways identified

by MSigDB ranked in order of significance are shown in Figure 11, which were similar to

the DAVID analysis. Among the top 20 canonical pathways upregulated from MSigDB

the most enriched were in the immune system, RNA metabolism, and the p53 pathway.

Contrarily, among the most enriched downregulated clusters were related to ECM and

glucose metabolism. This data is in agreement with our previous microarray data which

identifies FoxO as a regulator in multiple pathways during cancer cachexia.

FoxO1 is Necessary for Cancer-induced Downregulation of Genes Encoding Collagen and Sarcomeric Proteins

In the microarray there were 1,047 FoxO1 target genes downregulated in skeletal

muscle of cachectic mice. Analysis of these genes using DAVID identified the

contractile fiber to be among the most highly enriched annotations (Figure 10A). For a

list of genes see Figure 4-14. Several of these genes have previously been shown to be

44

downregulated during cancer cachexia (Bonetto et al, 2011), though this is the first

evidence demonstrating the requirement of FoxO1 for their regulation.

Several small leucine-rich repeat (SLRP) proteoglycans were identified as FoxO1

targets including fibromodulin, lumican, asporin, and osteoglycin. The SLRP family was

originally thought to be only structural genes, but have recently been identified to be a

network of signal regulation. They are upstream of multiple signaling cascades including

those driving by TGF-beta superfamily members, IGF-1 receptors, and Toll-like

receptors. For example, asporin and fibromodulin bind to TGF-beta pathway through

LRP1 and regulates three-dimensional characteristics of collagen matrices and skeletal

muscle differentiation (Cabello-Verrugio & Brandan, 2007). While our interest is not

directly in SLRPs, it would be interesting to see what role they play during cancer

cachexia.

Collagen formation was not identified as a highly enriched annotation through

DAVID or MSigDB but still had several genes that fell within the cutoffs to be a FoxO1

target gene. Among these genes were some encoding for Type I, III, and VI collagens.

Indeed, we found collagen type 1 alpha was down 9.2 fold in cachectic muscle, but

attenuated with shFoxO1 AAV. Another highly downregulated gene was collagen type 3

which had a 3-fold decrease in cachectic muscle, but in the shFoxO1 muscle the

expression was brought back within 25% of control values. Collagen VI is produced by

fibroblasts and is critical to the structure of muscle. Indeed, muscle membranes of

collagen VI knockout mice show increased leakage and reduced stiffness of the muscle

(Canato et al, 2010). It is speculated that collagen VI deficiency introduces muscle

atrophy, which triggers satellite cell activation and regeneration (Paco et al, 2012).

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Also enriched among the downregulated genes regulated by FoxO1 were several

whose protein localize to the muscle sarcomere. Included among these is Cypher,

which is down 3.4-fold during cancer and increased by 100% in muscles injected with

shFoxO1 AAV. Another gene that was identified as a FoxO1 target gene was Homer1,

which was downregulated by 3.4-fold, but was increased 50% in muscle injected with

shFoxO1 AAV. Both of these have been shown to play pivotal roles in muscle fiber

function and integrity. Indeed, Stiber et al. showed mice lacking Homer1 exhibit skeletal

myopathy. Additionally Zhou et al. showed that ablation of cypher causes a severe form

of congenital myopathy. Therefore we believe FoxO1 regulating these genes plays an

important role in the downregulation of structural genes which could cause muscle

atrophy and warrants further investigation.

Validation of Transcripts Regulated by FoxO1

To validate a subset of genes identified on the microarray, we used qRT-PCR

using cDNA from the same RNA samples used in the microarray. Similar to our findings

using microarray analysis, we found Mef2c downregulated ~3.5 fold in C26 compared to

control. This also matches our previous microarray data. Furthermore, we found the

decrease in Mef2c to be attenuated ~3-fold by shFoxO1 in cachectic muscle. This data

matches that of the microarray and helps validate it.

Ubiquitin Proteasome Pathway Enriched Among FoxO1 Targets Upregulated in Cachectic Muscle

Analysis performed using DAVID and MSigDB identified genes involved in the

ubiquitin proteasome pathway to be enriched among the FoxO1 target genes in

cachectic muscle. Among those genes identified were those of the 19s subunit (PSMD4

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and PSMD11), those of the 20s core (PSMA1, PSMA4, and PSMA7), and those which

play a role in the 26s proteasome (PSMC6, PSMC4, and PSMC2).

A recently identified FoxO target gene in nutrient deprivation, MUSA1/Fbox31,

was also increased 4.9-fold in our microarray. This increase was attenuated 40% by

shFoxO1. MUSA1 was found to be a critical E3 ligase for muscle atrophy by the Sandri

lab in 2014. Expression of MUSA1 is thought to be regulated by Smad transcription

factors in some atrophy conditions. Furthermore, it has been shown that Smads require

FoxO for regulation of specific target genes.

Surprisingly, Atrogin-1 was not identified as a FoxO1 target gene but was

increased 3.7-fold in cachectic muscle. This data is in alignment with our previous

microarray using a pan-FoxO inhibitor. Furthermore, MuRF-1 was identified as highly

upregulated during C26 conditions (5.8 fold), but also did not meet criteria to be

identified as a FoxO1 target gene.

While we did not identify all of the classical atrophy related genes as FoxO1

targets this could be due to multiple reasons. First, we only inhibited ~60% of FoxO1

mRNA thus endogenous binding could still occur. There could also be alternative

factors that control the transcriptional regulation of many genes.

FoxO1 Mediates Part of the Autophagy and Apoptosis Systems

Autophagy is a process known to help cells undergoing stress and has been

linked to the apoptotic process. Thus, apoptosis is not the only way in which the cell can

go through program regulated processes. Ultimately, the cell decides which pathway to

use depending on the specific stimulus and the cell environment. Remarkably,

apoptosis and autophagy are not mutually exclusive pathways. They have been shown

to act in synergy or sometimes even counteract each other.

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FoxO controls several stress response pathways, one of which is autophagy.

Autophagy plays an important role in the turnover of muscle components. In fact,

skeletal muscle has one of the highest rates of autophagy when compared to all other

organs. The autophagy system creates autophagosomes which are then delivered to

lysosomes for degradation. One lysosomal protease, cathepsin-L, has been shown to

be upregulated during muscle atrophy (Deval et al, 2001) and is responsible for

degradation of membrane proteins. Indeed, our lab has previously identified Cathepsin-

L, Gabarapl1 and Bnip3 as FoxO target genes during cancer. Bnip3 is a central

mediator of autophagy and is increased in numerous models of muscle atrophy

(Mammucari et al, 2007).

We found a 2.8 fold increase in LC3 mRNA in cachectic muscle compared to

control. Furthermore, shFoxO1 attenuated this increase by 33%. LC3 is the homolog of

yeast Atg8, which is important for membrane growth (Nakatogawa et al, 2009).

Interestingly, knockdown of LC3 spares muscles mass in SOD1 transgenic mice.

Furthermore, Bnip3 and Bnip3L reportedly bind directly to LC3 and can recruit the

autophagosome (Hanna et al, 2012). This data could indicate a novel role of FoxO1 in

part of the autophagy system during cancer cachexia. Others have shown that FoxO3a

Is a critical factor that is sufficient and required to activate protein breakdown. Likewise

numerous autophagy genes are under FoxO3a regulation including LC3, Bnip3, Atg12

and Gabarap (Mammucari et al, 2007).

One of the upregulated genes previously identified as a FoxO target gene,

Ubiquilin-1, which encodes for ubiquitin-like protein. This protein interacts with both

ubiquitin ligases and proteasomes and is involved in protein degradation and

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autophagy. Ubiquilin 1 was increased 1.9-fold during cancer cachexia, but decreased

25% in cachectic muscles injected with shFoxO1. Interestingly, it was shown in HeLa

cells that ubiquilin binds to the autophagosome marker LC3 in a complex. Furthermore,

the reduction of ubiquilin expression reduces autophagosome formations (Rothenberg

et al, 2010).

Another known target of FoxO, Gabarapl1, was successfully identified as a

FoxO1 target gene in our current microarray and plays a role in the

autophagy/lysosomal pathway in muscle wasting conditions. However, other markers of

the autophagy pathway were upregulated during cachexia such as Cathepsin-L and

Bnip3 but were not identified as FoxO1 target genes. These genes may be regulated

through FoxO3a since they were identified as FoxO targets in our previous manuscript.

The autophagy-lysosome system is central to muscle mass regulation during

catabolic conditions. In addition, the autophagy system is also required for normal

muscle homeostasis and inhibiting it can lead to muscle damage.

FoxO1 May Alter Cytokine and Other Upstream Signaling

Cytokines play a key role as the main humoral factors involved in cancer

cachexia. A large number of these factors are involved in the metabolic changes

associated with muscle wasting during cancer cachexia. The inflammatory cytokines

that have been implicated in muscle wasting diseases include: interleukin-6 (IL-6),

tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ) among

others.

IL-6 has received significant attention for its role in cancer cachexia. It is a

member of a family that includes LIF and IL-8. Circulating levels of IL-6 are almost

undetectable under normal conditions but drastically increased in response to

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inflammatory conditions and exercise. Indeed, elevated circulating IL-6 levels have been

clearly associated with contracting muscle. Furthermore, circulating IL-6 increases

during exercise for several hours to days, yet cancer cachexia patients have elevated

levels for weeks. This chronic elevation of cytokines is believed to play a role in muscle

wasting. Indeed, circulatory IL-6 receptor expression is increased in patients with colon

cancer (Yeh Oncol et al. 2010). The membrane associated IL-6 receptor forms a

heterodimer with the gp130 receptor and activates downstream signaling. In this study

we found that IL-6 mRNA was increased 1.5 fold in cachectic muscle. Furthermore,

muscle expressing shFoxO1 reduced this increase back to baseline. The microarray

data indicates IL-6 as a FoxO1 target gene during cancer cachexia. In combination with

the increase in IL-6, there is also an increase in IL-6 receptor mRNA which is expressed

10-fold higher in cachectic muscle compared to control. While this increase was not

completely blunted by shFoxO1, it did attenuate the increase by ~40%. Many cell types

can respond to IL-6-initiated signaling which could be important for other tissues.

Cancer cachexia is multifactorial and we believe that it is not a singular factor that

causes the muscle atrophy seen in cachectic models. Finally, the systemic alterations

are important for understanding these complex events.

There is some evidence that TNF-α plays a role in muscle loss during cancer

cachexia, though its role in human conditions is more questionable. TNF-α has been

shown to activate NF-kB which leads to induction of the UPP pathway. However, in this

study two canonical members of the UPP (atrogin-1 & MuRF1) are not increased at the

mRNA level. Notably, we find the mRNA levels of TNF-α to be decreased 1.4-fold in

cachectic mice, with no change due to shFoxO1. In a previous microarray of C26 mice,

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we also found the mRNA of TNF-α to be decreased 1.2-fold. Therefore, while the serum

cytokine levels have been reported to increase in moderately and severely cachectic

mice (Bonetto et al, 2011), we believe there could be a negative feedback mechanism

controlling TNF-α mRNA production. TNF-α also inhibits myogenesis through an NF-kB

mechanism which leads to degradation of MyoD transcripts (Guttridge et al, 2000). This

could explain the 2-fold decrease in MyoD we see in our experiments.

Myostatin, while not a cytokine, is a transforming growth factor family member

that acts to limit muscle mass. Mutations in the myostatin gene have been shown to

result in significant muscle hypertrophy, while inhibitors of myostatin given systemically

can also increase muscle mass. We found myostatin mRNA to be increased 1.3-fold in

cachectic muscle. Interestingly, knocking down FoxO1 during cancer cachexia caused a

1.8-fold increase in myostatin mRNA production compared to control. We ha