UC Davis

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths, with chemotherapy as the standard of care for all stages of disease. Gemcitabine (GEM)-based chemotherapy regimens persist as front-line treatment options, but without substantial improvements in survival. Cancer-associated cachexia (CAC), a multifactorial disorder characterized by the involuntary and ongoing wasting of skeletal muscle with or without loss of adipose tissue, is a critical contributor to PDAC mortality. Thus, there is an urgent need for better therapeutics for PDAC and CAC; and the exploration of dietary interventions is a critical component. A ketogenic diet (KD), characterized by a very low carbohydrate and high fat composition, has shown anti-tumor and anti-cachectic potential. Still, there is limited understanding on the effect of KDs in PDAC and CAC progression, even less in combination with chemotherapeutics, such as GEM. Therefore, the overall aim of this thesis work was to evaluate the effect and mechanisms of a KD alone or in combination with GEM on morbidity and mortality in a clinically relevant genetically engineered LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) mouse model of PDAC.Chapter 1 provides a thorough review of the current literature regarding the use of KDs in pancreatic cancer and CAC, with a focus on cellular mechanisms and clinical perspectives. A summary of the evidence reported is that a KD can reduce tumor growth and act as an adjuvant therapy in various cancers, including pancreatic cancer. In addition, the chapter also discusses the limited research published on KDs effect on CAC mitigation. The main cellular mechanisms that may explain KD’s potential anti-tumor and anti-cachexia effects are described, focusing primarily on reprogramming of cell metabolism, epigenome, and the gut microbiome. Chapter 2 describes the effects of a KD with or without GEM in the pancreas/tumor of KPC mice, with the main objective of determining whether a KD plus GEM increases survival of KPC mice following PDAC detection. Following tumor size determination, male and female KPC mice were fed a control diet (CD; %kcal: 70% carb, 14% protein, 16% fat), a KD (%kcal: 14% protein, 1% carb, 85% fat), a CD + GEM (CG), or a KD + GEM (KG) group. GEM was administered to the CG and KG groups at 100 mg/kg by intraperitoneal injections twice per week for 3.5 weeks (7 total injections). Throughout the survival study, mice were monitored daily until an endpoint criterion was reached and they were euthanized. We observed that a KD plus GEM extended overall median survival in KPC mice when compared to a CD. Mechanistically, KG treatment significantly reduced AKT, ERK, IGFR and AMPK phosphorylation in pancreatic tumors compared to CG-treated mice. Furthermore, following KG treatment, palmitic acid, myristoleic acid, palmitoleic acid, asclepic acid and linoleic acid were reduced in pancreatic tumors. Moreover, data from the relative abundance of fecal bacteria using 16S rRNA sequencing analysis of the microbiota showed that KG treatment leads to increased relative abundance of Faecalibaculum and the reduction of Lactobacillus at one-month of treatment. In summary, results from chapter 2 of this thesis highlight that a KD has a synergistic effect with GEM that benefits survival in KPC mice and that such response is multifactorial. The aim of chapter 3 was to evaluate whether feeding a strict KD alone or in combination with GEM mitigates CAC in the autochthonous KPC mouse model, and to elucidate the potential mechanisms involved. In the survival study, KD alone or combined with GEM resulted in the mitigation in the decline of muscle strength over time, as determined with a forelimb grip strength dynamometer. When analyzed by sex, in females, both KD and KG had significantly higher grip strength force than CD-fed groups. In addition, female KPC mice fed a KD had higher gastrocnemius weights compared CD fed mice. Caloric intake was recorded to evaluate anorexia, which was diminished in the KG groups when compared to CG and lessened in KG-treated female mice. A cohort of mice was allocated to either the CG or the KG groups after tumor detection and euthanized at two months post-interventions to collect the gastrocnemius muscle, which was used to evaluate potential mechanisms involved in muscle strength preservation. Mechanistically, findings from this chapter signal towards sex-specific differential effects of KG treatment, including the inhibition of autophagy in KPC female mice, increased total acetyl-lysine levels and reduced phosphorylation levels of eIF2α in the KG-fed female KPC mice when compared to CG-treated mice. Findings from chapter 3 contribute to the beneficial potential of a KD in combination with GEM for the preservation of skeletal muscle mass in KPC mice, deserving further evaluation. Finally, Chapter 4 delves into the safety evaluation of a KD in combination with GEM. In particular, we examined the hepatic safety profile of a KD in combination with GEM in KPC mice. Feeding a strict KD in combination with GEM failed to significantly affect mouse body weight, liver weight, liver aminotransferases, liver markers of inflammation and oxidative stress, or liver enzymes involved in ketone bodies and glucose metabolism. In addition, KG did not increase markers of liver-lipid accumulation nor serum cholesterol and triglyceride levels. In summary, results of chapter 4 indicate that a KD in combination with GEM appears safe in KPC mice with no apparent hepatotoxicity. These safety data support the evaluation of a KD as an adjuvant dietary treatment for pancreatic cancer. Overall, a KD in combination with GEM has the potential to be beneficial as a treatment strategy for PDAC in KPC mice. Our findings indicate a beneficial effect of a KD in combination with gemcitabine in survival and in the preservation of skeletal muscle function in KPC mice with PDAC and CAC. The mechanisms of the favorable effect appears to be multifaceted, including decreased autophagy and increased cellular response and acetylation. Furthermore, we demonstrate that a KD in combination with GEM used for the treatment of PDAC and CAC in KPC mice, appears safe with no deleterious effects on liver physiopathology or function. Additional research is warranted to further investigate how such diet-treatment combination can be optimized for clinical advantage in PDAC-patients.