Titolo della tesi: ANALYSIS OF THE EPI-METABOLIC CROSS-TALK SUPPORTING THE PANCREATIC DUCTAL ADENOCARCINOMA TUMORIGENESIS
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with poor prognosis
and therapeutic outcome. Several epidemiological studies show that chronic metabolic diseases,
including obesity and type II diabetes, are associated with an increased risk of PDAC incidence
and worse clinical outcome. In these conditions, the prolonged exposure to altered metabolism
induces aberrant metabolite fluctuations, possibly affecting epigenetic enzyme reactions and
consequently modifying the epigenome, thus supporting cancer development. Here, it has been
investigated the correlation between metabolism and epigenetics associated with PDAC onset
and progression upon dysmetabolic condition.
First, the link between dysmetabolism and PDAC was examined in LSL-KrasG12D; PDX-1
Cre mice (KC mice) exposed to high-fat diet (HFD). Untargeted metabolomics on pancreatic
samples highlighted free fatty acid level alteration during pancreatic tumorigenesis upon
dysmetabolic condition. Moreover, targeted metabolomics showed higher S-adenosyl
methionine (SAM) level and a ratio between α-ketoglutarate (α KG) and succinate (SA) lower
than 1 in HFD mice, prompting to focus attention on ten-eleven dioxygenase 1 (TET1)/ thymine
DNA glycosylase (TDG) DNA demethylation complex. Interestingly, epi-metabolite level
alterations were paralleled by TET1/TDG complex dissociation and consequent accumulation
of iterative cytosine modifications, including 5-formylcytosine (5fC). These findings were
validated in an in vitro model of human pancreatic ductal epithelial cells bearing Kras mutation
(HPDEmut) exposed to a combination of high glucose and free fatty acid, mimicking
dysmetabolic condition. Specifically, decreased α-KG/SA balance, TET1/TDG complex
dissociation, 5fC accumulation and, furthermore, increased levels of abasic (AP) sites were
detected in dysmetabolic HPDEmut cells, pointing out TDG malfunctioning. In this light, we
investigated the role of SA on TDG activity modulation. Molecular dynamics, SPR assay and
mutational analyses highlighted that SA directly binds TDG at residue Arg275, inducing its
hyperactivation. Accordingly, HPDEmut cells exposed to SA recapitulated the alterations
induced by dysmetabolic conditions in our in vivo and in vitro model. Furthermore, αKG
administration to dysmetabolic HPDEmut cells restored DNA demethylation cycle, reducing
5mC, 5hmC and 5fC, as well as AP site levels. Our in vivo and in vitro models exposed to
dysmetabolic conditions showed a decreased levels of LIG1 and LIG3, two proteins involved
in base excision repair, suggesting that dysmetabolism might lead to an impairment of BER
process through the alteration of DNA methylation/demethylation machinery.
In conclusion our results demonstrate that metabolic alterations affect the DNA
methylation/demethylation machinery as consequence of increased intracellular SA levels,
leading to impaired TDG activity, iterative cytosine modification and AP site accumulation,
consequent to BER machinery impairment. These epi-metabolic alterations might support
PDAC tumorigenesis, suggesting that they should be taken into consideration for diagnostic or
therapeutic purposes.