MARIA FERNANDA MONTENEGRO ARCE¹, Luis Sánchez del Campo Ferrer², Román Martí Díaz², JUAN CABEZAS HERRERA³, JOSE NEPTUNO RODRIGUEZ LOPEZ³

(1) NEUROPATOLOGÍAS DEL SISTEMA COLINÉRGICO, IMIB-Arrixaca, España
(2) Departamento de Bioquímica y Biología Molecular A. Universidad de Murcia, 30100, España (Región de Murcia)
(3) TERAPIAS MOLECULARES Y BIOMARCADORES DE TUMORES SÓLIDOS, IMIB-Arrixaca, España

Pancreatic ductal adenocarcinoma (PDAC) is a public health problem worldwide, as evidenced by its high incidence and mortality rates; therefore, the discovery of new molecules or therapies to prevent its progression and increase the quality and life expectancy of patients with PDAC is a highly desired objective. Recent studies, conducted in our laboratory and others, suggest that the control of protein methylation could be a novel and efficient epigenetic strategy for the treatment of cancer. While therapies focused on regulating DNA methylation are widely known, the control of protein methylation and its effects on some aspects of cancer biology, such as growth, progression or metastasis, are much less known. Concretely, in pancreatic cancer, we observed that hypomethylating treatments inhibited basal autophagy and induced consistent apoptosis in PDAC cultured cells.

PDAC cells were treated with a hypomethylating treatment (HMT) and the effects on protein methylation and autophagy inductions were examined by Western blot analysis, electron and confocal microscopy. Apoptosis was determined using appropriate methods.

Studies in our laboratory and others have shown that pancreatic cancer cells require a high level of autophagy for their growth and development. When PANC1 cells were cultured under normal culture conditions, these underwent a time-dependent phenotypic transformation characteristic of autophagy. However, treatment of PANC1 cells with a combination of TMCG and DIPY (HMT treatment] was able to inhibit the phenotypic changes associated with basal autophagy. Although epigenetic regulation is known to be a fundamental mechanism for various cellular processes, the epigenetic factors mediating the response of cancer cells to autophagy are less known. Therefore, we analyzed the time course of SAM concentration during the basal autophagy of PANC1 cells. We observed a consistent and significant increase in intracellular SAM during basal autophagy, which was clearly abolished in the presence of HMT. Next, we aimed to determine whether the accumulation of intracellular SAM during basal autophagy affected the protein methylation status in PANC1 cells, as well as the impact of HMT on protein methylation. First, we focused in the epigenetic regulation of PP2A, a trimeric serine/threonine phosphatase regulated by protein methylation. Although during basal autophagy the methylation of PP2A at its catalytic subunit was significantly augmented, we observed that treatments with HMT clearly induced demethylation of this protein. Consequently, HMT-dependent demethylation of PP2Ac was also accompanied by a substantial reduction of PP2A activity in vivo. These results could be important in the context of pancreatic cancer since a recent investigation revealed that “autophagy-addicted” cells are dependent on a high phosphatase activity and that the regulation of autophagy could be coordinated by the action of mTORC1 and PP2A. Interestingly, inhibition of autophagy by HMT induced consistent apoptosis in pancreatic cancer cells.

PP2A is essential for the activation of autophagy in pancreatic cancer, but in turn, it also controls several components of the KRAS pathway and AKT activation. Understand the epigenetics of autophagy in pancreatic cancer could generate new therapies against this type of aggressive cancer.