Abstract
The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA.
Highlights
Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies and is typically diagnosed in an advanced state [1]
The treatment of the spheroidal cultures with sulforaphane for 24 h increased the expression level of Cx43 phosphorylated at Ser 368, E-cadherin and active caspase 3, whereas the levels of c-Met and CD133 decreased. These results demonstrate that Cx43 expression is low in primary cancer stem cells (CSCs), but can be increased by sulforaphane treatment, which is associated with inhibition of CSC and epithelialmesenchymal transition (EMT) markers and induction of apoptosis
We examined gap junctional intercellular communication (GJIC) in human pancreatic cells and tissues and found a direct correlation among low Cx43 protein levels, blocked GJIC, gemcitabine resistance, CSC features and EMT marker expression
Summary
Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies and is typically diagnosed in an advanced state [1]. Our recent results demonstrated that sulforaphane sensitizes pancreatic CSCs to chemotherapy by inhibiting their self-renewal potential, apoptosis resistance and NFκB activity [8,9,10,11]. The phosphorylation of Cx43, the most ubiquitously expressed connexin, has been implicated in the regulation of GJIC at several stages, e.g., the export of the protein to the plasma membrane, the formation and activity of GJs and connexin degradation [24]. Epigenetic mechanisms may be involved in the regulation of Cx43 protein levels, because previous findings demonstrated that the HDAC inhibitor 4-phenylbutyrate increased the expression of Cx43 in pancreatic cancer cells and promoted the growth inhibition of xenograft tumors [26]. The hypermethylation of the Cx43 promoter was correlated with low Cx43 expression in human gliomas and lung cancer [23, 27]
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