Critical alterations in cellular bioenergetics and epithelial‐mesenchymal transition mediated by crosstalk between tumor cells and lymphatic vasculature augments tumor progression in cholangiocarcinoma

Abstract

Cholangiocarcinoma is a rare but highly aggressive cancer of bile duct cells with a dismal 5-year survival rate following surgical tumor resection of 2%. Despite the presence of a high degree of lymphangiogenesis in the prominently inflamed CCA tumor microenvironment (TME), the role of lymphatic-CCA crosstalk in CCA metastasis has been unexplored. Increased glucose metabolism and oxidative phosphorylation (OXPHOS) in CCA cells increase CCA metastasis and therapeutic resistance. Further mitochondrial dysfunction is a driver of epithelial-mesenchymal transition (EMT), critical for tumor progression. We hypothesized that CCA-LEC interactions in the tumor microenvironment alters CCA metabolism and drives EMT and lymphangiogenesis. We used CCA (HuCCT1, Mz-ChA1 and CCLP1), LEC (human dermal and lymph node LECs) and normal human cholangiocyte (H69 and HiBEC) cell lines as well as human CCA tissue arrays. Conditioned medium (CM) was collected from CCA, control and inflamed LECs (LECs stimulated with lipopolysaccharide (LPS, 100ng/ml for 24hr). To define the CCA-LEC crosstalk, both CCA and LECs were treated with LEC/inflamed LEC CM and CCA-CM respectively, and alterations of mitochondrial gene expression, alterations in cellular metabolism, and migration and proliferation of CCA and LECs was determined. CCA cells showed enhanced migration and proliferation in response to LPS-LEC-CM. Real time PCR revealed that this was associated with altered expression of EMT genes such as Gli1, SNAI1, SLUG and TWIST1 or changes in matrix metalloproteinases (MMP) as MMP2, MMP9 and MMP21. The seahorse metabolic data showed that stimulation of CCA cells by LPS-LEC-CM, caused a distinct shift towards higher glycolysis and oxidative phosphorylation (OXPHOS), along with increased ROS and ATP production. This was associated with dysregulation of some critical metabolic genes such as PFKP and ATP6 involved in increasing glycolysis and OXPHOS rate critical for aggressive tumor growth and metastasis. Further, analysis of mitochondrial genes in CCA-CM treated LECs showed increased expression of PFKP, GLUT3, FASN, CO I, HK2 and ATP6, critical genes involved in glycolysis, OXPHOS and fatty acid synthesis, key pathways known to drive tumor-lymphangiogenesis. Dysregulation of these mitochondrial genes was also observed in clinical samples of CCA. siRNA mediated silencing of a number of these genes including SLUG1 and ATP-6 show decreased CCA migration, EMT and reversal of the increased glycolysis as well as significantly reduced LEC tube formation. Thus, alterations in cellular metabolism due to tumor-lymphatic crosstalk alter oncogenic pathways and regulates lymphangiogenesis in progression of CCA.