Adaption to hypoxia and nutrient depletion determines therapy sensitivity of cholangiocarcinoma

Wappler, Jessica; Cramer, Thorsten (Thesis advisor); Blank, Lars Mathias (Thesis advisor); van Dongen, Joost Thomas (Thesis advisor)

Aachen (2018)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018


A key challenge in the development of effective therapies for cholangiocarcinoma (CCA) is the natural anatomic diversity and molecular heterogeneity of the tumor. The majority of CCA patients present with unresectable tumors and palliative chemotherapy with gemcitabine and/or cisplatin is the current gold standard at this stage. A better understanding of the underlying resistance mechanisms is mandatory to improve therapy strategies. One factor known to profoundly influence susceptibility to anti-cancer drugs is the tumor microenvironment. A co-occurrence of oxygen and nutrient deprivation is a common phenomenon in core regions of solid tumors and both are known to stimulate aggressiveness and treatment resistance. As the non-essential amino acid glutamine plays a central role for the metabolism of a variety of solid tumors, the aim of this thesis was to investigate its general importance for CCA as well as its effect on cytostatic drug therapy in a setting with coincident hypoxia. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) analysis revealed glutamine to be strongly depleted in CCA patient tissue. Moreover, in vitro experiments showed that CCA cell lines are highly dependent on glutamine supply. By using gradual reduction of external glutamine, two extrahepatic CCA (eCCA) cell lines (EGI-1 and TFK-1) were able to overcome glutamine addiction. The glutamine-deprived cell lines obtained in the selection process (termed EGI-1 GD and TFK-1 GD) were capable of glutamine-independent growth. Bioenergetic analysis showed that glutamine deprivation affected mitochondrial respiration of TFK-1 cells, suggesting a shift to glycolysis during the adaption process. Since the effects of coincident hypoxia and long-term glutamine starvation remain broadly unknown, GD cell lines were treated with cisplatin or gemcitabine under normoxia and hypoxia. Under normoxic conditions, GD cells displayed decreased sensitivity to both drugs. Under hypoxia, both parental cell lines showed enhanced chemoresistance, a phenomenon well in line with published literature. Strikingly, hypoxia was not able to increase therapy resistance in GD cells. These results argue for an interconnection between the molecular mechanisms that govern adaptation to glutamine deprivation and the hypoxic response that was not previously appreciated.The oncoprotein c-Myc plays an important role in glutamine metabolism of cancer cells and glutamine deprivation resulted in a reduction of c-Myc protein levels in eCCA cell lines. Glutamine-deprived GD cells showed normalisation of c-Myc to the level of glutamine-supplemented parental cells. When treated with cisplatin, both parental cell lines showed equal levels of c-Myc independent of oxygen availability, whereas distinct hypoxia-mediated downregulation was observed in both GD cell lines. These findings indicate, that c-Myc could be involved in the different cisplatin-induced DNA damage response between cells adapted to glutamine deprivation and their parental counterparts.Given the challenging anatomic diversity of CCA, another aim was to comparatively analyse the response of eCCA and intrahepatic CCA (iCCA) cell lines to cytostatic drug treatment. Whereas both eCCA cell lines showed significant susceptibility to hypoxic conditions, the tested iCCA cell lines (CC-LP-1 and SNU-1079) showed no difference in proliferation. The experiments also revealed a higher tolerance to cisplatin and gemcitabine treatment in iCCA compared to eCCA cell lines with two to ten times higher IC50 values. Moreover, hypoxia-mediated resistance to cisplatin was not detectable in iCCA cell lines. γH2AX is a DNA damage marker which can help to predict tumor response particularly to cisplatin treatment. Analysis of a CCA patient cohort revealed a significant correlation between cytostatic drug treatment and γH2AX-positive tumors as well as a trend towards an improved survival rate in patients with Klatskin tumors that was not detectable in iCCA patients. In summary, the findings suggest that long-term glutamine deprivation does not intensify the resistance-mediating effect of hypoxia, indicating that the adaption to tumor microenvironmental changes might be more complex for therapy outcomes than expected. Moreover, the results indicate that the reliance on oxygen might be dependent on the anatomic origin of the CCA. Since both subclasses showed different susceptibility to anti-cancer drugs, it is reasonable to expect that personalised treatment options are able to improve outcomes.