¿ALTERATIONS OF CHOLINE PHOSPHOLIPID METABOLISM IN OVARIAN CANCER: STUDY OF THE ACTIVITY AND EXPRESSION OF CHOLINE KINASE AS A PROGNOSTIC VALUE¿

Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to late diagnosis and early relapse associated with development of chemoresistance. So far, the origin of drug resistance is not completely understood and several mechanisms seem to be responsible for this process including the ability of cancer cells to modify/reprogram cellular metabolism. Aberrant choline metabolism, characterized by increased phosphocholine (PCho) levels sustained by enhanced choline kinase (ChoK) activity, is a new cancer metabolic hallmark that can be monitored by Magnetic Resonance Spectroscopy (MRS) and that reflects the interactions between oncogenic signaling and cellular metabolism. EOC possesses an altered MRS-choline profile, characterized by increased PCho content to which mainly contribute over-expression and activation of Choline kinase-alpha (ChoKα). The project aimed to investigate the biological and clinical relevance of increased ChoK expression and activity in EOC. We showed that transient CHKA silencing induced: a significant reduction in PCho accumulation as assessed by MRS analysis; an inhibition of in vitro cell growth; down modulation of genes related to inflammation and EOC aggressiveness; a significantly reduced cell motility and invasion and a significant increase of sensitivity to platinum, paclitaxel and doxorubicin even in a drug-resistant context. Since the dynamics of biological effects related to CHKA silencing could not possibly be detectable by transient RNAi, we stably downregulated ChoKα mRNA expression using a lentiviral vector, expressing GFP and specific CHKA shRNA, in two EOC cell lines, INTOV11 and SKOV3. In both transduced cell lines, we confirmed the data obtained with the transient downmodulation, as reduction of PCho content, inhibition of in vitro cell growth, reduction of invasive potential and migration capability. Furthermore, a significant decrease of in vivo tumor growth (sub-cutaneous injection) was evident in both sh-CHKA cellular models as compared to their controls. The global metabolic profiles of sh-CHKA and control EOC cell lines, performed with the Metabolon technology platform, revealed in both cellular models an alteration of glutathione metabolism, commonly characterized by a decrease of reduced glutathione (GSH) content in shCHKA cells as compaired to their controls. Interestingly, sh-CHKA showed an increased sensitivity to drug treatment (platinum and doxorubicin) suggesting that CHKA knocking down may change antioxidant cellular defense thus increasing drug sensitivity. Since the GSH intracellular levels are well known to interfere with treatment efficacy of several DNA damaging drugs, we have investigated whether the alteration of GSH/GSSG levels dependent upon CHKA silencing is the mechanism by which response/resistance to DNA-damaging drugs is affected. Artificial reduction of intracellular GSH in wild type EOC cells phenocopied CHKA silencing effects while restoration of GSH levels in CHKA silenced cells caused a recovery of the wt phenotype by rescuing drug resistance. The concurrent modulation of active ROS species following CHKA silencing demonstrates the direct involvement of oxidative stress pathway regulation by ChoKα expression. Finally, in order to investigate the putative role of CHKA in EOC pathogenesis we performed an in silico analysis on seven public data sets of gene expression profile, including 698 EOC patients and we found a progressive increase of CHKA expression along EOC grading. Interestingly the CHKB expression in the same samples showed an opposite trend. Given these premises, we propose that a metabolic approach to EOC treatment might have the potential to address some of the issues that contribute to the high EOC lethality and open the way for the validation of ChoKα as a new therapeutic target to be used alone or in combination with conventional drugs for EOC treatment.