Changes in the status of p53 affect drug sensitivity to thymidylate synthase (TS) inhibitors by altering TS levels

Abstract

Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of several genes, it may modulate TS activity, and changes in the status of p53 might be responsible for chemoresistance. Therefore, this study was aimed to investigate TS levels and sensitivity to TS inhibitors in wild-type (wt) and mutant (mt) p53 CRC cells, Lovo and WiDr, respectively, transfected with mt and wt p53. Lovo 175X2 cells (transfected with mt p53) were more resistant to 5-fluorouracil (5-FU; 2-fold), nolatrexed (3-fold), raltitrexed (3-fold) and pemetrexed (10-fold) in comparison with the wt p53 parental cells Lovo 92. Resistance was associated with an increase in TS protein expression and catalytic activity, which might be caused by the loss of the inhibitory effect on the activity of TS promoter or by the lack of TS mRNA degradation, as suggested by the reversal of TS expression to the levels of Lovo 92 cells by adding actinomycin. In contrast, Lovo li cells, characterized by functionally inactive p53, were 3-13-fold more sensitive to nolatrexed, raltitrexed and pemetrexed, and had a lower TS mRNA, protein expression and catalytic activity than Lovo 92. However, MDM-2 expression was significantly higher in Lovo li, while no significant differences were observed in Lovo 175X2 cells with respect to Lovo 92. Finally, mt p53 WiDr transfected with wt p53 were not significantly different from mt p53 WiDr cells with respect to sensitivity to TS inhibitors or TS levels. Altogether, these results indicate that changes in the status of p53, can differently alter sensitivity to TS inhibitors by affecting TS levels, depending on activity or cell line, and might explain the lack of clear correlation between mutations in p53 and clinical outcome after chemotherapy with TS inhibitors.

Upregulated p53 expression activates apoptotic pathways in wild-type p53-bearing mesothelioma and enhances cytotoxicity of cisplatin and pemetrexed

“…21 Activities of enzymes involved in PEM metabolisms may play a crucial role in the anti-tumor effects, but the relationship between the p53 status and PEM sensitivity has been unclear. 22,23 An administration schedule of an anticancer agent and Ad-p53 might be influential to the cytotoxic activity, but our preliminary data did not show any significant difference in the cytotoxicity irrespective of the administration order (data not shown).…”

Modulation of thymidilate synthase and p53 expression by HDAC inhibitor vorinostat resulted in synergistic antitumor effect in combination with 5FU or Raltitrexed

“…Previous studies, demonstrated that TS mRNA and protein are both downregulated by HDAC-I through two independent mechanisms: at the transcriptional level and through modulation of protein degradation by a mechanism involving acetylation of the chaperone protein Hsp90. 14,15 Moreover, in our study we demonstrated that p53 protein, whose functional wild-type expression is critical for drug sensitivity to TS inhibitors such as 5FU and RTX, 35 is upregulated by vorinostat in wt-p53 cells but downregulated in mut-p53 cells, as single agent or in combination treatment. It as been previously reported that p53 protein acetylation, upon HDAC inhibition, is essential for preventing the degradation and for leading to an open conformation that allowed the protein to bind DNA.…”

“…33 More prominently, it was recently reported, in colon cancer cells transfected with mut-p53, an increased TS mRNA and protein expression as well as activity, associated with decreased sensitivity to 5-FU and antifolates, compared to the wild-type parental cells. 35 On these basis we proposed that the opposite modulation of wt-and mut-p53 protein by vorinostat could be an additional mechanism explaining the synergistic interaction with TS inhibitors.…”

“…31,32 On the other hand, expression of wild-type p53 has been shown to be required for 5FU-and RTX-induced antitumor effects. 28,29,[33][34][35] Therefore new strategies overcoming these molecular mechanisms of chemo-resistance are required.…”

TS gene polymorphisms are not good markers of response to 5-FU therapy in stage III colon cancer patients

“…Our results do not support this thesis. However, the regulation of TS expression and function remains quite complex and most likely is influenced by many still unknown factors [11,37]. Thus, ideally to explain the biological role of TS in the resistance to 5-FU, other techniques to objectively study protein expression and preferably function, would probably be more accurate.…”