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Page 720 Bijnsdorp et al. Cancer Drug Resist 2021;4:719-27 https://dx.doi.org/10.20517/cdr.2021.21

Conclusion: Rapamycin resistance in TP-expressing cancer cells may therefore be related to thymidine-mediated
autophagy activation.

Keywords: Thymidine phosphorylase, mTOR, rapamycin, thymidine, thymidine phosphorylase inhibitor, autophagy

INTRODUCTION
Thymidine phosphorylase (TP) is also known as platelet derived-endothelial cell growth factor, which plays
[1]
a role in angiogenesis . TP is often overexpressed in cancer, including colorectal, breast and bladder
cancer . When upregulated, TP is related to a poor prognosis for the patient, induces metastasis, and leads
[2]
[3,4]
to a higher microvessel density . The enzymatic activity of TP has been demonstrated to be essential for
stimulating angiogenesis in various studies . Therefore, inhibitors of TP have been developed as anti-
[5]
[6]
angiogenesis therapy, including the thymidine phosphorylase inhibitor (TPI) . TPI inhibits TP very
potently and specifically and has demonstrated anti-angiogenic activity. However, its primary application is
in the combination with trifluorothymidine (TFT) in TAS-102 (Lonsurf), which is registered as third line
[7,8]
therapy in the treatment of advanced colorectal cancer .
[9]
It is not clear whether angiogenesis plays a role in the efficacy of TAS-102 . However, various studies
clearly indicated a role of TP in angiogenesis, although the exact mechanism behind TP-mediated
angiogenesis remains unclear. TP converts thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-
[10]
1-P) . dR-1-P can be converted to deoxyribose-5-phosphate or deoxyribose (dR). dR is often thought to be
the main regulator behind TP-mediated angiogenesis because it is the main product that can be secreted by
the cells . Moreover, dR was demonstrated to be pro-angiogenic in various in vitro and in vivo studies [11-16] .
[10]
In addition to dR, various angiogenic factors may also be involved in the anti-angiogenic effects of TP, such
as Interleukin-8 (IL-8) and Vascular Endothelial Growth Factor [17-19] .

Several mechanistic studies on TP-mediated angiogenesis indicated that p70/S6k phosphorylation was
involved in migration and invasion [11,20] . P70/S6k is a kinase that is directly downstream of mTOR
[21]
(mammalian target of rapamycin) and plays a role in signaling to proliferation and angiogenesis . Various
inhibitors of mTOR have been developed, next to rapamycin, which is an immunosuppressant and is used
to prevent rejection after organ transplantation. Since it has strong antiproliferative activity, rapamycin and
other potent novel analogs (temsirolimus and everolimus) have been evaluated for their anticancer effects.
Both drugs have been registered for treatment of renal cell cancer. mTOR now represents an attractive anti-
tumor target, either alone or in combination [21,22] . One of the mechanisms of action of rapamycin is the
induction of autophagy. Autophagy may result in the induction of cell death, but it can also cause
[23]
protection against cell death . Autophagy may also provide protection against cytotoxicity induced by 5-
fluorouracil (5-FU), but not by TFT [24-26] . Inhibition of autophagy with 3-methyl-adenine (3-MA) enhanced
the sensitivity to 5-FU. In addition, for other anticancer drugs, it has been demonstrated that autophagy
may protect against their action, which can be reversed by inhibitors such as (hydroxy)chloroquine .
[27]
Several clinical studies have been initiated to reverse resistance due to autophagy protection [28,29] . Therefore,
autophagy appears to be an interesting target to increase chemosensitivity.

To determine to what extent TP plays a role in the p70/S6k signaling pathway, Colo320 and Colo320TP1
cancer cells (deficient and with high TP expression, respectively) were exposed to rapamycin, combined
with TdR and TPI. In the present study, we show that TdR addition results in rapamycin resistance.
Downstream effects of mTOR and autophagy may play a role in this protective effect. These data provide
important information for the application of the mTOR inhibitors in the clinic.