Human dCTP pyrophosphatase 1 promotes breast cancer cell growth and stemness through the modulation on 5-methyl-dCTP metabolism and global hypomethylation

Abstract

Human DCTPP1 (dCTP pyrophosphatase 1), also known as XTP3-transactivated protein A, belongs to MazG-like nucleoside triphosphate pyrophosphatase (NTP-PPase) superfamily. Being a newly identified pyrophosphatase, its relevance to tumorigenesis and the mechanisms are not well investigated. In the present study, we have confirmed our previous study that DCTPP1 was significantly hyperexpressed in breast cancer and further demonstrated its strong association with tumor progression and poor prognosis in breast cancer. Knockdown of DCTPP1 in breast cancer cell line MCF-7 cells remarkably retarded proliferation and colony formation in vitro. The capacity of mammosphere formation of MCF-7 was suppressed with the silence of DCTPP1, which was consistent with the enhanced mammosphere-forming ability in DCTPP1-overexpressed MDA-MB-231 cells. To further dissect the mechanisms of DCTPP1 in promoting tumor cell growth and stemness maintenance, its biochemical properties and biological functions were investigated. DCTPP1 displayed bioactive form with tetrameric structure similar to other MazG domain-containing pyrophosphatases based on structure simulation. A substrate preference for dCTP and its methylated or halogen-modified derivatives over the other canonical (deoxy-) NTPs was demonstrated from enzymatic assay. This substrate preference was also proved in breast cancer cells that the intracellular 5-methyl-dCTP level increased in DCTPP1-deficient MCF-7 cells but decreased in DCTPP1-overexpressed MDA-MB-231 cells. Moreover, global methylation level was elevated in DCTPP1-knockdown MCF-7 cells or mammosphere-forming MCF-7 cells but decreased significantly in DCTPP1-overexpressed MDA-MB-231 cells and its mammospheres. Our results thus indicated that human DCTPP1 was capable of modulating the concentration of intracellular 5-methyl-dCTP. This in turn affected global methylation, contributing to a known phenomenon of hypomethylation related to the cancer cell growth and stemness maintenance. Our current investigations point to the pathological functions of DCTPP1 overexpression in breast cancer cells with aberrant dCTP metabolism and epigenetic modification.

DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by up-regulating MDR1 expression epigenetically

“…The expressions of DCTPP1 and MDR1 in GC tissue microarray were tested by immunohistochemistry described in our previous report using anti-DCTPP1 and anti-MDR1 antibodies [21]. Slides were observed under the light microscope and data were analyzed using the Leica Qwin V3 image analysis system (Leica Microsystems GmbH, Wetzlar, Germany).…”

Serum profiling identifies ibrutinib as a treatment option for young adults with B‐cell acute lymphoblastic leukaemia

“…HEK293T cell line was used to demonstrate we could immunolabel BMX (van Oosterwijk et al , ) and VGLL4 (Jiao et al , ), but only showed a moderate cytoplasmic expression of BMX and were negative for VGLL4; weak immunolabelling of survivin was also detected. The K562 cell line was used to demonstrate the immunolabelling of DCTPP1 (Song et al , ) and survivin (Schmidt et al , ), and showed moderate cytoplasmic levels of both. MDA‐MB‐231 cell line was used to demonstrate we could immunolabel DCTPP1 (Song et al , ), while MDA‐MB‐468 was chosen as an immunolabelling control for VGLL4 (Zhang et al , ), but both were negative for the detection of these antigens.…”

“…The K562 cell line was used to demonstrate the immunolabelling of DCTPP1 (Song et al , ) and survivin (Schmidt et al , ), and showed moderate cytoplasmic levels of both. MDA‐MB‐231 cell line was used to demonstrate we could immunolabel DCTPP1 (Song et al , ), while MDA‐MB‐468 was chosen as an immunolabelling control for VGLL4 (Zhang et al , ), but both were negative for the detection of these antigens. Of note VGLL4 was not found in any cell line, MDA‐MB‐231 and MDA‐MB‐468 did not express DCTPP1 or survivin, while each had transcripts from these antigens that were detectable by qPCR.…”

“…The other two antigens investigated were DCTPP1 an intracellular regulator of 5‐methyl‐dCTP metabolism that has been associated with DNA hypermethylation and gene silencing (Song et al , ). DCTPP1 is downregulated when the PI3K/AKT/mTOR pathway is activated and autophagy activity is suppressed (Lu et al , ) with its’ expression being associated with poor prognosis and lower overall survival of solid cancer patients (Zhang et al , ; Morisaki et al , ; Song et al , ; Lu et al , ). DCTPP1 has also been shown to increase chemotherapy resistance in gastric cancer suggesting a role as an indicative biomarker of chemoresistance in patients (Xia et al , ).…”

Chemical Genetics Reveals a Role of dCTP Pyrophosphatase 1 in Wnt Signaling

“…[9] It catalyzes the hydrolysis of dNTPs to their corresponding monophosphates and displays higher affinity towards 5-modified non-canonical dNTPs (e.g., 5-I-dCTP > 5-Br-dCTP > 5-Me-dCTP > dCTP), thereby decreasing their intracellular levels and hence subsequently their insertion into nascent DNAd uring replication. [9,10] dCTPP1 is considered an attractive anticancer target, because it is overexpressed in many carcinomas (e.g.,b reast and gastric). [11] Reversible binding of dCTPP1 to probe 2 was confirmed by means of concentration-dependent competitive pull-down using immunoblotting for dCTPP1 ( Figure 2b).…”