Inhibition of DTYMK significantly restrains the growth of HCC and increases sensitivity to oxaliplatin

Abstract

Most patients with hepatocellular carcinoma (HCC) are in the middle or advanced stage at the time of diagnosis, and the therapeutic effect is limited. Therefore, this study aimed to verify whether deoxythymidylate kinase (DTYMK) increased in HCC and was an effective therapeutic target in HCC. The findings revealed that the DTYMK level significantly increased and correlated with poor prognosis in HCC. However, nothing else is known, except that DTYMK could catalyze the phosphorylation of deoxythymidine monophosphate (dTMP) to form deoxythymidine diphosphate (dTDP). A number of experiments were performed to study the function of DTYMK in vitro and in vivo to resolve this knowledge gap. The knockdown of DTYMK was found to significantly inhibit the growth of HCC and increase the sensitivity to oxaliplatin, which is commonly used in HCC treatment. Moreover, DTYMK was found to competitively combine with miR-378a-3p to maintain the expression of MAPK activated protein kinase 2 (MAPKAPK2) and thus activate the phospho-heat shock protein 27 (phospho-HSP27)/nuclear factor NF-kappaB (NF-κB) axis, which mediated the drug resistance, proliferation of tumor cells, and infiltration of tumor-associated macrophages by inducing the expression of C-C motif chemokine ligand 5 (CCL5). Thus, this study demonstrated a new mechanism and provided a new insight into the role of mRNA in not only encoding proteins to regulate the process of life but also regulating the expression of other genes and tumor microenvironment through the competing endogenous RNA (ceRNA) mechanism.

Fig. 1. Increased expression of DTYMK in HCC.

A Gene analysis of patients with HCC from TCGA. B Eight genes were upregulated and had the most significant survival differences (C) DTYMK expression increased in multiple cancer types, especially in HCC. D Expression of DTYMK was related to cancer stages. E Expression of DTYMK was related to cancer grades. F Increased RNA level of DTYMK in HepG2, Huh7, and Hep3B HCC cell lines. G Increased RNA level of DTYMK in most patients with HCC. H Increased protein level of DTYMK in most patients with HCC.

Fig. 2. Decreased expression of DTYMK inhibited proliferation and increased sensitivity to oxaliplatin in HCC cells.

A DTYMK catalyzed the phosphorylation of dTMP to form dTDP. B and C Decreased DTYMK significantly inhibited the proliferation of Huh7 and Hep3B cells, while the supplement of dTDP partly rescued the proliferation ability. D and E Decreased expression of DTYMK could lead to cell cycle arrest and reduce the proportion of cells entering the G1 phase. F Decreased expression of CDK2, CDK4, Cyclin A2, and Cyclin D1 after DTYMK knockdown. G and H Increased sensitivity to oxaliplatin after DTYMK knockdown. I Reduced Bcl-2 and increased cleaved-PARP and Bax in the sh-DTYMK group than in the other groups. DTY, DTYMK; OXA, Oxaliplatin. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Fig. 3. DTYMK acted as ceRNA to affect the function of MAPKAPK2.

A Decreased expression of miR-378a-3p in HCC from TCGA and GEO. B and C Decreased expression of miR-378a-3p in HCC from RNA-seq of 6 paired HCC tissues and qPCR of 20 paired HCC tissues. D Expression of DTYMK, MAPKAPK2, AKT1, CART, and NRF1 could be inhibited by miR-378a-3p. E Decreased expression of MAPKAPK2, AKT1, CART, and NRF1 after DTYMK knockdown. F Heatmap (RNA-seq) showed a clear correlation between DTYMK and MAPKAPK2. G Positive correlation between DTYMK and MAPKAPK2 (RNA-seq). H and I Opposite relation between miR-378a-3p, DTYMK, and MAPKAPK2 (RNA-seq). J Significant correlation between DTYMK and MAPKAPK2 in multiple cancer types. K and L Increased expression of MAPKAPK2 in 16 of 20 patients with HCC, which correlated with the expression of DTYMK. M An opposite expression trend existed between miR-378a-3p and DTYMK. N An opposite expression trend existed between miR-378a-3p and MAPKAPK2. O Subcellular locations experiments showing that DTYMK and MAPKAPK2 were enriched in the cytoplasm. P Decreased expression of MAPKAPK2 and p-hsp27, and decreased nuclear translocation of NF-κB (p65) after DTYMK knockdown. Q Potential binding sites between DTYMK, MAPKAPK2, and miR-378a-3p. R MiR-378a-3p bound and significantly inhibited the expression of DTYMK and MAPKAPK2. S MiR-378a-3p inhibited the expression of DTYMK, thus inhibiting MAPKAPK2/p-hsp27/NF-κB (p65). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Fig. 4. Tumor formation rate and growth speed were significantly inhibited after DTYMK knockdown in vivo.

A and B A stronger inhibitory effect on tumor formation rate was exhibited in transplanted carcinoma in nude mice (n = 5 per group). C Tumor growth speed was significantly inhibited after DTYMK knockdown (p = 0.039). D Tumor sizes in the negative control group decreased partially after oxaliplatin treatment, while no tumors were formatted in the sh-DTYMK group (n = 5 per group). E Schematic diagram of tumor xenotransplantation model. F The results of immunohistochemistry showed that the expression of DTYMK and MAPKAPK2 reduced in the sh-DTYMK group compared with the negative control group. Moreover, higher CCL5 expression and more CD163⁺ TAMs were found in the negative control group. Scale bar = 50 μm. G Statistical analysis of the mice IHC results. H ELISA showed an inhibited level of CCL5 after DTYMK knockdown. I CCL5 could stimulate the chemotactic migration of monocytes. NC negative control, DTY DTYMK. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Fig. 5. DTYMK expression was significantly upregulated and associated with a poor prognosis.

A Immunohistochemistry tests showing that DTYMK expression was significantly upregulated in HCC tissues. Scale bar = 50 μm. B Increased expression of DTYMK was associated with poor OS and DFS among 105 patients with HCC from the Sun Yat-sen University Cancer Center. C Higher expression of DTYMK was associated with poor OS and DFS in HCC from TCGA. D and E DTYMK correlated with the infiltration of M2 macrophages and predicted the prognosis of patients with HCC more accurately. OS overall survival, DFS disease-free survival.