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. 2015 Apr 7;112(14):4411-6.
doi: 10.1073/pnas.1421365112. Epub 2015 Mar 23.

Enhanced expression of DNA polymerase eta contributes to cisplatin resistance of ovarian cancer stem cells

Amit Kumar Srivastava  1 , Chunhua Han  1 , Ran Zhao  1 , Tiantian Cui  1 , Yuntao Dai  2 , Charlene Mao  3 , Weiqiang Zhao  4 , Xiaoli Zhang  5 , Jianhua Yu  6 , Qi-En Wang  7
Affiliations

"V体育安卓版" Enhanced expression of DNA polymerase eta contributes to cisplatin resistance of ovarian cancer stem cells

Amit Kumar Srivastava et al. Proc Natl Acad Sci U S A. .

Abstract

Cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance are believed to be responsible for treatment failure and tumor relapse in ovarian cancer patients VSports手机版. However, it is still unclear how CSCs survive DNA-damaging agent treatment. Here, we report an elevated expression of DNA polymerase η (Pol η) in ovarian CSCs isolated from both ovarian cancer cell lines and primary tumors, indicating that CSCs may have intrinsically enhanced translesion DNA synthesis (TLS). Down-regulation of Pol η blocked cisplatin-induced CSC enrichment both in vitro and in vivo through the enhancement of cisplatin-induced apoptosis in CSCs, indicating that Pol η-mediated TLS contributes to the survival of CSCs upon cisplatin treatment. Furthermore, our data demonstrated a depletion of miR-93 in ovarian CSCs. Enforced expression of miR-93 in ovarian CSCs reduced Pol η expression and increased their sensitivity to cisplatin. Taken together, our data suggest that ovarian CSCs have intrinsically enhanced Pol η-mediated TLS, allowing CSCs to survive cisplatin treatment, leading to tumor relapse. Targeting Pol η, probably through enhancement of miR-93 expression, might be exploited as a strategy to increase the efficacy of cisplatin treatment. .

Keywords: DNA polymerase eta; cancer stem cell; cisplatin; miR-93; translesion synthesis. V体育安卓版.

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"V体育2025版" Conflict of interest statement

The authors declare no conflict of interest.

"VSports在线直播" Figures

Fig. 1.
Fig. 1.
Enhanced TLS in ovarian CSCs. (A-C) Expression of various TLS Pols in ovarian cancer cell lines 2008 (A), C13 (B), and SKOV3 (C), as well as their corresponding CSC populations were determined using quantitative RT-PCR (qRT-PCR). n = 3; Bar, SD; **, P < 0.01. (D) The mRNA expression level of POLH was determined in primary tumor cells isolated from five freshly removed ovarian tumors and their corresponding spheroid cells, using qRT-PCR. n = 3; Bar, SD. Analysis by the linear mixed model indicates that POLH expression increased significantly in spheroid cells compared with bulk cancer cells (P < 0.0001). (E) Protein levels of Nanog and Pol η in ovarian cancer cell lines and their corresponding CSC populations were determined using immunoblotting. (F) Monoubiquitylated PCNA in ovarian cancer cell lines and their corresponding CSC populations were determined using immunoblotting. Results shown here are from one of three experiments with identical results.
Fig. 2.
Fig. 2.
Down-regulation of Pol η in ovarian cancer cells blocked cisplatin-induced enrichment of CSCs in vitro. (A, C, and E) Ovarian cancer cell lines 2008 (A), C13 (C), and SKOV3 (E) were transiently transfected with either POLH siRNA or control siRNA for 24 h, and the expression of Pol η was determined using immunoblotting. (B, D, and F) The siRNA transfected 2008 (B), C13 (D), and SKOV3 (F) cells were treated with cisplatin for 3 d and stained with anti–CD44-FITC and anti–CD117-PE antibodies. The percentage of CD44+CD117+ cells was analyzed using FACS. n = 3; Bar, SD; *, P < 0.05; **, P < 0.01.
Fig. 3.
Fig. 3.
Down-regulation of Pol η in ovarian cancer cells blocked cisplatin-induced enrichment of CSCs in vivo and sensitized xenografts to cisplatin treatment. (A) The 2008 cell lines with stable transfection of either shCtrl or shPOLH were established. (B) The 2008-shCtrl and 2008-shPOLH cells were injected into the flank of athymic nude mice s.c. (n = 8). Mice were treated with cisplatin (7 mg/kg) once every week for 2 wk after tumors were about 0.5 cm in diameter. Tumor sizes were recorded, and tumor growth curves were plotted. Red arrows indicate the cisplatin treatment. (C and D) Tumors were harvested after 2 d of the second treatment. Tumor cells were isolated, and the percentage of CD44+CD117+ cells was analyzed using FACS (C) and plotted (D). n = 7; Bar, SD; *, P < 0.05; **, P < 0.01.
Fig. 4.
Fig. 4.
Down-regulation of Pol η-sensitized CSCs to cisplatin treatment. (A–C) The 2008-CD44+CD117+ (A), C13-CD44+CD117+ (B), and SKOV3-spheroid (C) cells were transfected with either POLH siRNA or control siRNA, followed by treatment with cisplatin for 3 d. Cell survival was determined using the MTT assay. n = 4; Bar, SD; *, P < 0.05; #, P < 0.01 compared between control and POLH knockdown cells at each time point. (D) SKOV3-spheroid cells were transfected with either control or POLH siRNA for 48 h and then treated with cisplatin for 24 h. Apoptotic cells were stained with Annexin V and detected by FACS. n = 3; Bar, SD; *, P < 0.05.
Fig. 5.
Fig. 5.
Reduced miR-93 expression in ovarian CSCs. (A) Predicted binding of miR-93 to the 3′UTR of POLH. (B and C) Expression of miR-93 was determined in various ovarian cancer cell lines and their corresponding CSCs by qRT-PCR. n = 3; Bar, SD; **, P < 0.01. (D) Expression of miR-93 in bulk primary tumor cells and their corresponding spheroid cells was analyzed using qRT-PCR. n = 3, Bar, SD. Analysis indicates that there was significantly decreased miR-93 expression in the spheroid cells compared with bulk cells (P = 0.047).
Fig. 6.
Fig. 6.
Reduced expression of miR-93 contributes to the elevated POLH expression and enhanced cisplatin resistance in ovarian CSCs. (A and B) Western blot analyses of the protein level of Pol η in ovarian cancer cell lines with miR-93 inhibition (A) or their corresponding CSCs with miR-93 overexpression (B). The results are representative of three experiments with similar results. (C–E) MTT assay was used to determine the effect of enhanced miR-93 expression on the sensitivity of 2008 (C), C13 (D), and SKOV3 (E) CSCs to cisplatin. n = 4; Bar, SD; *, P < 0.05; **, P < 0.01 compared between control and miR-93 overexpressed cells at each concentration. (F–H) Effect of enhanced miR-93 expression on cisplatin-induced apoptosis in 2008 (F), C13 (G), and SKOV3 (H) CSCs was determined with Annexin V staining and FACS. n = 3; Bar, SD; *, P < 0.05, **, P < 0.01.
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