Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is involved in cervical cancer stemness and can be a target of immunotherapy

Abstract

Cervical cancer is a major cause of cancer death in females worldwide. Cervical cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are resistant to conventional radiotherapy and chemotherapy, and CSCs/CICs are thought to be responsible for recurrence. Eradication of CSCs/CICs is thus essential to cure cervical cancer. In this study, we isolated cervical CSCs/CICs by sphere culture, and we identified a cancer testis (CT) antigen, CTCFL/BORIS, that is expressed in cervical CSCs/CICs. BORIS has 23 mRNA isoform variants classified by 6 subfamilies (sfs), and they encode 17 different BORIS peptides. BORIS sf1 and sf4 are expressed in both CSCs/CICs and non-CSCs/CICs, whereas BORIS sf6 is expressed only in CSCs/CICs. Overexpression of BORIS sf6 in cervical cancer cells increased sphere formation and tumor-initiating ability compared with those in control cells, whereas overexpression of BORIS sf1 and BORIS sf4 resulted in only slight increases. Thus, BORIS sf6 is a cervical CSC/CIC-specific subfamily and has a role in the maintenance of cervical CSCs/CICs. BORIS sf6 contains a specific c-terminal domain (C34), and we identified a human leukocyte antigen (HLA)-A2-restricted antigenic peptide, BORIS C34_24(9) encoded by BORIS sf6. A BORIS C34_24(9)-specific cytotoxic T cell (CTL) clone showed cytotoxicity for BORIS sf6-overexpressing cervical cancer cells. Furthermore, the CTL clone significantly suppressed sphere formation of CaSki cells. Taken together, the results indicate that the CT antigen BORIS sf6 is specifically expressed in cervical CSCs/CICs, that BORIS sf6 has a role in the maintenance of CSCs/CICs, and that BORIS C34_24(9) peptide is a promising candidate for cervical CSC/CIC-targeting immunotherapy.

Keywords: BORIS; CTL; cancer stem cells; cervical cancer; peptide vaccine.

Figure 1. Spheroids have characteristics of CSCs

Representative pictures of CaSki cells in a serum cultured condition (A) and sphere culture condition (B). Magnifications are 40 × and 100 ×, respectively. (C and D) Resistance to radiotherapy and resistance to chemotherapy. WST-1 analysis showed that spheroid cells derived from cervical cancer cell lines are resistant to irradiation and chemotherapy. Data represent means ± SE. *P < 0.05. (E) Cell cycle analysis of spheroids and adherent cultured cells of CaSki and TC-S. (F) Expression of Stemness genes in serum cultured cells and spheroids derived from cervical cancer cell lines was evaluated by quantitative RT-PCR. Data represent relative quantity means ± SD. Asterisks indicate statistically significant difference (p < 0.01) between serum and sphere cells.

Figure 2. The cancer/testis gene BORIS is preferentially expressed in CSCs and BORIS expression is a marker of poor prognosis for patients with advanced cervical cancer

(A) Quantitative RT-PCR (qRT-PCR) of BORIS in normal organs. (B) QRT-PCR of BORIS in cervical cancer cell lines. Serum: serum culture sells. Sphere: sphere culture cells. Each value is mean relative quantity. Immunohistochemistry of 38 stage III/IV cervical squamous cell carcinoma samples with a BORIS-specific antibody. Immunohistochemical staining of BORIS in a normal testis tissue as a positive control (C), a BORIS^high case (D) and a BORIS^low case (E). Original magnification is 100 × and the size bar is 50 μm. (F) Kaplan-Meier survival estimates were performed according to immunohistochemistry positivity of BORIS. The median survival times of the BORIS^high group (n = 12) and BORIS^low group (n = 26) were 10.5 months and 48.0 months, respectively. The log-rank test revealed a significantly worse prognosis for BORIS^high cases (p = 0.0212). The hazard ratio of BORIS^high cases was 2.407 (95% confidence interval: 1.190–8.567). (G) BORIS sf6 expression in cervical cancer tissue specimens of BORIS^high group by IHC. Tissue samples were analyzed by RT-PCR with BORIS sf6-specific primers. RNA is extracted from formalin-fixed, paraffin-embedded (FFPE) samples.

Figure 3. BORIS sf6 is involved in sphere forming ability and cancer initiation ability

(A) QRT-PCR of CaSki cells transfected with BORIS siRNAs. BORIS mRNA expression in CaSki cells transfected with BORIS-specific siRNA and scrambled siRNA, detected by qRT-PCR analysis. Each value is the mean ± SD of relative quantity (RQ). *P < 0.001. (B) Sphere formation of BORIS-knocked-down cells. Each value is the mean ± SD. *P < 0.001, **P = 0.003. (C) Images of spheres. Magnification is 10 ×, size bar = 100 μm. (D) RT-PCR using specific primers for BORIS subfamilies. (E) Sphere formation of BORIS variants. Cells with overexpression of each of four different BORIS variants, B0, B3, B6 and C7, were established by using aretroviral vector. Sphere formation assays were performed using stable transfectants. Each value is the mean ± SD. (F) Growth curves of tumors derived from CaSki cells transfected with mock and BORIS B0, B3, B6 and C7. Ten, 10² and 10³ tumor cells were injected into BALB/C nude mice, respectively. Each value is the mean tumor volume + SE. *P < 0.05. (G) Tumor incidence and estimated frequency of cancer stem cells. The number indicates tumor-initiation incidence in BALB/C nude mice. Cancer stem cell frequency was calculated by Extreme Limiting Dilution Analysis (ELDA) software. CI = confidence interval.

Figure 4. Schema of BORIS variants expressed in cercvical cancer stem cells

BORIS variants were divided into two groups according to the expression in spheres derived from CaSki and MS751 cells. Asterisks indicate variants for which we examined the functions of by overexpression. We designed HLA-A2-restricted peptides from the C-terminus domain of BORIS sf6. Aa: amino acids Sf: subfamily ZF: zinc finger.

Figure 5. BORIS sf 6-specific CTL response can suppress sphere formation

(A) Peptide binding assay. Binding affinity was evaluated by comparing mean fluorescence intensity of HLA-A2 expression in the presence of peptide pulsation to mean fluorescence intensity in the absence of the peptide. CMV and influenza peptides were used as positive controls, and GK12 peptide was used as a negative control. (B) ELISPOT assay. BORIS C34_24(9) peptide-specific cytotoxic T cell (CTL) induction was performed and assessed using the interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay. HLA-A*0201-positive PBMCs were obtained from four healthy donors. Donors A, B and C were HLA-*A0201-positive and donor D was HLA-*A0206-positive. (C) Tetramer assay of BORIS C34_24(9)-specific CTLs. Fluorescence-activated cell sorting (FACS) was performed with PE-conjugated BORIS C34_24(9) peptide/HLA-A*0201 tetramer and anti-CD8-FITC antibody. BORIS C34_24(9) peptide/HLA-A*0201 tetramer-positive cells were directly sorted and a CTL clone was established. (D) Tetramer assay of a BORIS C34_24(9) peptide-specific CLT clone, E1. CTL clone E1 and negative CTL clone were stained by PE-conjugated BORIS C34_24(9) peptide/HLA-A*0201 tetramer and anti-CD8-FITC antibody and analyzed. (E) ELISPOT assay of CTL clone E1. BORIS C34_24(9) peptide specificity of CTL clone E1 and negative clone evaluated by the ELISPOT assay. (F) LDH release cytotoxicity assay. Specific cytotoxicity for peptide-pulsed T2 cells was aexamined (left panel). Influenza peptide-pulsed T2 cells, peptide (−) T2 cells and K562 cells were uses as negative controls. Specific cytotoxicity for CaSki and CaSki/BORIS B6 cells was examined (right panel). K562 cells were used as a negative control. Each value is the mean ± SE. (G) Blocking by anti-HLA-class I antibody. Cytotoxicity of the E1 clone for CaSki cells and CaSki/BORIS B6 cells was examined using anti-HLA class I mAb W6/32 and anti MHC-class II mAb L243. Each value is the mean ± SE. (H and I) Sphere formation in the presence of BORIS C34_24(9)-specific CTL clone. Five × 10³ CaSki cells and 5 × 10⁴ E1 CTL clone or CTL negative clone were co-cultured in a 96-well ultra low attachment plate. On co-culture day 8, a microscope photograph was taken (H). Original magnification is × 100. Size bar is 100 μm. The numbers of spheres were counted (I). Each value is the mean ± SE. (J) Tumor growth of CaSki cells in a therapeutic adoptive transfer model. Each value is the mean ± SE. Asterisks indicate statistically significant difference (p < 0.05).