Identification of characteristic molecular signature of Müllerian inhibiting substance in human HPV-related cervical cancer cells

Abstract

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone (AMH), is a member of the transforming growth factor-β (TGF-β) superfamily that plays an important role in the mesenchymal-epithelial interaction, cell growth and proliferation, extracellular matrix production and tissue remodeling. Previously, we demonstrated that MIS suppressed ovarian cancer cell growth and suggested large-scale genetic elements that could be responsible for anti-neoplastic effects of MIS on ovarian cancer cells. In this study, we demonstrated the expression of MIS type II receptor (MISRII) in the human papillomavirus (HPV)-16-related cervical cancer cell lines CaSki and SiHa, and a non-HPV-related cervical cancer cell line, C33A. We also showed that MIS inhibited growth of cervical cancer cells, and induced cellular apoptosis of C33A. In addition, we identified a characteristic molecular signature of MIS in CaSki cells by using whole genome expression analysis. Of the 1,690 genes that showed significant expression changes by MIS, 21 genes were related to cell cycle; 13 genes to apoptosis; and 52 genes to the cancer pathway. On performing a search for cell cycle pathways in the KEGG pathway database, several gene expressions at the G1/S checkpoint were found. In particular, the expression of p16 and p107 increased and that of E2F2 and E2F3 decreased at an early stage, whereas the expression of E2F4 and E2F5 decreased at a later stage after MIS treatment. These data suggest that MIS produces activity against HPV16-related cervical cancers in vitro, and MIS may also be an effective targeted therapy for HPV16-related cervical cancer. Genetic data obtained here could be useful in determining the treatment strategy of MISR-expressing cervical tumors in the future.

Figure 1

Immunohistochemistry using MISRII antibody from the human kidney epithelial cell line, 293 cells (A) and cervical cancer cell line, C33A (B), CaSki (C), and SiHa cells (D). Compared to 293 cell line showing negative staining pattern for MISRII, all cervical cancer cell lines showed strong specific staining for MISRII; staining in C33A was the most intense (×200). At higher magnification (×400) of the boxed area of (B, C, and D), MISRII was localized specifically and exclusively in the cell membrane and occasionally in the Golgi’s complex of the cytoplasm. The nucleus does not stain for MISRII.

Figure 2

Effect of MIS on the viability of 293 (A) and of C33A (B), CaSki (C), and SiHa cells (D). Cells were treated with MIS at 10 μg/ml 24 h after plating. At 24 and 48 h after treatment, cells were stained MTT, and the absorbance was read at 550 nm. Results were presented as percentage of control which was calculated using the equation: (mean absorbance of treated cells/mean absorbance of control cells) × 100. Data were expressed as mean ± standard deviation (SD) from three independent experiments. *P<0.05 as compared to corresponding control cells.

Figure 3

Cell cycle distribution after exposure to MIS in 293 (A) and of C33A (B), CaSki (C), and SiHa cells (D). Cells were treated with 10 μg/ml MIS for 24 and 48 h, respectively. Then they were trypsinized and fixed in 70% ethanol. After washing, cells were exposed to propidium iodide/RNase solution. Histograms of cellular DNA content were obtained by flow cytometry.

Figure 4

Induction of appoptosis by MIS in 293 (A) and of C33A (B), CaSki (C), and SiHa cells (D). Cells were treated with 10 μg/ml MIS for 24 and 48 h, respectively. For apoptosis, the externalization of phosphatidylserine was assessed by measuring Annexin-V-FITC binding using propidium iodide as a counterstain. Quadrant rectangular dot grams from a representative of 3 independent experiments are shown.

Figure 5

Differential gene expression profiling and identification of large-scale molecular changes in the CaSki cervical cancer cell line that was treated with MIS at 10 μg/ml. (A), Unsupervised hierarchical clustering of 14,930 genetic elements with minimum selection and filtering criteria resulted in two divided subcultures on dendrogram. (B), A significant subset of outlier genes was further narrowed by filtering genes showing expression changes due to MIS treatment of cells. Briefly, the mathematical comparisons of genes between 0 h and the 4- to 96-h time points were performed by selection of genes showing at least 1.5-fold changes induced by MIS treatment compared to non-treated control (0 h). This analysis resulted in the identification of 1,690 outlier genes that were significantly differentially expressed in MIS-treated cells. These elements were then visualized as a heat map where the red color indicates that expression levels of genetic elements are higher than the mean value of non-treatment, and green color indicates that expression levels of genetic elements are lower than the mean value of non-treatment.

Figure 6

Outlier expression profile of cancer pathway-related genetic elements changed significantly by MIS in the CaSki cells. Of 1,690 outlier genes, 52 genes were mapped into pathway in cancer which included most genetic elements (Table I) using the KEGG pathway database. The expression patterns of genes were then visualized as a heat map. The red color indicates that expression levels of genetic elements are higher than the mean value of non-treatment, and green color indicates that expression levels of genetic elements are lower than mean value of non-treatment.

Figure 7

Outlier expression profile of cell cycle-related genetic elements changed significantly by MIS in the CaSki cells. Of 1,690 outlier genes, 21 genes were mapped into cell cycle through the KEGG pathway database.

Figure 8

Outlier expression profile of apoptosis genetic elements changed significantly by MIS in the CaSki cells. Of 1,690 outlier genes, 13 were mapped to apoptotic pathways in the KEGG database.

Figure 9

Validation of genes previously known to be changed by MIS treatment.