Transformation of human ovarian surface epithelial cells by Krüppel-like factor 8

Abstract

We have previously demonstrated that Krüppel-like factor 8 (KLF8) participates in oncogenic transformation of mouse fibroblasts and is highly overexpressed in human ovarian cancer. In this work, we first correlated KLF8 overexpression with the aggressiveness of ovarian patient tumors and then tested if KLF8 could transform human ovarian epithelial cells. Using the immortalized non-tumorigenic human ovarian surface epithelial cell line T80 and retroviral infection, we generated cell lines that constitutively overexpress KLF8 alone or its combination with the known ovarian oncogenes c-Myc, Stat3c and/or Akt and examined the cell lines for anchorage-independent growth and tumorigenesis. The soft agar clonogenic assay showed that T80/KLF8 cells formed significantly more colonies than the mock cells. Interestingly, the cells expressing both KLF8 and c-Myc formed the largest amounts of colonies, greater than the sum of colonies formed by the cells expressing KLF8 and c-Myc alone. These results suggested that KLF8 might be a weak oncogene that works cooperatively with c-Myc to transform ovarian cells. Surprisingly, overexpression of KLF8 alone was sufficient to induce tumorigenesis in nude mice resulting in short lifespan irrespective of whether the T80/KLF8 cells were injected subcutaneously, intraperitoneally or orthotopically into the ovarian bursa. Histopathological studies confirmed that the T80/KLF8 tumors were characteristic of human serous ovarian carcinomas. Comparative expression profiling and functional studies identified the cell cycle regulators cyclin D1 and USP44 as primary KLF8 targets and effectors for the T80 transformation. Overall, we identified KLF8 overexpression as an important factor in human ovarian carcinoma pathogenesis.

Figure 1

The aberrant overexpression of KLF8 protein is highly correlated with the aggressiveness of human ovarian tumors. A, Representative IHC staining images of patient specimens. Human ovarian tissue array (OV1005, US Biomax) was stained with anti-KLF8 antibody (brown). The nuclei were counterstained with hematoxylin (blue). B. Summary of the staining result from A. Tumor scoring was described in Supplemental Table 1. *p-value was analyzed by Fisher's exact test, comparing KLF8 expression to Normal. Normal, normal ovarian tissue; NAT, cancer adjacent normal ovarian tissue; Benign, mucinous or serous cystadenoma; Borderline, borderline mucinous of serous papillary cystadenoma; Invasive, Invasive ovarian carcinoma, adenocarcinoma or cystadenocarcinoma; Metastatic, metastasized ovarian cystadenocarcinoma or adenocarcinoma.

Figure 2

Overexpression of KLF8 transforms human ovarian epithelial cells. A, Confirmation of T80 cell lines (shown on the top) constitutively overexpressing KLF8 (K), c-Myc (M), Akt (A), Stat3c (S) or their combinations. The cell lines were generated as described in Materials and Methods. Whole cell lysates were used for western blotting for the indicated proteins shown to the left. B & C, KLF8 promotes anchorage-independent growth. The cell lines described in A were grown in soft agar culture and colonies were photographed and counted. D, KLF8 accelerates G1 to S phase progression of the cell cycle. After 48 h serum starvation, the cells were stimulated with serum containing BrdU for 12 h and subject for BrdU incorporation analysis. E, KLF8 increases cell proliferation. The cells were grown and subject to WST-1 assay as described in Material and Methods. The numbers for the lanes and columns in all the panels label the same cell lines as indicated on the top of panel A. *P < 0.05 compared to Mock by X2-test.

Figure 3

KLF8 alone is sufficient to induce T80 cells to form subcutaneous tumors resulting shortened life. A, Representative subcutaneous tumors formed by the indicated cell lines. Mock and SKOV3-ip1 were used as negative and positive controls, respectively. Tumorigenesis experiments were carried out as described in Materials and Methods. Photos were taken on 30 days (for SKOV3-ip1) or 105 days after injection. B, Tumor formation rates Tumor volumes were recorded at the indicated time points (see tumor incidence in Supplemental Table 1). C, T80/KLF8 tumor formation shortened mouse survival time. Survival distribution was presented by Kaplan-Meier curve. D, The T80/KLF8 tumors are highly similar to tumors of ovarian cancer patients. H & E and IHC staining of the tumors were performed as described in Materials and Methods for expression of the KLF8 and human ovarian cancer marker proteins. a, H & E (100X); b, KLF8 (100X); c, pan-cytokeratins (100X); d, CA 125 (100X); e, mesothelin (100X); f, HE4 (100X). Inlets, 400X

Figure 4

KLF8 alone is sufficient to induce to induce T80 cells to form orthotopic ovarian tumors. A, Representative ovarian tumors formed by KLF8 expressing T80 cells. Mock and SKOV3-ip1 were used as negative and positive controls, respectively. 5 x 10⁵ cells per cell lines were injected into the ovarian bursa. Photos of representative mice (top) and tumors (bottom) were taken 60 days or (SKOV3-ip1) 90 days (mock and KLF8) after injection. B, Tumor formation rate. The ovarian tumor volume recorded at the time of euthanasia is presented by box-plot (see tumor incidence in Supplemental Table 1). C. The T80/KLF8 ovarian tumors are highly similar to tumors of ovarian cancer patients. . H & E and IHC staining of the tumors were performed as described in Materials and Methods for expression of the KLF8 and human ovarian cancer marker proteins. a, H & E (100X); b, KLF8 (100X); c, pan-cytokeratins (100X); d, CA 125 (100X); e, mesothelin (100X); f, HE4 (100X). Inlets, 400X.

Figure 5

KLF8 promotes proliferation by up-regulating cyclin D1 and down-regulating USP44 expression in the human ovarian cells. A-D, KLF8 up-regulates cyclin D1 and down-regulates USP44 expression. mRNA and whole cell lysates were prepared from T80 cells stably expressing empty vector (Mock) or KLF8 (KLF8) (lanes or columns 1 & 2) or T80 cells transiently expressing wild type (wtKLF8) or transactivation-defective mutant (mKLF8) for 48 h (lanes or columns 3 & 4), or the SKOV3ip1 cells transiently transfected with control siRNA (siCtrl) or siRNA against KLF8 (siKLF8) for 48 h (lanes or columns 5 & 6) for RT-PCR (A), qRT-PCR (B & C) or western blotting (D). E-H, Knockdown of cyclin D1 or re-expression of USP44 reverses the increased proliferation induced by KLF8 back to the Mock level. T80 cells stably expressing KLF8 (KLF8) were transfected with non-targeting siRNA or siRNA against cyclin D1 for 72 h (lanes or columns 3 & 4, siCtrl & siCycD1) or with tet-on regulated USP44 vector for 24 h followed by induction of USP expression for 48 h (lanes or columns 5 & 6, USP44-U & USP44-I). A fraction of the cells were used for western blotting to confirm the cyclin D1 knockdown (A) and induced expression of USP44 (B). Two other fractions of the cells were used for analysis of cell proliferation (G) and BrdU incorporation (H), respectively. Mock and untreated KLF8 expressing T80 stable cells (lanes or columns 1 & 2, Mock & untreated) were included as controls. U, expression uninduced; I, expression induced. *P < 0.05.