ZBRK1 acts as a metastatic suppressor by directly regulating MMP9 in cervical cancer

Abstract

The BRCA1-interacted transcriptional repressor ZBRK1 has been associated with antiangiogenesis, but direct evidence of a tumor suppressor role has been lacking. In this study, we provide evidence of such a role in cervical carcinoma. ZBRK1 levels in cervical tumor cells were significantly lower than in normal cervical epithelial cells. In HeLa cervical cancer cells, enforced expression inhibited malignant growth, invasion, and metastasis in a variety of in vitro and in vivo assays. Expression of the metalloproteinase MMP9, which is known to be an important driver of invasion and metastasis, was found to be inversely correlated with ZBRK1 in tumor tissues and a target for repression in tumor cells. Our findings suggest that ZBRK1 acts to inhibit metastasis of cervical carcinoma, perhaps by modulating MMP9 expression.

Figure 1. Expression level of ZBRK1 is attenuated in various cervical cancer cell lines and clinical cervical cancer patients

(A) ZBRK1 expression was examined in various cervical cancer cell lines by RT-PCR. GAPDH serves as an internal control. (B) ZBRK1 expression was determined in normal and tumor areas of surgical biopsies from 12 cervical cancer patients by RT-PCR. GAPDH transcript level was used as the load control. “N” and “T” respectively denote “normal” and “tumor” areas of the same patients. The bottom panels show the amount of ZBRK1 mRNA, relative to GAPDH by RT-PCR.

Figure 2. Overexpression of ZBRK1 inhibits cell proliferation and anchorage-independent activity of cancer cells

(A) ZBRK1 attenuated the growth rate of cells. Equal amounts of HeLa cells stably expressing EGFP (G) or EGFP-ZBRK1 (GZ#1 and GZ#6) were seeded, and then the viable cell number was determined at the indicated times. Stably expressed levels of ZBRK1 in HeLa cells were analyzed by Western blot. (B) An increase in ZBRK1 expression attenuated cell proliferation. A focal formation assay was performed with the stable cell lines of HeLa cells. Values are the relative cell number ± S.E.M. (C) Overexpression of ZBRK1 attenuated focal formation of cancer cells. A soft agar assay was performed with HeLa cell lines stably expressing EGFP (G) or EGFP-ZBRK1 (#1 and #6). Values are the relative cell number ± S.E.M. (D) Overexpression of ZBRK1 reduced the formation of tumors in nude mice. Tumor volumes were measured every day after a subcutaneous injection of EGFP or EGFP-ZBRK1 HeLa cells. Data from six mice in each group are presented as the mean ± SD.

Figure 3. ZBRK1 inhibits cell migration

(A) Wound-healing migration was performed with EGFP- and EGFP-ZBRK1-expressing cells. Representative images of wound sealing were taken on the day of the laceration and day 2 after the wound scratch. The level of cell migration into the wound scratch was quantified as the percentage of wound sealing. Values represent the average ±S.E.M. of three independent measurements. (B) Overexpression of ZBRK1 inhibits invasion of cancer cells. The cells were seeded in ECMatrix layer and the level of cell invasion was determined using QCM^™ 96-well cell invasion assay as described in Materials and Methods. (C) Inactivation of ZBRK1 increased migration of cancer cells. Left panel: mRNA and protein levels of ZBRK1 in A431, HeLa, and SiHa cervical cancer cell lines. Right panel: A431 cells were treated with lentiviral shZBRK1 or shGFP in the QCM^™ 96-well cell migration assay. The top panel shows the amounts of ZBRK1 and GAPDH. (D) Equal amounts of EGFP- and EGFP-ZBRK1-#6 HeLa cells were injected into the tail vein of scid mice. The experimental mice were sacrificed to calculate the metastatic nodes on lung tissues after 4 weeks. Data from six mice in each group are presented as the mean ± SD. Representative pictures taken at the time of sacrifice are shown (left panel).

Figure 4. ZBRK1 represses MMP9 promoter activation

(A) Left panel: ZBRK1 inhibited the MMP9 transcripts in xenogenic tumor. Expression levels of multiple genes of tumor sample from mice resulting after subcutaneously injected with EGFP (TS-C) or EGFP-ZBRK1#6 (TS-#6) HeLa cells were analyzed by RT-PCR. The transcripts of human GAPDH served as a control. Right panel: The loss-of-function ZBRK1 enhanced MMP9 transcripts. Stable ZBRK1-expressing cells were incubated with lentiviral shRNA of ZBRK1 or the control. The lysates and total RNA of infected cells were harvested for Western blot and RT-PCR analyses, respectively. (B) ZBRK1 inhibited the MMP9 reporter. EGFP-ZBRK1-expressing HeLa cells transfected with the −1940/+113 and −940/+113 MMP9 reporters contained wild-type or loss-of-function ZBRK1 responsive element, respectively. Lysates of the transfectants were harvested after 12 h of transfection for luciferase assay. (C) ZBRK1 regulates MMP9 transcription through binding to ZBRK1 motifs. The heterologous reporters bearing the wild-type or mutant ZBRK1 motifs of the MMP9 promoters were transfected in EGFP-ZBRK1-expressing HeLa cells. The results shown are averages from three independent transfection assays and are plotted as relative activities to cells transfected by the backbone reporter, pGL2-promoter (pGL2-p). The latter activity was considered to be 100. Data are shown as the mean ± SEM. * p < 0.05, by Student’s t-test. (D) Left panel: ZBRK1 suppresses TNFα- and EGF-induced MMP9 transcription. HeLa cells transfected the region of −1940/+113 of the MMP9 promoter and stimulated with or without TNF-α or EGF. Right panel: An increase in ZBRK1 attenuated EGF-induced enzyme activity of MMP9. The concentrated lysates for gelatin zymography were performed by the supernatants harvested from the cells stably expressing EGF or EGF-ZBRK1 upon EGF treatment or not.

Figure 5. ZBRK1 binds to the MMP9 promoter in vivo

The sheared formaldehyde-cross-linked chromatins, extracted from HeLa cells stably expressing EGFP (G) or EGFP-ZBRK1 (GZ), were immunoprecipitated with the ZBRK1 or GFP antibody. The figure represents PCR products obtained using specific primers on the MMP9 promoter region as shown in the upper panel. Very similar results were obtained from two independent experiments.