HOXB13 promotes ovarian cancer progression

Abstract

Deregulated expression of HOXB13 in a subset of estrogen receptor-positive breast cancer patients treated with tamoxifen monotherapy is associated with an aggressive clinical course and poor outcome. Because the ovary is another hormone-responsive organ, we investigated whether HOXB13 plays a role in ovarian cancer progression. We show that HOXB13 is expressed in multiple human ovarian cancer cell lines and tumors and that knockdown of endogenous HOXB13 by RNA interference in human ovarian cancer cell lines is associated with reduced cell proliferation. Ectopic expression of HOXB13 is capable of transforming p53(-/-) mouse embryonic fibroblasts and promotes cell proliferation and anchorage-independent growth in mouse ovarian cancer cell lines that contain genetic alterations in p53, myc, and ras. In this genetically defined cell line model of ovarian cancer, we demonstrate that HOXB13 collaborates with activated ras to markedly promote tumor growth in vivo and that HOXB13 confers resistance to tamoxifen-mediated apoptosis. Taken together, our results support a pro-proliferative and pro-survival role for HOXB13 in ovarian cancer.

Fig. 1.

HOXB13 is expressed in a subset of human ovarian carcinoma cell lines and primary tumors, and its inhibition is associated with decreased proliferation. (A and B) RT-PCR analysis of HOXB13 expression in (A) SKOV-3, IOSE80, CAOV-3, OVCAR-3, OVCAR-5, OVCAR-8, OV-30, OV-90, ES-2, and TOV112D cell lines and in (B) human ovarian carcinoma tissues. Case 1 is a clear cell carcinoma and cases 2–9 are papillary serous carcinoma subtype. Case 10 is a serous papillary carcinoma of the fallopian tube. Amplification of β-actin cDNA was used as a normalizing control. NTC denotes no template control PCR. (C) Endogenous HOXB13 inhibition in OVCAR-5 and SKOV-3 cells by lentiviral RNAi. shHOXB13-1 and shHOXB13-2 represent two different shRNA constructs targeting HOXB13. GFP-targeting shRNA was used as a control. Cell proliferation was assessed by measuring absorbance of the cell-associated dye at 595 nm. Error bars represent the standard deviation of three independent lentiviral infections and cultures.

Fig. 2.

Ectopic expression of HOXB13 in murine T1 ovarian cancer cells. (A) RT-PCR detection of endogenous and ectopic HOXB13 in T1 cells infected with the control RCAS-GFP retrovirus (T1-GFP) and T1 cells infected with the RCAS-HOXB13 retrovirus (T1-HOXB13). (B and C) Western blotting (B) and immunofluorescence (C) detection of the human HOXB13 protein by using a rabbit polyclonal antibody to HOXB13. (D and E) The morphology of T1-GFP and T1-HOXB13 cells in monolayer culture grown at low (D) and high (E) cell densities. (F) Representative images of duplicate independent assays of cell proliferation in soft agar. (G) Representative images of TUNEL staining in T1-GFP and T1-HOXB13 cells grown in monolayer culture at high cell density. (H and I) Growth curve plot of cells (×10⁴) (H) and cell cycle analysis (I) of T1-GFP and T1-HOXB13 cells; data are representative of duplicate independent experiments.

Fig. 3.

HOXB13 induces oncogenic transformation of p53^−/− MEFs as well as rapid progression of mouse ovarian epithelial tumors in the presence of activated ras. (A) Tumor formation in mice injected s.c. with p53^−/− MEFs that were infected with RCAS-HOXB13. The control p53^−/− MEFs were infected with RCAS-GFP. (B–D) Enhanced tumor growth in mice that were injected s.c. (B) or i.p. (C and D) with T1 mouse ovarian cancer cells that were infected with RCAS-HOXB13 in comparison with the T1 cells that were infected with RCAS-GFP. (E–H) Influence of HOXB13 on tumor growth in mice that were injected s.c. with T22 cells (E), vector-transduced T22 cells (F), and T22 cells with activated K-ras (G) or H-ras (H).

Fig. 4.

Effect of estradiol and tamoxifen on T1-GFP and T1-HOXB13 cells. (A) Western blot analysis of ER-α and β-actin in T1-GFP and T1-HOXB13 cells. (B) Estradiol responsiveness of T1-GFP and T1-HOXB13 cells. (C) ERE-luc responsiveness requires ER expression. T1-GFP and T1-HOXB13 cells were treated with vehicle or 200 nM Falsodex for 24 h before and 48 h after cells were cotransfected with pERE-luc reporter plasmid and pRL normalization plasmid. Results presented are average of triplicate determinations from a representative assay of three independent experiments. Inset demonstrates Western blot analysis of ER-α and GAPDH for the corresponding samples. (D) Effect of tamoxifen on ERE-luc responsiveness. T1-GFP and T1-HOXB13 cells were cotransfected with pERE-luc and pRL and then treated with vehicle or tamoxifen at the doses shown for 48 h. Luciferase assays were performed, and results are representative of three independent experiments. (E) Quantitation of apoptosis in T1-GFP and T1-HOXB13 ovarian cancer cell lines in the indicated doses of tamoxifen. Apoptotic cell death was determined by FACS analysis of phycoerythrin-conjugated annexin V staining. The error bars represent standard deviation in triplicate experiments.