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, 7 (5), e37076

Increased Expression of PITX2 Transcription Factor Contributes to Ovarian Cancer Progression

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Increased Expression of PITX2 Transcription Factor Contributes to Ovarian Cancer Progression

Frederic K C Fung et al. PLoS One.

Abstract

Background: Paired-like homeodomain 2 (PITX2) is a bicoid homeodomain transcription factor which plays an essential role in maintaining embryonic left-right asymmetry during vertebrate embryogenesis. However, emerging evidence suggests that the aberrant upregulation of PITX2 may be associated with tumor progression, yet the functional role that PITX2 plays in tumorigenesis remains unknown.

Principal findings: Using real-time quantitative RT-PCR (Q-PCR), Western blot and immunohistochemical (IHC) analyses, we demonstrated that PITX2 was frequently overexpressed in ovarian cancer samples and cell lines. Clinicopathological correlation showed that the upregulated PITX2 was significantly associated with high-grade (P = 0.023) and clear cell subtype (P = 0.011) using Q-PCR and high-grade (P<0.001) ovarian cancer by IHC analysis. Functionally, enforced expression of PITX2 could promote ovarian cancer cell proliferation, anchorage-independent growth ability, migration/invasion and tumor growth in xenograft model mice. Moreover, enforced expression of PITX2 elevated the cell cycle regulatory proteins such as Cyclin-D1 and C-myc. Conversely, RNAi mediated knockdown of PITX2 in PITX2-high expressing ovarian cancer cells had the opposite effect.

Conclusion: Our findings suggest that the increased expression PITX2 is involved in ovarian cancer progression through promoting cell growth and cell migration/invasion. Thus, targeting PITX2 may serve as a potential therapeutic modality in the management of high-grade ovarian tumor.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PITX2 is upregulated in ovarian cancer samples and cell lines.
(A) Quantitative RT-PCR analysis was performed in normal ovaries (n = 54) and ovarian cancer samples (n = 97) using PITX2 specific primer. 18S and TATA-box binding protein (TBP) were used as the internal loading controls. *P<0.001. (B) Western blot analysis using anti-PITX2 antibody to evaluate the expression level of PITX2 (isoforms A, B and C)) (35 kDa) in ovarian cancer cell lines (n = 9) and HOSE cell lines (n = 2). β-actin was used as a loading control. (C) Immunohistochemical analysis of PITX2 expression (nuclear staining) in borderline cystadenoma and high-grade (3) serous cystadenocarcinoma on an ovarian cancer tissue array (OVC1021). Magnification: 20×.
Figure 2
Figure 2. PITX2 promotes ovarian cancer cell growth.
(A) PITX2A stable expressing clones were established in SKOV3 and OVCA433 cells. Western blot analysis using anti-HA antibody showed the expression levels of HA-tagged PITX2A in C4 and C5 clones of SKOV-3, and C33 and C34 clones of OVCA 433. β-actin was used as loading control. (B) Western blot analysis using anti-PITX2 antibody showed the reduced expressions of endogenous PITX2 in stable knockdown clones generated by 2 shRNA constructs (K1 and K2). Scrambled is the non-specific shRNA control. The numerical units represent the relative expressions of PITX2 reduction in each stable clone as compared with the scrambled controls. β-actin was used as loading control. (C) Ectopic expression of PITX2A stimulated cell proliferation in ovarian cancer cells. Both C4 and C5 of SKOV-3 cells demonstrated 1.5- fold increase of cell growth (* P<0.05), C33 and C34 of OVCA 433 cells had 3- fold increase of cell growth (** P<0.01) as compared with their vector control. (D) Depletion of endogenous PITX2 reduced cell proliferation in ovarian cancer cells. A 3- to 4- fold of reduction on cell viability in both knockdown clones (K1 and K2) of OV2008 cells (** P<0.01) and 2- to 3- fold decrease on cell proliferation in knockdown clones (K1 and K2) of OVCA 433 cells (* P<0.05) were observed.
Figure 3
Figure 3. PITX2 enhances anchorage-independent growth ability of ovarian cancer cells.
(A)Enforced expression of PITX2A enhanced anchorage independent growth ability of ovarian cancer cells. The bar chart shows that PITX2A stably expressing clones (C33 and C34) of OVCA 433 (**P = 0.05) and (C4 and C5) of SKOV-3 (*P = 0.01) had increased number of colonies in soft agar compared with their vector controls. Representative pictures show larger colony size in PITX2A stable expressing clones under microscopy. (B)Depletion of PITX2 by shRNA inhibited anchorage independent growth ability of ovarian cancer cells. The number of colonies in PITX2stable knockdown clones (K1 and K2) of OVCA 433 and OV2008 showed significantly reduction as compared with the scrambled controls (* P<0.01). Representative pictures shows that the reduced colony size of PITX2stableknockdown clones and their scrambled controls. The above experiment was repeated at least three times independently and the data was calculated with mean ± SD.
Figure 4
Figure 4. PITX2 promotes ovarian cancer cell migration and invasion.
(A) Wound healing assay showed that PITX2 stably expressing cells (C4 of SKOV-3) exhibited faster wound closure rate than the vector control in the time course of 8 hr (**P<0.05). (B) PITX2 stable knockdown clones (K1 and K2 of OVCA 433) showed significant reduction on wound closure rate compared with vector control in the time course of 12 hrs (*P<0.05). The arrows indicate the width of wound and the relative cell migration rate is expressed as relative width of the wounds/time. The assay was repeated three times independently. (C) Transwell migration and invasion assay demonstrated that PITX2 stably expressing clones in SKOV3 (C4 and C5) cells migrate faster through the membrane (*P<0.01) and invade faster through the matrigel (C4, *P = 0.04 and C5, **P = 0.01) compared with the vector control. (D) PITX2 stable knockdown clone (K2 of OVCA 433) exhibited remarkable reduction in cell penetration through the membranes and cell invasiveness compared with the scrambled controls (**P = 0.01). Three views were randomly picked in each inserts and the numbers of invaded cell were counted. The results of three independent experiments were plotted with error bar.
Figure 5
Figure 5. PITX2 elevates the expressions of Cyclin-D1 and C-myc in ovarian cancer cells.
(A) Q-PCR analysis revealed that the levels of Cyclin-D1 and C-myc were elevated in PITX2A stably expressing clones (C4, C5 of SKOV-3 and C33, C34 of OVCA 433). (B)Western blot analysis showed that the protein levels of Cyclin-D1 and C-myc were increased by PITX2 in PITX2A stably expressing clones (C4, C5 of SKOV-3 and C33, C34 of OVCA 433). (C) Knockdown of PITX2 significantly reduced the levels of Cyclin-D1 and C-myc in PITX2 knockdown stable clones (C6, C8 of OVCA 433 and C1, C4 of OV2008). The levels of HA-tagged PITX2A were detected using anti-HA antibody, while endogenous PITX2 expression was examined by anti-PITX2 antibody, β-actin was used as loading control. The numerical value under each panel represents the relative expression of Cyclin-D1 and C-myc to their vector controls.
Figure 6
Figure 6. PITX2 promotes the tumor growth in nude mice.
(A) PITX2 stably expressing clones in SKOV3 (C4 and C5) showed faster in the tumor growth in nude mice as compared with the vector control. The tumor size was represented by the mean ± SE of five mice and was measured for 36 days.*, P<0.01, significantly different from vector control group. (B) Photograph illustrates the dissected tumors taken from nude mice subcutaneously injected by two PITX2 stable expressing clones in SKOV3 (C4 and C5) and the vector control on Day 36. (C) Western blot analysis showed the expressions of PITX2, Cyclin-D1 and C-myc from the mice subcutaneous tumor tissues. The numerical value under each panel represents the relative expression of Cyclin-D1 and C-myc to their vector controls.

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