IQGAP3 promotes cancer proliferation and metastasis in high-grade serous ovarian cancer

Abstract

Ovarian cancer is a type of gynecological cancer with the highest mortality rate worldwide. Due to a lack of effective screening methods, most cases are diagnosed at later stages where the survival rates are poor. Thus, it is termed a 'silent killer' and is the most lethal of all the malignancies in women. IQ motif containing GTPase Activating Protein 3 (IQGAP3) is a member of the Rho family of GTPases, and plays a crucial role in the development and progression of several types of cancer. The aim of the present study was to investigate the oncogenic functions and mechanisms of IQGAP3 on the proliferation and metastasis of high-grade serous ovarian cancer (HGSOC). Therefore, the expression levels of IQGAP3 in HGSOC and normal tissue samples were compared, and IQGAP3 knockdown was performed to examine its functional role using various in vitro and in vivo experiments. It was demonstrated that the expression of IQGAP3 was upregulated in HGSOC tissues compared with the healthy tissues; this differential expression was also observed in the ovarian cancer cell lines. Functional experimental results suggested that IQGAP3 silencing significantly reduced proliferation, migration and invasion in ovarian cancer cell lines. Moreover, in vivo experimental findings validated the in vitro results, where the tumorigenic and metastatic capacities of IQGAP3-silenced cells were significantly lower in the nude mice compared with the mice implanted with the control cells. Furthermore, knockdown of IQGAP3 resulted in increased apoptosis, and the effects of IQGAP3 expression on various epithelial-mesenchymal transition markers were identified, suggesting a possible mechanism associated with the role of IQGAP3 in metastasis. The effect of IQGAP3 silencing on chemosensitivity towards olaparib was also assessed. Collectively, the present results indicated that IQGAP3 is a potential diagnostic and prognostic marker, and a putative therapeutic target of HGSOC.

Keywords: IQ motif containing GTPase activating protein 3; cell division cycle 42; metastasis; ovarian cancer; proliferation.

Figure 1.

IQGAP3 expression is upregulated in HGSOC and its increased expression is associated with a poorer prognosis. (A) Survival analysis based on immunohistochemical analysis identified significantly improved overall survival (log rank P=0.0149) and progression-free survival (log rank P=0.0044) in patients with lower expression levels of IQGAP3. (B) Kaplan-Meier survival plots based on data obtained from the Gene Expression Omnibus database had improved overall and progression-free survival in cases with lower expression levels of IQGAP3. (C) Western blotting results demonstrated higher protein expression levels of IQGAP3 in HGSOC tissues (T1-T5) compared with healthy FTE (F1-F3). Expression of IQGAP3 between the two groups was significantly different. (D) Reverse transcription-quantitative-PCR analysis indicated higher mRNA expression levels of IQGAP3 in HGSOC tissues (n=26) compared with FTE (n=24). (E) Immunohistochemical staining of IQGAP3 in HGSOC and FTE samples. Magnification, ×40. *P<0.05; **P<0.01; ***P<0.001. IQGAP3, IQ motif containing GTPase Activating Protein 3; HGSOC, high-grade serous ovarian cancer; FTE, fallopian tube epithelium.

Figure 2.

IQGAP3 promotes the proliferation of HGSOC in vitro and in vivo. (A) MTT assay identified significantly reduced proliferation in A2780 and HEY cells following knockdown of IQGAP3 using si-IQGAP3−1 and si-IQGAP3−2 compared with the corresponding control. (B) Silencing IQGAP3 reduced colony formation in A2780 and HEY cells compared with the respective controls. (C) Knockdown of IQGAP3 with sh-IQGAP3 in HEY cell line. (D) Knockdown of IQGAP3 resulted in reduced tumor forming capacity in nude xenograft mice compared with the corresponding controls. Control cells were injected in the right armpit and sh-IQGAP3-transfected cells were injected in the left armpit. The weight and the volume of the xenograft tumors were significantly different between the control group and the sh-IQGAP3-transfected group. (E) Bioluminescence imaging identified the effects of IQGAP3 silencing on tumor formation, as well as metastasis in vivo. Control cells were injected in the left armpit and IQGAP3 knockdown cells were injected in the right. Metastatic foci were more visible in the nude mice injected intraperitoneally with control (left) compared with sh-IQGAP3-transfected cells (right). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 vs. NC. IQGAP3, IQ motif containing GTPase Activating Protein 3; siRNA, small interfering; shRNA, short hairpin RNA; NC, negative control; shRNA, short hairpin RNA.

Figure 3.

IQGAP3 potentiates the migratory and invasive capacities of the ovarian cancer cells in vitro and in vivo. Knockdown of IQGAP3 using two siRNAs significantly decreased the (A) migration and (B) invasion of A2780 and HEY cells. Quantitative analysis of migration and invasion. Magnification, ×10. (C) In vivo experiments demonstrated significantly lower numbers of metastatic foci (arrows) in the nude mice injected with sh-IQGAP3-transfected cells compared with the control. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 vs. NC. IQGAP3, IQ motif containing GTPase Activating Protein 3; shRNA, short hairpin RNA; NC, negative control; si, small interfering RNA.

Figure 4.

Western blot analysis revealed the changes in protein expression of several proteins following IQGAP3 knockdown using two siRNAs. Both A2780 and HEY cells had an altered protein expression following the knockdown. The alteration in the protein expression included EMT-related proteins (E-CAD, N-CAD, ZEB-1, Vimentin and Snail), apoptosis-related proteins (Caspase-3, Caspase-9, Bcl2 and Bax), proteins associated with DNA damage and chemoresistance (Rad51, p-ATM, ATM, CHK2 and Pgp), and proteins involved in the regulation and mechanism of the effect of IQGAP3 (CDC42, PI3K, p-AKT, AKT, p-mTOR and mTOR). β-actin was used as the internal control. IQGAP3, IQ motif containing GTPase Activating Protein 3; EMT, epithelial-to-mesenchymal transition; p-, phosphorylated; E-CAD, E-cadherin; N-CAD, N-cadherin; Pgp, phosphoglycolate phosphatase; Rad51, RAD51 recombinase; CHK2, checkpoint kinase 2; NC, negative control; si, small interfering RNA; ZEB-1, zinc finger E-Box binding homeobox 1.

Figure 5.

IQGAP3 increases apoptotic potential in cancer cell lines. Flow cytometry analysis indicated increased early, as well as late apoptosis in (A) A2780 and (B) HEY cells following knockdown of IQGAP3. ***P<0.001 vs. NC. IQGAP3, IQ motif containing GTPase Activating Protein 3; NC, negative control; si, small interfering RNA; PI, propidium iodide.

Figure 6.

IQGAP3 silencing increases the sensitivity of ovarian cancer cells to olaparib and the regulatory role of CDC42 on IQGAP3. (A) Cell viability assay demonstrated increased sensitivity of olaparib in A2780 and HEY cells following IQGAP3 knockdown using two siRNAs. (B) Knockdown of CDC42 by transient transfection. (C) CDC42 silencing reduces HEY cell proliferation in a time-dependent manner. (D) Silencing of CDC42 significantly reduces the migration and invasion in HEY cells. Magnification, ×10. (E) Downregulation of CDC42 increases apoptosis in HEY cells. **P<0.01; ***P<0.001; ****P<0.0001 vs. NC. IQGAP3, IQ motif containing GTPase Activating Protein 3; siRNA, small interfering RNA.