Reduced IQGAP2 expression promotes EMT and inhibits apoptosis by modulating the MEK-ERK and p38 signaling in breast cancer irrespective of ER status

Abstract

IQGAP2, a member of the IQGAP family, functions as a tumor suppressor in most of the cancers. Unlike IQGAP1 and IQGAP3, which function as oncogenes in breast cancer, the role of IQGAP2 is still unexplored. Here we report a reduced expression of IQGAP2, which was associated with lymph node positivity, lymphovascular invasion, and higher age in breast cancer patients. We found an inverse correlation of IQGAP2 expression levels with oncogenic properties of breast cancer cell lines in estrogen receptor (ER) independent manner. IQGAP2 expression enhanced apoptosis via reactive oxygen species (ROS)-P38-p53 pathway and reduced epithelial-mesenchymal transition (EMT) in a MEK-ERK-dependent manner. IQGAP2-IQGAP1 ratio correlated negatively with phospho-ERK levels in breast cancer patients. Pull-down assay showed interaction of IQGAP1 and IQGAP2. IQGAP2 overexpression rescued, IQGAP1-mediated ERK activation, suggesting the possibility of IQGAP1 sequestration by IQGAP2. IQGAP2 depletion, in a tumor xenograft model, increased tumor volume, tumor weight, and phospho-ERK expression. Overall, our findings suggest that IQGAP2 is negatively associated with proliferative and metastatic abilities of breast cancer cells. Suppression of IQGAP1-mediated ERK activation is a possible route via which IQGAP2 restricts oncogenic properties of breast cancer cells. Our study highlights the candidature of IQGAP2 as a potent target for therapeutic intervention.

Fig. 1. Reduced expression of IQGAP2 in breast cancer tissues.

A The comparison of Allred scores of IQGAP2 expression between tumor (n = 226) and adjacent normal tissue (n = 63) of breast cancer patients. The Allred score for IQGAP2 is significantly (Mann–Whitney U test) reduced in tumor tissues (median = 3) compared to normal (median = 8). B Representative images and distribution frequency of normal versus cancer tissues according to the Allred score of IQGAP2. For statistical analyses, the Allred scores of 0–2 were treated as negative or weak, 3–6 as moderate, and 7–8 as strong expression levels. y-axis represents the percentage of patients positive with IQGAP2 low, intermediate, or high Allred score. x-axis represents two groups; normal and tumor. C The representative images of tumor tissue and adjacent normal tissue of two breast cancer patients, showing IQGAP2 expression and localization. C-a Indicates the glandular cells and C-b shows the stromal region. The images were captured using a ×10 objective lens of bright field microscope. Scale bar in all images is 50 microns, AS Allred score, ****p ≤ 0.0001.

Fig. 2. Reduced IQGAP2 expression promotes cell proliferation in breast cancer cell lines independent of ER status.

A The upper panels show representative Western blot images of IQGAP2 expression in stable MCF7_Control_EV, MCF7_IQGAP2_Ex, MCF7_Control_Sc, and MCF7_IQGAP2_KD, respectively. Lower panels showing the relative densitometry bar graph of respective images. B, C The graph shows cell proliferation (MTS assay) of MCF7 IQGAP2_KD and MCF7 IQGAP2_Ex, respectively, at different time intervals. B Knockdown of IQGAP2 shows significant increase in cell proliferation. C Overexpression of IQGAP2 shows significant decrease in cell proliferation. D Left panel shows the images of colony formation assay in MCF7 IQGAP2_KD. Representative bar graph in the right panel showing a significantly higher number of colonies in the IQGAP2 knockdown group than in the control group. E Image showing the number of colonies in MCF7 IQGAP2_Ex group and MCF7 Control_EV group (left panel). The right panel bar graph showing the differences of colony numbers between both the groups. Overexpression of IQGAP2 shows significantly low colony number compared to control. F Western blot image showing (upper panel) knockdown of IQGAP2 in MDA-MB-468 cell lines and its relative densitometry bar graph (lower panels). G Bar graph shows the comparison between cell number (MTS assay) in MDA-MB-468 IQGAP2_KD group and Control_Sc group, from 24 to 96 h of cell plating. Knockdown of IQGAP2 shows higher cell proliferation rate than control. H Representative images of colony formation assay (left panel) in MDA-MB-468 IQGAP2_KD group and Control_Sc group. In the right panel, the bar graph shows the difference of colony numbers between both the groups. Knockdown of IQGAP2 shows significantly more number of colonies. Experiments were performed in triplicate and data were presented as mean ± SEM. Student’s t test, two-tailed unpaired was used for the comparison of means. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 3. IQGAP2 expression levels show dual effect on EMT and apoptosis in opposite manner.

A Wound healing assay in MCF7 IQGAP2_KD and MCF7 Control_Sc group showing higher recovery rate of wound in IQGAP2 knockdown group, compared to control. B Transwell migration assay in MCF7 IQGAP2_KD and MCF7 Control_Sc groups. Cells with IQGAP2 knockdown show enhanced transwell migration, compared to control. C Wound healing assay of MDA-MB-468 IQGAP2_KD and Control_Sc cells showing higher wound recovery rate of IQGAP2 knockdown group, compared to empty vector control. D Transwell migration assay in MDA-MB-468 IQGAP2_KD and Control_Sc groups shows enhanced transwell migration with IQGAP2 knockdown, compared to control. E Wound healing assay of MCF7 IQGAP2_Ex and MCF7 Control_EV cells showing reduced wound recovery with IQGAP2 overexpression, compared to control. F MCF7 cells with ectopic IQGAP2 expression (IQGAP2_Ex) show reduced transwell migration, compared to control (Control_EV). G MCF7 cells with IQGAP2 knockdown (IQGAP2_KD) show increased transwell invasion, compared to control (Control_Sc). H MDA-MB-468 cells with IQGAP2 knockdown (IQGAP2_KD) demonstrate enhanced transwell invasion, compared to control (Control_Sc). I MCF7 cells with ectopic IQGAP2 expression (IQGAP2_Ex) showing reduced transwell invasion, compared to control (Control_EV). J Cell apoptosis was measured by flow cytometry using 488 nm excitation and 647 nm emission filters in the MDA-MB-468 IQGAP2_KD and Control_Sc groups. The x-axis represents the cells positive for Annexin V stain and y-axis shows positive cells for propidium-iodide (PI) stain. Experiments were performed in triplicate and data are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Student’s t test, two-tailed unpaired was used for the comparison of means. Scale bar in all images is 50 microns.

Fig. 4. IQGAP2 modulates apoptosis by affecting the p38-p53 pathway triggered by increase in ROS.

A IQGAP2 knockdown (IQGAP2_KD) in MCF7 shows reduction of ROS production, compared to control (Control_Sc). B IQGAP2 knockdown in MDA-MB-468 (IQGAP2_KD) shows reduction of ROS production, compared to control (Control_Sc). C IQGAP2 overexpression (IQGAP2_Ex) in MCF7 shows an increase of ROS production than control (Control_EV). D Representative images of Western blots of phospho-p38 and phospho-p53 in MCF7 with IQGAP2 depletion (IQGAP2_KD) and control (Control_Sc) groups (left panel). Graph showing reduced phospho-p53 and phospho-p38 level in IQGAP2 knockdown. E Representative images of Western blots of phospho-p38 and phospho-p53 in MDA-MB-468 with IQGAP2 depletion (IQGAP2_KD) and control (Control_Sc) group (left panel). Graph showing reduced phospho-p53 and phospho-p38 level in IQGAP2 knockdown (right panel). F Representative images of Western blots of phospho-p38 and phospho-p53 in MCF7 cells with IQGAP2 overexpression (IQGAP2_Ex) and control (Control_EV) group (left panel). Graph showing elevated phospho-p53 and phospho-p38 level in IQGAP2 overexpression group (right panel). G Graph showing caspase 3/7 levels in MCF7 with IQGAP2 depletion (IQGAP2_KD) and control (Control_Sc) groups. H Graph showing caspase 3/7 levels in IQGAP2 knockdown (IQGAP2_KD) in MDA-MB-468 and control (Control_Sc) groups. I Western blot images showing the expression level of phospho-p38 and phospho-p53 upon treatment with p38 inhibitor (SB202190, 1 µM) or vehicle control (DMSO) in MCF7_IQGAP2_Ex (IQGAP2 overexpression) group. J Western blot images showing the expression level of phospho-p38 and phospho-p53 upon treatment with p38 activator (anisomycin, 0.5 ng/ml) or vehicle control (DMSO) in MCF7_IQGAP2_KD (IQGAP2 depletion) group. Experiments were performed in triplicate and data are presented as mean ± SEM. ^#p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Student’s t test, two-tailed unpaired was used for the comparison of means. Scale bar in all images is 100 microns.

Fig. 5. IQGAP2 affects EMT via activation of ERK pathway.

Western blot showing elevated expression of N-cadherin, Snail, and Twist, and reduced level of E-cadherin in A, MCF7 with IQGAP2 depletion (IQGAP2_KD) compared to respective control (Control_Sc), and, in B, MDA-MB-468 with IQGAP2 depletion (IQGAP2_KD) compared to respective control (Control_Sc). C Western blot showing reduced expression of N-cadherin, Snail, and Twist and elevated level of E-cadherin in MCF7 with IQGAP2 overexpression (IQGAP2_Ex) compared to respective control (Control_EV). Western blot showing elevated expression of phospho-MEK and phospho-ERK in D, MCF7 with IQGAP2 depletion (IQGAP2_KD) compared to respective control (Control_Sc) and in E, MDA-MB-468 with IQGAP2 depletion (IQGAP2_KD) compared to respective control (Control_Sc). F Western blot showing reduced expression of phospho-MEK and phospho-ERK in MCF7 with IQGAP2 overexpression (IQGAP2_Ex) compared to respective control (Control_EV). G Western blot showing rescue of phospho-ERK in MCF7 with IQGAP2 depletion (IQGAP2_KD) upon IQGAP2 expression (IQGAP2_KD + IQGAP2_Ex). H Western blot showing rescue of Twist (phospho-ERK downstream target) after treating MCF7 with IQGAP2 depletion (IQGAP2_KD) with phospho-ERK inhibitor II. I Cell invasion assay after treating MCF7 with IQGAP2 depletion (IQGAP2_KD), using phospho-ERK inhibitor II (left panel). Right panel, bar graph showing no significant difference in no. of cells invaded in control (Control_Sc), and IQGAP2 depletion group (IQGAP2_KD) treated with phospho-ERK inhibitor II. Experiments were performed in triplicate and data are presented as mean ± SEM. ^#p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Student’s t test, two-tailed unpaired was used for the comparison of means.

Fig. 6. Reduced expression of IQGAP2 promotes tumor growth in mouse model.

Two groups of nude mice were injected with MCF7 cells having IQGAP2 knockdown (IQGAP2_KD) or its control vector (Control_Sc). A Shows control (Control_Sc) and IQGAP2 knockdown (IQGAP2_KD) groups of nude mice and their tumors (n = 4). B Tumor volume of IQGAP2 knockdown (IQGAP2_KD) group and control (Control_Sc) group at the interval of 6 days (n = 4). Tumor volume was calculated using equation: volume = length × width². C Tumor weight of IQGAP2 knockdown (IQGAP2_KD) and control (Control_Sc) mice groups at day 30 (n = 4). D Representative images of immunohistochemical staining for phospho-ERK in sections of control (C1, C2, C3, and C4 derived from MCF7_Control_Sc) and IQGAP2 knockdown (KD1, KD2, KD3, and KD4 derived from MCF7_IQGAP2_KD) nude mice xenograft-derived tumors (upper panel). Images were taken at ×40 with an upright bright field microscope. Graphs (lower panel) showing percentage of cells positive for low, moderate, or high phospho-ERK expression in xenografts derived from MCF7 with IQGAP2 knockdown (IQGAP2_KD) and its control (Control_Sc) from nude mice. The expression intensity was calculated using IHC profiler tool of ImageJ. Y-axis represents the percentage cell positivity for low, weak, or high phospho-ERK expression. X-axis shows mice in each group. Data are presented as mean ± SEM. Student’s t test, two-tailed unpaired was used for the comparison of means. *p ≤ 0.05, **p ≤ 0.01. The scale bar is 20 micron.

Fig. 7. IQGAP2 is negatively correlated with phospho-ERK and interaction of IQGAP2 with IQGAP1 modulates IQGAP1 function.

A Pearson correlation between the expression of IQGAP2 and IQGAP1 in tissues of breast cancer (r = −0.56, n = 38). B Pearson correlation between the expression of IQGAP2 and phospho-ERK (r = −0.63, n = 10) in tissues of breast cancer. C Pearson correlation between the expression of IQGAP1 and phospho-ERK (r = 0.49, n = 10) in tissues of breast cancer. D Pearson correlation between the expression of the ratio of IQGAP2/IQGAP1 and phospho-ERK (r = −0.64, n = 10) in tissues of breast cancer. In A–D solid and dashed line show regression line and standard error, respectively. E Representative IHC images of IQGAP2, phospho-ERK, and IQGAP1 in the normal and tumor region of a breast cancer patient. F Western blot images showing status of IQGAP2 in pull-down assay performed with IQGAP1_GST and GST only in MCF7 cells. The blots were probed with anti-IQGAP1, anti-IQGAP2, and GST antibodies. G Western blot images showing status of IQGAP2 in pull-down assay performed with IQGAP1_GST and GST only in Hela cells. The blots were probed with anti-IQGAP1, anti-IQGAP2, and GST antibodies. H Left panel shows Western blot of phospho-ERK in MCF7 cell having IQGAP2 overexpression (IQGAP2_Ex), IQGAP2 empty vector (Control_EV), IQGAP2 overexpression with IQGAP1 empty vector (IQGAP2_Ex + G1_EV), overexpression of IQGAP2 on IQGAP1 overexpression (IQGAP2_Ex + IQGAP1_Ex) background, and IQGAP1 overexpression (IQGAP1_Ex). Right panel shows corresponding densitometry data (n = 3). I Western blot of phospho-ERK in MCF7 cell having depletion of IQGAP1 and IQGAP2 (IQGAP2_KD + IQGAP1_KD), depletion of IQGAP2 with control vector for IQGAP1_KD (IQGAP2_KD + Control_Sc), IQGAP2 depletion (IQGAP2_KD), and its control vector (Control_Sc). J Western blot (left) showing expression of IQGAP1 in MCF7 with IQGAP2 reduction (IQGAP2_KD) and its control (Control_Sc). The bar graph (right panel) shows corresponding densitometry data (n = 3). K Western blot (left) image for IQGAP1 expression in MCF7 with IQGAP2 overexpression (IQGAP2_Ex) and its control (Control_EV). The bar graph (right panel) shows corresponding densitometry data (n = 3). Data are presented as mean ± SEM. Student’s t test, two-tailed unpaired was used for the comparison of means. ^#p > 0.05, *p ≤ 0.05.