IQGAP3 in clear cell renal cell carcinoma contributes to drug resistance and genome stability

Human myocardial tissue collection

Seven pairs of tumor and adjacent normal tissues were collected from the department of urology, Henan Provincial People’s Hospital. The study was approved by the medical ethics committee of Henan Provincial People’s Hospital (No. 2019074) following the Declaration of Helsinki. The experiments were undertaken with the understanding and written consent of each subject. The participants allowed the researchers to use their tissue during the tumor resection and conduct the study accordingly. The patient information was listed in Table 1.

Cell culture and RNAi transfection

Human clear cell adenocarcinoma cell lines 786-O and ACHN were seeded at 37 °C and 5% CO2 in RPMI-1640 and DMEM medium, respectively. All mediums were supplemented with 10% FBS and 1% Penicillin/Streptomycin. The cell lines were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology and were went through mycoplasma testing every month. The Lipofectamine RNAiMAX reagent (Invitrogen) was used to transfect siRNAs (50 nM) for 72 to 96 h. The siRNA sequences targeting IQGAP3 were as follows:

siIQGAP3-1#: 5′-CGUCCGAACUGGCCAAAUA-3′;

siIQGAP3-2#: 5′-GGGUGUGGCUGUCAUGAAA-3′.

Antibodies

The human IQGAP3 antibody was obtained from Proteintech (25930-1-AP, 1:1000). Human GAPDH antibody was obtained from Proteintech (10494-1-AP, 1:1000). Human Integrin Alpha 6 antibody was obtained from Proteintech (27189-1-AP, 1:1000). Human Twist antibody was obtained from Proteintech (11752-1-AP, 1:1000). Human Slug antibody was obtained from Proteintech (12129-1-AP, 1:1000). Human Vimentin antibody was obtained from Proteintech (10366-1-AP, 1:1000). Human Phospho-H2AX-S139 antibody was obtained from Abclonal (AP0687, 1:1000).

Western blotting

The collected cells were centrifuged and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris–HCl (pH 7.4), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) with 1% PMSF for 30 min. The supernatant was separated by 13,000 g centrifugation at 4 °C for 20 min. The protein samples were denatured at 100 °C for 10 min and loaded in SDS-PAGE gel. After being transferred to a PVDF membrane, the blocking was performed with 5% skimmed milk for at room temperature 1 h. The membrane was incubated overnight at 4 °C with the primary antibodies and incubated at room temperature with the secondary antibody for 1 h. Signal detection was performed by enhanced chemiluminescence (PerkinElmer).

Cell proliferation and survival assay

Cell proliferation was detected by MTT assay. Briefly, 2 × 103 cells were seeded in 96 well plate for 1 to 6 days. MTT reagent (Sigma-Aldrich) was added at the concentration of 5 mg/ml, followed by 4 h incubation at 37 °C. The culture supernatant was discarded and 100 µl DMSO (Sigma-Aldrich) was added. After 10 min of oscillation, 490 nm wavelength was selected on the enzyme-linked immunosorbent monitor to measure the light absorption value.

For the ionizing radiation sensitivity test, 200 to 5000 cells were seeded in six well plate, followed by several doses of X-ray irradiation. The number of clones was counted after 14 days of cell culture. For drug sensitivity test, 2000 cells were seeded in 96 well plate with several dosed of cisplatin, camptothecin and doxorubicin (Selleck). The surviving cells were measured by MTT method after 48 h of culture.

3D spheroid cell growth assay

For the 3D spheroid cell growth assay, 1 ×10³ cells were seeded in 24 well plates with ultra-low protein adsorption. The cell images were taken and recorded by light microscope after 6 days, 12 days and 14 days.

Immunohistochemistry (IHC)

IHC staining was performed on renal cell carcinoma tissue. Tissue sections were dewaxed in xylene and rehydrated in graded ethanol (100%, 95%, 80% and 70% ethanol for 10 min). The antigen was recovered by heat induced epitope recovery method. The slices were treated by 10 mmol/l EDTA (pH 8.0) at 98 °C for 15 min. 3% hydrogen peroxide were used in methanol at 37 °C for 15 min to quench the endogenous peroxidase activity, followed by blocking with 5% bovine serum albumin. The incubation condition of primary antibody was 4 °C overnight.

Immunofluorescence

The growing cells were inoculated on the sterilized coverslips and cultured at 37 °C and 5% CO2 for 24 h. The medium was removed when the cell grew to about 70% and washed with PBS. The cells were fixed at room temperature with 4% paraformaldehyde for 15 min. After washing with PBS for three times, Triton was added at the final concentration of 0.3% to treat the cell at room temperature for 20 min. After blocking in 5% BSA for 30 min, the cells were stained with primary antibodies (diluted in 1% BSA) at room temperature for 2 h. Cells were washed with PBST (PBS with 0.1% Tween-20) three times and incubated with fluorescence-conjugated secondary antibodies at room temperature for 1 h. After being washed three times with PBST, cells were mounted with antifade mounting medium with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI). The slides were observed by confocal microscope, and the fluorescence intensity were calculated by ImageJ software.

TCGA database analysis

The sequencing results (HTSeq FPKM data) were obtained and analyzed from TCGA database (https://portal.gdc.cancer.gov/). IQGAP3 expression between normal and tumor tissues was analyzed by UALCAN database (http://ualcan.path.uab.edu). Survival analysis between patients with low and high IQGAP3 expression was performed by Kaplan Meier database (https://kmplot.com/analysis/). Timer database (http://timer.cistrome.org/) was used to explore the relationship between IQGAP3 expression and immune infiltration. Student t-test and log rank test were used for data statistics.

Transcriptome sequencing and analysis

Total RNA isolation by Trizol, mRNA enrichment with oligo DT magnetic beads, mRNA fragmentation and cDNA synthesis were all processed according to the manufacturer’s protocol. The cDNA was complemented and repaired. After amplification, the RNA library was sequenced by Illumina pe150 in Shenzhen Haplox company. The reference genome used was GRCh37 (hg19). The FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) of each gene was calculated according to gene length. Differential expression analysis was performed using the DESeq R package (Yu et al., 2012).

Statistical analysis

Each experiment was validated by three independent replicates. Unpaired two tailed Student t-test was used to analyze the statistical significance. The experimental values are expressed as the mean ± standard deviation (SD). Statistical significance was analyzed by GraphPad Prism 6.0 software (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

IQGAP3 was highly expressed in most cancer types compared with normal tissues

The expression analysis of TCGA database showed that there was no significant difference in the expression of IQGAP1 in the cancer and adjacent tissues of the three renal cancer subtypes (data were not shown). IQGAP2 was downregulated in tumor tissues of kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP), and upregulated in tumor tissues of kidney chromophobe (KICH) (data were not shown). The mRNA sequencing data of IQGAP3 from 730 adjacent normal tissues and 10,363 tumor tissues in TCGA pan cancer database were extracted. Comparing unpaired samples and paired samples, IQGAP3 was highly expressed in most tumor types (Figs. 1A, 1B). All the three subtypes of renal cell carcinoma showed significantly enhanced IQGAP3 expression in tumor tissues than that in normal tissues (Fig. 1C). Due to the small number of samples contained in KICH, the followed bioinformatics analysis mainly focused on the common subtypes KIRC and KIRP. Gene expression profiles were obtained by high-throughput gene array analysis in GEO database (https://www.ncbi.nlm.nih.gov/geoprofiles/). The expression of IQGAP3 in 27 pair (Series: GSE66272) and 72 pair (Series: GSE53757) of ccRCC tumor tissues and matched normal tissues at different disease stages were extracted and analyzed. IQGAP3 increased significantly in tumor tissues at different stages (Figs. 1D, 1E). Interestingly, IQGAP3 was highly expressed in both cancer and adjacent paired samples with (13 pairs of samples, Series: GSE66271, Fig. 1F) or without metastasis (14 pairs of samples, Series: GSE66270, Fig. 1G). In patients with metastatic ccRCC, the expression is higher in tumor tissue than normal tissue. Immunohistochemistry also showed that the expression of IQGAP3 was high in tumor tissues, which is consistent with the analysis of the database (Fig. 1H).

Correlation between IQGAP3 expression and clinical features

According to clinical features in TCGA database, the higher the TNM (tumor-node-metastasis) stage, histological grade and pathological stage, the higher IQGAP3 expression in KIRC and KIRP (Figs. 2A, 2B). 265 ccRCC tumor samples from GEO database were compared (Series: GSE73731). The expression of IQGAP3 was higher in high-grade samples (Fig. 2C). The diagnostic value of IQGAP3 mRNA level was evaluated by ROC (receiver operating characteristic) curve and the area under the ROC curve (AUC). The AUC value of IQGAP3 were 0.934 and 0.939 in KIRC (Fig. 2D) and KIRP (Fig. 2E) respectively, which showed high diagnostic value. The mRNA expression of IQGAP3 have similar diagnostic value in different stages and grades.

Correction between IQGAP3 expression with prognosis and immune cell infiltration in two subtypes of renal cell carcinoma

Kaplan–Meier analysis showed that the high expression of IQGAP3 was correlated with low OS (Overall Survival), DSS (Disease Specific Survival) and PFS (Progression Free Survival) both in KIRC and KIRP (Figs. 3A, 3B). The “immune gene” module of Timer database was used to explore the relationship between IQGAP3 expression and immune infiltration. In KIRC, IQGAP3 expression was positively correlated with infiltrated Th2 cells, Treg cells, NK (CD56+) cells, Th1 cells, aDC cells, T cells, macrophages and B cells, but negatively correlated with iDC cells, NK cells, Tgd cells, pDC cells, Th17 cells and mast cells (Fig. 3C). In KIRP, IQGAP3 expression was positively correlated with infiltrated Th2 cells, aDC cells, pDC cells and T helper cells, as well as a negative correction with DC cells, cytotoxic cells, neutrophils, Tem cells, CD8 T cells, mast cells, iDC cells, eosinophils and macrophages (Fig. 3D).

The deletion of IQGAP3 in ccRCC inhibit cell proliferation

IQGAP3 depletion was performed by siRNA transfection in ccRCC cell lines 786-O and ACHN (Fig. 4A). Cell proliferation was reduced after IQGAP3 depletion in both cell lines (Fig. 4B). Cell clone formation and cell metastasis were not affected (data were not shown). The cells present cell agglomerates in the 3D cell culture dish (Fig. 4C). At the early stage of culture (day 6), IQGAP3 knockdown had no significant effect on the growth of cell spheres. At the late stage of culture (day 12), deletion of IQGAP3 significantly inhibited the growth of 3D cell spheres (Fig. 4D). Cell growth in 3D culture was significantly suppressed after IQGAP3 depletion after 14 days both in 786-O and ACHN cells (Fig. 4E).

The depletion of IQGAP3 increased genomic instability

In the process of cell mitosis, some chromosome breaks will produce centromere-free chromosome fragments, which are wrapped in the nuclear membrane to form a micronucleus (MN) structure with a diameter less than 1/3 of the normal nucleus. In order to determine the effect of IQGAP3 on genomic stability in ccRCC, the formation of MNs was counted after 4 Gy ionizing radiation (IR) and recovery for 4 h (Fig. 5A). After IQGAP3 knockdown, the number of MNs in cells increased regardless of IR treatment (Fig. 5B). Phosphorylation of serine (Ser) at position 139 of histone H2AX (γ H2AX) is considered to be a marker of DNA breakage (Fig. 5C). As the initial signal molecule of damage induction, γ H2AX recruits a series of DNA damage repair proteins at the damage site to start the DNA repair cascade. After IR irradiation, the intensity and quantity of γH2AX were increased in 786-O and ACHN cells (Figs. 5D, 5E). With the increase of recovery time after radiation, DNA damage in IQGAP3 knockdown cells accumulated continuously (Fig. 5F). These results show that IQGAP3 can promote genome stability.

The depletion of IQGAP3 increases the sensitivity of ccRCC cells to radiation and chemotherapy drugs

The cells were treated with different doses of IR, and the cell survival was counted. After IQGAP3 knockdown, the cell survival rate decreased significantly and the cell radiosensitivity increased (Fig. 6A). The three common chemotherapeutic drugs, cisplatin, camptothecin and doxorubicin were selected to test IC50 value. The sensitivity of cells to these drugs increased after IQGAP3 knockdown (Fig. 6B). The results show that IQGAP3 promotes genome stability, which may be the reason for chemoradiotherapy resistance of ccRCC.

Enrichment of migration and drug metabolism related genes after IQGAP3 depletion

In order to study the signal pathways of IQGAP3 stabilizing genome and then participating in the inhibition of 3D growth in ccRCC, the IQGAP3 knockdown cells were sequenced to compare the global gene expression profile. In total of 462 differentially expressed genes under the condition of fold change ≥ 2 and p < 0.05 were found, including 278 upregulated genes and 184 downregulated genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for gene function research of these differentially expressed genes. The bubble chart displayed the affected genes were enriched in extracellular matrix (ECM)-receptor interaction, drug metabolism, regulation of actin cytoskeleton and cell adhesin molecules after IQGAP3 depletion (Fig. 6C). These results provide potential mechanistic insights into the promotion role of IQGAP3 in cell 3D growth and drug resistance in ccRCC. However, the interacting proteins and specific regulatory mechanisms need to be further studied.