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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Biochim Biophys Acta. 2016 Apr 12;1862(6):1172–1181. doi: 10.1016/j.bbadis.2016.03.012

AKAP-9 promotes colorectal cancer development by regulating Cdc42 interacting protein 4

Zhi-Yan Hu 1,2,3, Yan-Ping Liu 1,2,3, Lin-Ying Xie 1,2,3, Xiao-Yan Wang 1,2,3, Fang Yang 1,2,3, Shi-You Chen 4, Zu-Guo Li 1,2,3
PMCID: PMC4846471  NIHMSID: NIHMS777732  PMID: 27039663

Abstract

Our previous studies have shown that PRKA kinase anchor protein 9 (AKAP-9) is involved in colorectal cancer (CRC) cell proliferation and migration in vitro. However, whether or not AKAP-9 is important for CRC development or metastasis in vivo remains unknown. In the present study, we found that AKAP-9 expression was significantly higher in human colorectal cancer tissues than the paired normal tissues. In fact, AKAP-9 level correlated with the CRC infiltrating depth and metastasis. Moreover, the higher AKAP-9 expression was associated with the lower survival rate in patients. In cultured CRC cells, knockdown of AKAP-9 inhibited cell proliferation, invasion, and migration. AKAP-9 deficiency also attenuated CRC tumor growth and metastasis in vivo. Mechanistically, AKAP-9 interacted with cdc42 interacting protein 4 (CIP4) and regulated its expression. CIP4 levels were interrelated to the AKAP-9 level in CRC cells. Functionally, AKAP-9 was essential for TGF-β1-induced epithelial-mesenchymal transition of CRC cells, and CIP4 played a critical role in mediating the function of AKAP-9. Importantly, CIP4 expression was significantly up-regulated in human CRC tissues. Taken together, our results demonstrated that AKAP-9 facilitates CRC development and metastasis via regulating CIP4-mediated epithelial-mesenchymal transition of CRC cells.

Keywords: AKAP-9, Colorectal cancer, Metastasis, Epithelial-mesenchymal transition, Cdc42 interacting protein 4

1. Introduction

Colorectal Cancer (CRC) is the third leading cause of cancer death. Colorectal carcinogenesis is a multistep process involving progressive disruption of epithelial cell proliferation, apoptosis, and differentiation [1–2]. Metastasis is the major cause of mortality in patients with colorectal tumors [3]. Since metastasis of tumor is a complex process, understanding the key mechanisms and molecules involved in the complex process of tumor invasion and metastasis is likely to contribute to the development of effective therapeutics for treating CRC patients.

The A-kinase anchor proteins (AKAPs) are a group of structurally diverse proteins that have the common function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. Previous studies have reported that AKAP-9 is involved in the development or metastasis of several cancers, including breast cancer [4], lung cancer [5], melanomas [6], thyroid carcinomas [7–8]. AKAP-9 is also identified as a novel putative cancer gene in human Oral Squamous Cell Carcinoma (OSCC) datasets [9]. One single nucleotide polymorphism AKAP9 M463I has been identified to be significantly associated with CRC risk in human [10]. Our previous studies have shown that AKAP-9 is expressed in CRC cells and plays a role in MALAT1-mediated CRC proliferation, migration and invasion in vitro [11]. However, the role of AKAP-9 in CRC development or metastasis in vivo and the underlying mechanism remains to be determined.

By manipulating AKAP9 expression in CRC cells, we have demonstrated that AKAP-9 plays a critical role in the proliferation, migration and invasion of CRC in vitro as well as the tumorigenesis in vivo. Importantly, we found that AKAP-9 interacts with cdc42 interacting protein 4 (CIP4) and modulates its expression in CRC cells. Moreover, AKAP-9 appears to mediate TGF-β-induced epithelial-mesenchymal transition (EMT) via CIP4. Collectively, our study has provided a novel mechanism by which AKAP-9 regulates CRC tumorigenesis and metastasis.

2. Materials and Methods

2.1. Cell culture

Colorectal cancer cell lines Lovo, HT29, M5, LS174T, HCT116, DLD1, SW480, SW620 and a normal human fetal colonic mucosa cell line (FHC) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and maintained in standard conditions (5% CO2 and 95% atmosphere, 37°C).

2.2. Reverse transcriptase-polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR)

Total RNAs were isolated from the cells using TRIzol procedure (Takara). 1 μg of RNA was added to 20 μl of reaction mixture, and cDNA was synthesized by PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). The housekeeping gene glyceraldehyde-3 phosphate dehydrogenase (GAPDH) fragment was used as an internal quantitative control. qPCR was used to calculate the messenger RNA (mRNA) expression With SYBR® Premix Ex Taq™ (TaKaRa). The primers for qPCR were designed with Primer 5 and the primer sequences were as follows: human AKAP-9: 5′-ACT CAA GGC ACA GCA TAA ACA C-3′ (forward) and 5′-GTT CTT CAC TGC GTC CCAA-3′ (reverse); human CIP4: 5′-ACA CGG AGT TTG ATG AGG AT-3′ (forward) and 5′-ATG GTG GAA CGA TGG TAG AA-3′ (reverse); human GAPDH: 5′-ACA GTC AGC CGC ATC TTC TT-3′ (forward) and 5′-GAC AAG CTT CCC GTT CTC AG-3′ (reversed). The thermal cycle was defined at 95°C, 10min, followed by denaturing at 95°C for 10 s and annealing at 60 °C for 30 s and extension at 72 °C for 30 s for 35 cycles. AKAP-9 and CIP4 mRNA expression was normalized to GAPDH level.

2.3. Western blotting

Cell lysates were prepared using the RIPA buffer. Protein concentration was measured using a BCA protein assay kit. Equal amount of protein was separated by electrophoresis on a 10% SDS-polyacrylamide gel. The proteins were electrotransferred from the gel to nitrocellulose membrane. The membrane was blocked with 5% non-fat milk solution for 1 h, and then was incubated with primary monoclonal antibody against AKAP-9 (Abcam), E-cadherin (Cell signaling), vimentin (Cell Signaling), N-cadherin (Proteintech) and CIP4 (Santa Cruz) at 4°C overnight. α-Tubulin was used as an internal control. After washing with TBS-T, the membrane was incubated with secondary antibodies against goat or mouse Ig G. The membrane was washed and detected by the enhanced chemiluminescence (ECL) detection system (Thermo) according to the manufacturer’s instructions.

2.4. Coimmunoprecipitation

Cells were washed twice with cold PBS and lysed in RIPA buffer (1× PBS, 1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate, and 1 mM sodium orthovanadate) containing protease inhibitors at 4 °C followed by vortex and centrifugation at 14,000 rpm at 4 °C for 10 min. Total proteins (500 ug/sample) were pre-cleaned with 40 μl A-G beads (Santa Cruz) before immunoprecipitation with 3 μg control IgG (Santa Cruz Biotechnology), AKAP-9, or CIP4 antibody at 4°C overnight. After incubation with 40 μl A-G beads at 4°C for 6 hours, the immunoprecipitates were washed with PBS containing 0.2% NP-40 for 5 times. The immunoprecipiated protein complexes were then released by boiling in 2×SDS-PAGE sample buffer for 5 minutes and used for immunoblotting with both AKAP-9 (Abcam) and CIP4 antibodies (Santa Cruz).

2.5. Immunofluorescent co-localization

Cells plated on poly-L-lysine-coated glass coverslips were fixed with 4% paraformaldehyde, and washed with PBS. Cells were then permeabilized with 0.1% Triton X-100/PBS for 10 min and subsequently incubated with AKAP-9 and CIP4 antibodies followed by incubation with fluorescein isothiocyanate-tagged secondary antibodies. AKAP-9 expression localization was labeled green fluorescence, and CIP4 expression localization was labeled red fluorescence. 4′, 6-Diamidino-2-phenylindole (DAPI) was used for the nuclear counterstain. The co-localization of AKAP9 and CIP4 was observed using an confocal laser scanning microscope (Leica, Germany).

2.6. Lentiviral vector preparation

AKAP-9 was knocked down with RNA interference (RNAi) targeting on mRNA or promoter region of AKAP-9 gene. Stealth RNAiTM negative control with medium GC content was purchased from Invitrogen. RNAi cDNA sequence was cloned into the GV115 lentiviral expression vector according to the manufacturer’s instruction (Shanghai Genechem Co).

2.7. Plasmid transfection

Plasmids were transfected into cells using lipofectamine 2,000 (Invitrogen) according to the manufacturer’s instructions. The full length human wild-type CIP4 cDNA was inserted into CMV-MCS-3FLAG-SV40-Neomycin expression vector from shanghai Genechem Co. All constructs were verified by sequencing. Efficacy of cDNA expression was verified by western blotting and RT-RCR detection of cellular CIP4 expression.

2.8. Immunohistochemistry and Immunofluorescent staining

2 μm-thick tissue sections were incubated with primary antibody at 4 °C overnight. Before incubation with AKAP-9, CIP4, and Ki-67 (Abcam) antibodies, the sections were antigen-retried by citric acid hydrochloric acid buffer followed by heating in a pressure cooker. For negative control, the primary antibody was replaced with normal nonimmune serum. AKAP-9 and CIP4 cytoplasmic staining was considered as a positive signal. The degree of staining in the sections was observed and scored independently by 2 pathologists. The percentage of AKAP9 and CIP4 positive cells varied from 0% to 100%, which was recorded on the following 4-point scale: 1 (0–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The intensity of cytoplasmic staining varied from weak to strong. The cells at each intensity of staining were recorded on the following 4-point scale: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown). Tumor tissues with an intensity score of 2 or higher in which more than 50% of malignant cells were stained positive for AKAP-9 were classified as tumors with high expression (or overexpression), and tumor tissues with an intensity score less than 2 or of which less than 50% of malignant cells were stained positive for AKAP9 were classified as tumors with low expression.

For immunofluorescent staining, cells plated on poly-L-lysine-coated glass coverslips were fixed with 4% paraformaldehyde, and washed with PBS. Cells were then permeabilized with 0.1% Triton X-100/PBS for 10 min and subsequently incubated with primary antibodies followed by incubation with fluorescein isothiocyanate-tagged secondary antibodies. 4′, 6-Diamidino-2-phenylindole (DAPI) was used for the nuclear counterstain. The fluorescent staining was recorded using an inverted fluorescence microscope (Leica, Germany).

2.9. CRC cell proliferation, colony formation, cell migration, and invasion assays

The CRC cell proliferation, plate colony formation, migration, and invasion of CRC cells were determined as described previously [11].

2.10. Tumor growth and metastasis assay in vivo

CRC cells were harvested by trypsinization, washed twice, and then re-suspended with serum-free medium. To evaluate CRC tumor growth in vivo, 2 × 106 of Lovo and HT29 cells stably expressing control or AKAP-9 shRNA via lentiviral vector were separately injected subcutaneously into the left and right back flank of 4- to 6-week-old Balb/C-nu/nu athymic nude mice (n=6 per group), obtained from Animal Center of Southern Medical University. After injection, fluorescence emitted from the injected cells was collected and imaged with a whole-body GFP imaging system (Lighttools, Encinitas, California). IPP5.0 software (Cybermetics, Silver Spring, Maryland) was used for analysis of whole-body optical images which visualized the real-time tumor growth and tumor area. Tumor size was measured by a slide caliper, and tumor volume was calculated as follows: volume = (D × d2) / 2, where D meant the longest diameter and d meant the shortest. For testing the in vivo metastasis, 2×105 of Lovo and HT29 cells stably expressing control or AKAP-9 shRNA in a volume of 150 μl in the culture media were injected into 4- to 6-week-old Balb/C-nu/nu athymic nude mice (n=6 per group) via the tail vein, respectively. Lung metastases of tumor cells were observed 2 months post-injection. The experiments were performed according to institutional guidelines, and all procedures were approved by the Institution Animal Care and Use Committee of Southern Medical University.

2.11. Statistical analysis

All statistical analyses were performed using the SPSS 16.0 statistical software package (Abbott Laboratories, USA). All results were confirmed by statisticians in the Department of Health Statistics, Southern Medical University. Quantitative values of all experiments are expressed as the mean ± SD. The significance of correlation between the expression of AKAP9 and histopathological factors was determined using Pearson χ2 test. Survival curves were plotted by Kaplan-Meier method and compared by log-rank test. In vitro cell growth assay was tested using factorial design ANOVA. Comparisons between groups were performed with a 2-tailed paired Student’s t test. The Correlation between CIP4 and AKAP-9 expression was determined using Spearman’s correlation analysis. In all samples, P<0.05 was considered to be statistically significant.

3. Results

3.1. AKAP-9 expression correlated with human CRC development, metastasis and patient survival rate

Our previous studies have shown that AKAP-9 is expressed in CRC tissue samples [11]. To explore if AKAP-9 expression is associated with CRC development in human, we analyzed the AKAP-9 mRNA level in 54 paired human CRC tissue samples and found that AKAP-9 mRNA level in CRC tumor tissues was higher than the normal tissues in 45 out of 54 matching tissue samples (Fig. 1A–1B). Comparing with those without CRC metastasis, patients with CRC metastasis had significantly higher mRNA expression of AKAP-9 in CRC tissues (Fig. 1C). Moreover, analyses of the clinicopathologic characteristics of all the 54 matching tissue samples revealed that AKAP-9 mRNA expression level was closely associated with the depth of tumor cell infiltration (P<0.0057) and distant metastasis (P<0.007) (Table 1). To test if the altered AKAP-9 expression in CRC is related to patient’s prognosis, we performed bioinformatic analyses of NCBI GEO Database and found that AKAP-9 expression confidently correlated with patient survival rate and length. Patients with a higher level of AKAP9 expression had a shorter survival time compared to those with a lower level of AKAP-9 expression (P=0.043, Fig. 1D). These data suggested that the AKAP-9 may play an important role in CRC invasion and metastasis as well as the patient’s survival.

Figure 1. AKAP-9 mRNA expression level in human colorectal cancer tissues.

Figure 1

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(A) Quantitative analysis of AKAP-9 mRNA expression in 54 paired human colorectal cancer tissues. AKAP-9 mRNA expression was quantified by qPCR and normalized to the matched adjacent normal tissues. (B) Comparison of AKAP-9 abundance in 54 paired primary CRC tissues (Tumor) with paired adjacent normal tissues (Normal). *P<0.001. (C) Comparison of AKAP-9 expression between 21 paired primary CRC tissues without metastasis and 33 paired primary CRC tissues with metastasis, *P<0.01. (D) AKAP-9 expression positively correlated with patient survival time as shown by bioinformatic analysis.

Table 1.

Clinicopathological characteristics of patients and AKAP-9 mRNA expression in colorectal Cancer tissues.

Characteristic No. of Patients P
Age (y) ≧50 24 0.751
<50 30
Differentiation High 15 0.953
Moderate 17
Low 22
Infiltration Mucous layer 27 0.0057
Muscular layer 15
Full layer 12
Diameter ≧5cm 35 0.082
<5cm 19
Distant Metastasis Yes 33 0.007
No 21
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To further test the correlation between AKAP-9 with CRC development, immunohistochemistry (IHC) staining was performed to confirm the AKAP-9 protein levels in 113 paraffin-embedded CRC tissues. 73 out of 113 paraffin-embedded CRC samples had normal tissue adjacent to CRC, and we found that 63 of these samples exhibited a much higher AKAP-9 level in CRC tissues than the corresponding normal tissues (Supplemental Fig. 1A). In addition, we found that AKAP-9 level was associated with the depth of tumor infiltration (P<0.001) (Supplemental Table 1). These observations demonstrate that AKAP-9 may play a role in CRC invasion and metastasis.

3.2. AKAP-9 promoted CRC cells proliferation, migration, and invasion in vitro, and tumor growth and metastasis in vivo

In cellular level, we detected the protein expression of AKAP-9 in eight different CRC cells and found that Lovo and HT29 cells exhibited a much higher level of AKAP-9 expression than other cells (Supplemental Fig. 1B). Therefore, we chose Lovo and HT29 cells for the subsequent studies.

To study the potential role of AKAP-9 in CRC tumorigenesis and progression, we used lentiviral vector mediated shRNA knockdown of AKAP-9 in Lovo (Supplemental Fig. 2A) and HT29 cells (Supplemental Fig. 3A) and performed CCK8 proliferation assay and colony formation assay to detect CRC proliferation. As shown in Supplemental Fig. 2B–2C (Lovo cells) and 3B–3C (HT29 cells), knockdown of AKAP-9 inhibited both Lovo and HT29 cell growth and colony formation, indicating that AKAP-9 plays a role in CRC growth. Since AKAP-9 expression level was associated with the depth of tumor infiltration and patient’s metastasis (Table 1 and Supplemental Table 1), we tested if AKAP-9 affects the migration and invasion of Lovo and HT29 cells. As shown in Supplemental Fig. 2D–2F (Lovo cells) and 3D–3F (HT29 cells), knockdown of AKAP-9 indeed inhibited CRC cell migration (Supplemental Fig. 2D–2E and 3D–3E) and invasion (Supplemental Fig. 2F and 3F).

To assess the effect of AKAP-9 on tumor growth in vivo, we subcutaneously injected Lovo or HT29 cells that stably express scramble (control) or AKAP-9 shRNA into nude mice, and then monitored the growth of the resultant primary tumors. As shown in Fig. 2A–2B, the xenograft tumors developed at the injection site after 6 days. During a growing period of 26 days, primary tumors derived from AKAP-9 deficient CRC cells grew significantly slower than that derived from control cells (Fig 2A–2B). Moreover, the tumor volumes of the AKAP-9 deficient groups were significantly smaller than those of control groups (Fig 2A–2B (upper panels) and Fig 2C–2D). IHC staining confirmed that the tumors derived from CRC cells expressing AKAP-9 shRNA displayed a lower AKAP-9 expression and reduced cell proliferation index as shown by Ki-67 staining compared to the tumors derived from control cells (Supplemental Fig. 4A–4D). These data demonstrated that AKAP-9 plays a crucial role in CRC growth in vivo.

Figure 2. Knockdown of AKAP-9 inhibited tumor growth and metastasis in vivo.

Figure 2

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(A–B) GFP-labeled Lovo (A) or HT29 cells (B) with stable transfection of control (shCtrl) or AKAP-9 shRNA (shAKAP-9) were injected subcutaneously into nude mice as described in Methods. 26 days later, the tumors were removed and imaged (upper panels). Tumor growth curves were obtained by using a whole-body GFP imaging system during the growth of the tumors (lower panels). Tumors derived from cells expressing shAKAP-9 grew significantly slower than that from cells with shCtrl. *P<0.05 compared to shCtrl group in each corresponding time point for both A and B, n=6. (C–D) Scatter plot of the tumor sizes at 26 days post-injection. Tumors derived from Lovo (C) and HT29 (D) cells expressing shAKAP-9 were significantly smaller than that from cells with shCtrl. *P<0.05 compared to shCtrl groups, n = 6. (E–F) GFP-labeled Lovo (E) or HT29 cells (F) with stable transfection of control (shCtrl) or AKAP-9 shRNA (shAKAP-9) were injected via tail vein as described in Methods. Metastastic foci in the lung was observed in mice with injection of shCtrl-contained cells, but not with shAKAP-9 cells. Upper panels: whole lung images with the metastatic foci as indicated by arrows; middle panel: H&E staining of lung sections showing metastastic tumors; bottom panel: Mouse numbers with metastastic foci in their lung for both Lovo (E) and HT29 cells (F), respectively.

To test the effect of AKAP-9 on the metastasis of CRC in vivo, we performed a tail vein xenograft model to investigate lung metastases of CRC cell lines. Lovo or HT29 cells that stably express control or AKAP-9 shRNA were injected into 6 mice for each group. We found that five mice present lung colonization with control Lovo cells and four mice with control HT29 cells (Fig. 2E–2F). However, no lung colonization was observed in mice with either Lovo or HT29 cells expressing AKAP-9 shRNA (Fig 2E–2F). The tumor presence was also confirmed by histological examination (Fig. 2E–2F). These results demonstrated that AKAP-9 plays a role in CRC metastasis in vivo.

3.3. Knockdown of AKAP-9 induced a mesenchymal to epithelial transition (MET) of CRC cells

Since knockdown of AKAP-9 altered cell morphology of AKAP-9-overexpressed Lovo and HT29 cells to a flatten-shaped squamous cell morphology (Supplemental Fig. 5), and high levels of AKAP-9 correlated with CRC metastasis in human (Table 1), we hypothesized that AKAP-9 may be involved in the EMT of CRC cells, a crucial event during tumor invasiveness and metastasis [12]. The key characteristics of EMT are the reduction of membrane E-cadherin along with an increased expression of neuronal cadherin (N-cadherin) and other mesenchymal markers such as vimentin [13–16]. We found that knockdown of AKAP-9 in Lovo and HT29 cells significantly increased the expression of E-cadherin while decreased N-cadherin and vimentin expression (Fig. 3A–3F), suggesting that down-regulation of AKAP-9 promoted a MET of CRC cells, a reverse process of the EMT.

Figure 3. Knockdown of AKAP-9 facilitated a mesenchymal-epithelial transition.

Figure 3

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(A) Immunostaining of mesenchymal and epithelial markers in Lovo cells transfected with scramble (shCtrl) or AKAP-9 shRNA (shAKAP-9) as indicated. Magnification: 1800x. (B) The expression of mesenchymal and epithelial markers in Lovo cells transfected with scramble (shCtrl) or AKAP-9 shRNA (shAKAP-9) was detected by Western blotting. (C) Quantification of protein expression shown in B by normalized to α-Tubulin. *P<0.05 compared to the shCtrl groups, n=3. (D) Immunostaining of mesenchymal and epithelial markers in HT29 cells transfected with scramble (shCtrl) or AKAP-9 shRNA (shAKAP-9) as indicated. Magnification: 1800x. (E) The expression of mesenchymal and epithelial markers in HT29 cells transfected with scramble (shCtrl) or AKAP-9 shRNA (shAKAP-9) was detected by Western blotting. (F) Quantification of protein expression shown in E by normalized to α-Tubulin. *P<0.05 compared to the shCtrl groups, n=3. Knockdown of AKAP-9 in either Lovo or HT29 cells increased E-Cadherin while decreased N-Cadherin and vimentin expression.

3.4. AKAP-9 interacted with CIP4 and regulated CIP4 protein expression

Previous studies have shown that AKAP350, an alternate splicing isoform of AKAP-9 gene [17], interacts with cdc42 interacting protein 4 (CIP4) at the Golgi apparatus [18]. It has been shown that CIP4 promotes TGF-β1-induced EMT of renal proximal tubular epithelial cells [19] and mediates high glucose-induced EMT through PI3K-Akt signaling pathway in rat peritoneal mesothelial cells [20]. Moreover, knockdown of CIP4 strongly increases the formation of tubular E-cadherin vesicles at adherens junctions [21]. CIP4 can also indirectly regulate actin polymerization [22–24]. We hypothesized that AKAP-9 may interact with CIP4 protein to induce EMT. As shown in Fig. 4A, AKAP-9 and CIP4 colocalized in Lovo and HT29 cells. Co-immunoprecipitation assay revealed that AKAP-9 and CIP4 physically interacted with each other in Lovo and HT29 cells (Fig. 4B–4C).

Figure 4. AKAP-9 physically interacted with CIP4 in CRC cells.

Figure 4

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(A) AKAP-9 and CIP4 co-localized in Lovo and HT29 cells. DAPI stains nuclei. (B–C) AKAP-9 physically bound to CIP4 in Lovo and HT29 cells. Lovo (upper panels) and HT29 cells (lower panels) were lysed, and the lysates were subjected to immunoprecipitation (IP) with normal IgG, CIP4 (B), or AKAP-9 (C) antibody followed by immunoblotting (IB) with antibodies as indicated. Input was 5% of the total lysates used for the IPs.

To determine if AKAP-9 regulates CIP4 expression, we detected the expression of CIP4 in the Lovo and HT29 cells when AKAP-9 expression was blocked, and found that knockdown of AKAP-9 significantly decreased both the protein and mRNA levels of CIP4 in Lovo (Fig. 5A–5D and Supplemental Fig. 6A–6C) and HT29 cells (Fig. 5E–5H and Supplemental Fig. 6D–6F). CIP4 expression appeared to correlate with AKAP-9 level in CRC cells (Fig. 5A–5H). Since CIP4 is involved in the EMT of different cells, CIP4 may mediate the function of AKAP-9 in EMT.

Figure 5. AKAP-9 regulated CIP4 protein expression in CRC cells.

Figure 5

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(A) Knockdown of AKAP-9 attenuated CIP4 protein expression in Lovo cells. (B) Quantification of AKAP-9 and CIP4 protein levels shown in A by normalized to α-Tubulin. *P<0.01,**P<0.001 compared to shCtrl cells, n=3. (C) CIP4 expression in Lovo cells was detected by immunostaining. Magnification: 1800x. (D) Knockdown of AKAP-9 inhibited CIP4 mRNA expression in Lovo cells as detected by qRT-PCR. *P<0.001 compared to shCtrl cells, n=3. (E) Knockdown of AKAP-9 attenuated CIP4 protein expression in HT29 cells. (F) Quantification of AKAP-9 and CIP4 protein levels shown in E by normalized to α-Tubulin. *P<0.05,**P<0.01 compared to shCtrl cells, n=3. (G) CIP4 expression in HT29 cells was detected by immunostaining. Magnification: 1800x. (H) Knockdown of AKAP-9 inhibited CIP4 mRNA expression in HT29 cells as detected by qRT-PCR. *P<0.001 compared to shCtrl cells, n=3.

3.5. AKAP-9 was required for TGF-β-induced EMT and CIP4 rescued AKAP-9 function

It is known that TGF-β signaling plays a very important role in EMT [25]. Thus, we tested if TGF-β1 induces EMT of Lovo and HT29 cells. Indeed, TGF-β1 stimulation induced the EMT of Lovo and HT29 cells as shown by the decreased E-cadherin and increased N-cadherin and vimentin expression (Supplemental Fig. 7A–7B and 7D–7E). Importantly, TGF-β1 also enhanced AKAP-9 and CIP4 expression in Lovo and HT29 cells (Supplemental Fig. 7A–7B and 7D–7E). Interestingly, we knockdown of AKAP-9 inhibited TGF-β1 mRNA expression in both Lovo and HT29 cells (Supplemental Fig. 7C and 7F), suggesting that AKAP-9 regulated TGF-β1 expression, which may promote the EMT via an autocrine loop for TGF-β1 signaling.

To determine if AKAP-9 is required for TGF-β1-induced EMT, we knocked down AKAP-9 expression using shRNA in Lovo and HT29 cells treated with TGF-β1. As shown in Fig. 6A–6D, blockade of AKAP-9 expression restored E-cadherin expression while attenuated N-cadherin and vimentin expression in both Lovo and HT29 cells. These results suggested that AKAP-9 is required for TGF-β1-induced EMT of Lovo and HT29 cells.

Figure 6. AKAP-9 mediated TGF-β1-induced EMT via CIP4 in CRC cells.

Figure 6

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(A) TGF-β1 promoted AKAP-9 and CIP4 expression while inducing EMT of Lovo cells. Knockdown of AKAP-9 attenuated TGF-β1-induced EMT. CIP4 overexpression in AKAP-9 deficient cells rescued TGF-β1-induced EMT. (B) Quantification of protein levels shown in A by normalized to α-Tubulin. *P<0.001 compared to the untreated groups (blank); **P<0.01 compared to control shRNA (shCtrl)-treated groups (TGF-β1+shCtrl); ***P<0.001 compared to AKAP-9 shRNA (shAKAP-9)-treated groups (TGF-β1+shAKAP-9), n=3. (C) TGF-β1 promoted AKAP-9 and CIP4 expression while inducing EMT of HT29 cells. Knockdown of AKAP-9 attenuated TGF-β1-induced EMT of HT29 cells. CIP4 overexpression in AKAP-9 deficient cells recued TGF-β1-induced EMT of HT29 cells. (D) Quantification of protein levels shown in C by normalized to α-Tubulin. *P<0.001 compared to untreated groups (Blank); **P<0.01 compared to control shRNA (shCtrl)-treated groups (TGF-β1+shCtrl); ***P<0.001 compared to AKAP-9 shRNA (shAKAP-9)-treated groups (TGF-β1+shAKAP-9) for each individual protein, n=3.

Notably, knockdown of AKAP-9 also blocked TGF-β1-induced CIP4 protein expression (Fig. 6A–6D), consistent with the role of AKAP-9 in regulating CIP4 expression (Fig. 5A–5F). We therefore hypothesized that CIP4 mediates AKAP-9 function in TGF-β1-induced EMT, and thus CIP4 overexpression can rescue the function of AKAP-9 on the EMT. Indeed, overexpression of CIP4 attenuated the E-cadherin expression but restored N-cadherin and vimentin expression that was altered due to AKAP-9 knockdown in TGF-β1-treated cells (Fig. 6A–6D). Moreover, overexpression of CIP4 restored AKAP-9 deficiency-attenuated proliferation, migration, and invasion of Lovo (Supplemental Fig. 8) and HT29 cells (Supplemental Fig. 9). These results clearly showed that AKAP-9 mediates TGF-β-induced EMT of CRC cells via inducing CIP4 expression.

3.6. CIP4 was expressed in CRC cells and human CRC tumors

In order to determine if CIP4 is expressed in different CRC cells and in human CRC tissues, we detected CIP4 protein expression in control FHC cell and eight different CRC cell lines by western blot and in 38 paired paraffin-embedded human CRC tissue samples by IHC. As shown in Fig. 7A, CIP4 expression was expressed in all CRC cells detected, and CIP4 expression in CRC cells was higher than that in FHC cell. However, a high level of CIP4 expression was observed in Lovo and HT29 cells, consistent with the AKAP-9 expression (Supplemental Fig. 1B). Indeed, CIP4 expression correlated with AKAP-9 in most CRC cells (Fig 7A vs Supplemental Figure 1B). Moreover, both CIP4 and AKAP-9 expression were significantly elevated in human CRC tumors compared to the corresponding normal tissues in 30 out of 38 paired CRC samples (Fig. 7B–7C). Importantly, there was a spatial and protein level correlation between CIP4 and AKAP-9 in human CRC tumors (r=0.337, P=0.038; Fig. 7B and 7D), indicating that CIP4 may mediate AKAP-9 function in the colorectal cancer development in human.

Figure 7. CIP4 expressed in CRC cells and tumor tissues.

Figure 7

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(A) CIP4 protein expression in the normal fetal colonic mucosa cells (FHC) and eight CRC cell lines was detected by western blot and normalized to α-Tubulin level. (B) CIP4 and AKAP-9 expression in 38 paired paraffin-embedded CRC tissue samples was detected by immunohistochemistry staining. The CIP4 and AKAP-9 were spatially correlated. Representative CIP4 and AKAP-9 immuohistochemical staining photographs of normal tissue (Normal) and tumor tissue samples (Tumor 1, Tumor 2 and Tumor 3) as indicated. (C) Quantification of protein expression levels shown in B, CIP4 and AKAP-9 expression levels in CRC tumor (T) were significantly higher than the adjacent normal tissues (N). Magnification: 200x. *P <0.001 compared to normal tissue (Normal), n = 38. (D) Spearman’s correlation analysis showed a positive relationship between the CIP4 and AKAP-9 level in 38 human colorectal cancer tissues. r=0.337, P=0.038.

4. Discussion

In the present study, we have examined the role of AKAP-9 in CRC development. AKAP-9 appears to be important for CRC progression because blockade of AKAP-9 causes a decreased CRC proliferation in vitro (Supplemental Fig. 2B–2C and 3B–3C) and CRC tumor growth in vivo (Fig. 2A–2D). Moreover, AKAP-9 expression in human CRC tissues correlates with the depth of tumor infiltration and the distant metastasis (Table 1 and Supplemental Table 1), suggesting that AKAP-9 plays a role in CRC metastasis, which is supported by the decreased CRC cell migration and invasion in vitro (Supplemental Fig. 2D–2F and 3D–3F) and attenuated lung metastasis of CRC cells in vivo (Fig. 2E–2F) due to the AKAP-9 deficiency. Most importantly, AKAP-9 expression is associated with CRC patient prognosis or survival (Fig. 1D). These observations support the notion that AKAP-9 may serve as a valuable biomarker to monitor CRC development in humans.

Many different mechanisms have been implicated in tumor metastasis of different cancers. EMT is one of the mechanisms commonly believed to control the process of cancer cell invasion and metastasis [26]. AKAP-9 appears to regulate CRC tumor metastasis by mediating the EMT of CRC cells. Knockdown of AKAP-9 increases E-cadherin expression while decreasing N-cadherin and vimentin expression (Fig. 3A–3F), consistent with the key characteristics of EMT observed in cancer cells [27]. The role of AKAP-9 in EMT is further supported by its essential role in TGF-β1-induced EMT of CRC cells. Mechanistically, AKAP-9 mediates the EMT by inducing CIP4 expression. The correlation between AKAP-9 and CIP4 is supported by several lines of evidence. Firstly, CIP4 expression correlates with AKAP-9 level in most CRC cells (Fig. 7A and Supplementary Fig. 1B) and in human CRC tumors (Fig. 7B and 7D). Secondly, AKAP9 down-regulation causes a decreased expression of CIP4 (Fig. 5A–5H). Thirdly, TGF-β1 simultaneously induces both AKAP-9 and CIP4 expression in CRC cells (Supplemental Fig. 7). Finally, overexpression of CIP4 rescues the attenuation of TGF-β1-induced EMT by the blockade of AKAP-9 (Fig. 6A–6D). Interestingly, AKAP-9 not only regulates CIP4 expression, but also physically interacts with CIP4 (Fig. 4A–4C), suggesting that in addition to the expression, AKAP-9 may also regulate CIP4 function. Consistent with this notion, AKAP-9 regulates TGF-β1 expression. Thus, AKAP-9 may mediate an autocrine loop of TGF-β1, which may further enhance CIP4 function in mediating EMT. The detailed mechanisms regarding to how the interaction of AKAP-9 and CIP4 is involved in the EMT process and how AKAP-9 regulates CIP4 expression in CRC cells will be interesting subjects for the future study.

CIP4 has been shown to be involved in the metastasis of different cancers or poor patient prognosis including lung adenocarcinoma [28], triple-negative breast cancer [29] and osteosarcoma [30]. Our study suggests that CIP4 may also be involved in the metastasis of CRC tumor due to its role in mediating AKAP-9 function in the EMT of CRC cells. The CIP4 expression in human CRC tumors is significantly higher than the corresponding normal tissues (Fig. 7B–7D), further indicating its role in the development of CRC, which can be studied in the future.

Conclusion

We have demonstrated that AKAP-9 plays an important role in tumorigenesis and metastasis of CRC. AKAP-9 contributes to the CRC metastasis by regulating CIP4 expression and consequently EMT of CRC. Since AKAP-9 level is associated with the tumor infiltrating depth tumor metastasis, and lower survival rate of human patients, AKAP-9 may be used as a biomarker to monitor the progression of CRC cancer in human.

Supplementary Material

1

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NNSF81172054, NNSF81272408, NNSF81370227, NNSF81201664), and National Institutes of Health (HL123302, HL119053, HL107526). The authors are grateful to Pro. Ding YQ and the researchers at the Key Laboratory of Molecular Tumor Pathology, located in Guangdong, China for providing assistance in our experiments.

Abbreviations

AKAP-9

PRKA kinase anchor protein 9

CRC

colorectal cancer

CIP4

cdc42 interacting protein 4

EMT

epithelial-mesenchymal transition

MET

mesenchymal to epithelial transition

AKAPs

A-kinase anchor proteins

PKA

protein kinase A

OSCC

Oral Squamous Cell Carcinoma

MALAT1

Metastasis associated lung adenocarcinoma transcript 1

GAPDH

glyceraldehyde-3 phosphate dehydrogenase

FBS

fetal bovine serum

PCR

polymerase chain reaction

QPCR

quantitative polymerase chain reaction

RT-RCR

reverse transcription-PCR

RIPA

Radio-Immunoprecipitation Assay

BCA

bicinchonininc acid

SDS

dodecyl sulfate, sodium salt

TBS-T

triethanolamine buffered saline solution-Tween

ECL

enhanced chemiluminescence

PBS

Phosphate Buffered Saline

EDTA

Ethylene Diamine Tetraacetic Acid

NP40

NobleRyder 40

2×SDS-PAGE

2×dodecyl sulfate, sodium salt- Polyacrylamide gelelectrophoresis

DAPI

4′, 6-Diamidino-2-phenylindole

GEO

Gene Expression Omnibus

Footnotes

Conflict of Interest Statement: None declared.

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Supplementary Materials

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