S100A11 regulates renal carcinoma cell proliferation,invasion,and migration via the EGFR/Akt signaling pathway and E-cadherin

Abstract

S100A11 is a S100 protein family member that contributes to cancer progression. Upregulated in human renal cancer tissues, S100A11 may be a prognostic marker for clear cell renal cell carcinoma, but how it functions in cancer is uncertain. Thus, we studied S100A11 and noted knockdown of S100A11 using short hairpin RNA, which inhibited proliferation, invasion, and migration of renal carcinoma cells as well as increased expression of E-cadherin and decreased expression of epidermal growth factor receptor/Akt in renal carcinoma cells. Therefore, S100A11 may be a key molecular target for treating renal carcinoma.

Introduction

Renal carcinoma is the most frequent and lethal urological malignancy in adults, ¹ representing 2%–3% of all cancers worldwide; ² 5-year patient survival with metastatic renal carcinoma is less than 10%, ³ and in spite of surgical improvements, survival has changed little. Patient prognosis is poor ⁴ and renal carcinoma is largely resistant to radiation and chemotherapy. ⁵ Thus, understanding mechanisms of renal carcinoma development is critical for identifying molecular targets for treating this cancer.

S100A11, calgizzarin, or S100C is a S100 protein family member with roles in growth regulation of human keratinocytes. It mediates Ca²⁺-induced growth inhibition as well as stimulates growth by enhancing epidermal growth factor protein family members.^6,7 S100A11 is overexpressed in numerous cancers, including renal and papillary thyroid carcinomas, ⁸ colon cancer, ⁹ pancreatic cancer, ¹⁰ ovarian cancer, ¹¹ and breast cancer. ¹² How S100A11 influences renal carcinoma is unclear, so this was the focus of our investigation.

Increasing evidence suggests that the development of renal carcinoma is related to epidermal growth factor receptor (EGFR)/Akt and Wnt/β-catenin signaling.^13,14 Akt is an EGFR downstream target and effector of phosphatidylinositol 3-kinase (PI3K), ¹⁵ activation of which triggers a cascade of responses. In addition, S100A11 regulates renal carcinoma cells via the EGFR/Akt pathway.

E-cadherin is an important epithelial–mesenchymal transition (EMT) marker essential for cancer invasion and metastasis,^16,17 but its role in renal carcinoma development is unclear. Therefore, we assessed the role of S100A11 in the proliferation, migration, and invasion of renal carcinoma cells and a conceivable mechanism. We observed that S100A11 downregulation could inhibit the proliferation, migration, and invasion of renal carcinoma cells and this may be through the regulation of EGFR/Akt and E-cadherin.

Materials and methods

Cell culture and antibodies

ACHN and 786-O renal carcinoma cell lines were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12) containing 10% fetal bovine serum (FBS). Cells were grown at 37°C in a humidified atmosphere with 5% CO₂. Primary antibodies against EGFR, Akt, P-Akt, p21, E-cadherin, and actin were obtained from Cell Signaling Technology (Danvers, MA, USA).

S100A11 RNA interference

An S100A11 short hairpin (sh) RNA vector was compounded by Shanghai GenePharma Co., Ltd (Shanghai, China) with a target sequence of 5′-GGATGGTTATAACTACACT-3′. An sh-NC vector was used as a negative control. ACHN and 786-O cells were transfected with S100A11 or control shRNA using Lipofectamine LTX with PLUS reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA)

Cell proliferation and clonogenic assay

Cells (2 × 10⁴) were cultured in 96-well plates for 24 h and exposed to 100 µM EdU (RiboBio, Guangzhou, China) at 37 °C for additional 3 h. Cells were then fixed with 4% paraformaldehyde for 20 min and treated with 0.5% Triton X-100 for 10 min. After washing three times, cells were reacted with 100 µL 1× Apollo for 30 min. Subsequently, DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and visualized under fluorescent microscopy and photographed (Olympus, Tokyo, Japan). To determine long-term effects, ACHN and 786-O cells (~500) were seeded in six-well culture plates and then transfected with S100A11 or control shRNA, and a clonogenic assay was used to evaluate cells.

Wound healing assay

Cell migration was evaluated with a wound healing assay. 786-O cells transfected with S100A11 or control shRNA were lesioned using a plastic pipette tip, and cells were incubated in serum-free media in a six-well culture cluster. Immediately and after 24 h, five randomly selected fields at the lesion border were visualized under a microscope.

Transwell invasion and migration assays

To assess invasiveness, filters were pre-coated with Matrigel, and 786-O cells (~10⁵) in serum-free media that were transfected with S100A11 or control shRNA were added to the top chamber. The bottom chamber was filled with 10% FBS DMEM/F12. After 36 h of incubation, top chamber cells were removed with a cotton swab, and the membrane was fixed in 4% methanol for 15 min and stained with a 0.5% crystal violet solution for 20 min. Then, five fields of adherent cells in each well were photographed randomly. To measure migration, the same experiments were performed but filters were not pre-coated with Matrigel.

Cell cycle assays

To determine cell cycle effected by knockdown of S100A11, cell cycle analysis was carried out in 786-O cells transfected with S100A11 or control shRNA. Then, cells were collected and fixed in 70% ethanol. Afterwards, renal carcinoma cells were washed with phosphate-buffered saline (PBS) for twice, and finally, the cells were stained with PI solution that contained 50 µg/mL PI and 25 µg/mL RNase in the dark for 30 min. Subsequently, the cells were assayed on FACSCalibur (BD Biosciences, New Jersey, USA) and analyzed by CellQuest Pro software (BD Biosciences, New Jersey, USA).

Western blot

ACHN and 786-O cells were transfected with S100A11 or control shRNA, and after 24 h of incubation, total protein extracts from S100A11 or control shRNA cells were subjected to western blot; EGFR, Akt, P-Akt, p21, and E-cadherin expression was measured with antibodies. Actin was a loading control. Each experiment was carried out at least three times.

Tumor xenograft study

In experiments, 786-O cells were transfected with S100A11 or control shRNA. Cells were then harvested and resuspended in L15. The cell number was counted to be approximately 2 × 10⁶ cells/mL. A volume of 200 µL of cell suspension was injected into male BALB/c immunocompromised mice (5-week-old) bilaterally. After 20 days of injection, mice were sacrificed and the tumors were photographed and prepared for immunohistochemistry analysis.

Immunofluorescence staining

The subcutaneous tumor of the control and treated mice was fixed in 4% paraformaldehyde and dehydrated sequentially in 20% and 30% sucrose at 4°C until they sank. The frozen tumor tissues were serially sectioned at a thickness of 12 mm. The sections containing the tumor were incubated with 0.3% Triton X-100 followed by 10% goat serum. Next, the tumors were incubated overnight with Ki67 primary antibody. To visualize the Ki67-positive cells, the sections were incubated with Alexa-594-conjugated secondary antibody for 1 h at room temperature in the dark. DAPI was used to stain the cell nuclei. All sections were examined and photographed with a microscope with an attached fluorescence detector (IX71; Olympus).

Statistical analysis

Experiments were performed three times and data are presented as means ± standard deviation (SD). Student’s t-test was used for statistical analysis (p < 0.05).

Results

Expression of S100A11 in 786-O and ACHN cells is silenced by shRNA

To silence expression of S100A11 in renal carcinoma cells, 786-O and ACHN cells were transfected with S100A11 shRNA (sh) and western blot confirmed that S100A11 shRNA significantly repressed S100A11 expression (Figure 1). Therefore, these renal carcinoma cells were used in subsequent experiments.

Figure 1.

Silencing efficiency of S100A11 in 786-O and ACHN cells confirmed with western blot.

Knockdown of S100A11 inhibits renal carcinoma cell growth

EdU and plate colony formation assays were used to measure cell proliferation after knockdown of S100A11. Compared with the sh-NC group, EdU-positive cells in S100A11 knockdown groups were reduced (Figure 2(a), (c), (d), and (g)). Data from the plate colony formation assay showed that S100A11 knockdown decreased colony formation of renal carcinoma cells, and these data were consistent with EdU results (Figure 2(b), (d), (f), and (h)).

Figure 2.

Knockdown of S100A11 decreased proliferation of renal carcinoma cells. (a, c, e, and g) EdU assay confirming knockdown of S100A11 decreased 786-O and ACHN cell proliferation. (b, d, f, and h) Plate colony formation assay confirming S100A11 knockdown decreased 786-O and ACHN cell colony formation. Data are mean ± SEM from three independent experiments (*p < 0.05).

Knockdown of S100A11 inhibits invasion and migration of renal carcinoma cells via E-cadherin

We examined whether S100A11 silencing modified renal carcinoma cell migration with wound healing and Transwell migration assays. Figure 2(a) shows that immediately and 24 h after wounding, sh-NC cells healed and could fuse, but the sh-S100A11 group healed poorly. Compared with the sh-NC group, migratory sh-S100A11 cells decreased (Figure 2(d)). Furthermore, a three-dimensional cell migration assay with Transwell chambers yielded similar data to the wound healing assay. sh-S100A11 group migration decreased in 786-O cells (Figure 2(b) and (e)). To understand the role of S100A11 in renal carcinoma invasion, a Matrigel pre-coated Transwell chamber assay was used again, and Figure 3(c) shows that sh-S100A11 for 36 h reduced invasive 786-O cells compared with sh-controls (Figure 3(c) and (f)). Thus, S100A11 directly promotes renal carcinoma cell migration and invasion, and the gene(s) responsible for renal carcinoma cell invasion might be regulated by S100A11. Finally, S100A11 knockdown increased E-cadherin expression in 786-O cells (Figure 3(g)).

Figure 3.

Knockdown of S100A11 decreased invasion and migration of renal carcinoma cells and E-cadherin expression. (a, b, d, and e) Wound healing and Transwell assays show that S100A11 knockdown decreased 786-O cell migration (*p < 0.05). (c and f) Transwell assay shows that S100A11 knockdown decreased 786-O cell invasion (*p < 0.05). (g) Effect of S100A11 knockdown on E-cadherin expression. (h and i) knockdown of S100A11 induces cell cycle G1 arrest.

Knockdown of S100A11 induces cell cycle G1 arrest

We evaluated cell cycle distribution after transfected with S100A11 or control shRNA. As shown in Figure 3(h) and (i), the 786-O cells were arrested at G1 phase of the cell cycle in response to transfection with S100A11.

Knockdown of S100A11 decreased EGFR and Akt signaling

Previous studies indicated that EGFR and Akt signaling regulated cell growth and survival. Thus, we hypothesized that S100A11 regulated EGFR, Akt, and its downstream p21 to modulate renal carcinoma development and progression. Knocking down S100A11 in 786-O and ACHN cells with shRNA (Figure 4(a) and (b)) significantly decreased EGFR and P-Akt. However, total Akt did not change. Additionally, p21 expression was reduced. Therefore, S100A11 regulated renal carcinoma development and progression and this may be via EGFR and Akt signaling.

Figure 4.

S100A11 promoted EGFR/Akt signaling activity. (a) EGFR, Akt phosphorylation, and p21 were decreased by S100A11 knockdown in 786-O and ACHN cells, while total Akt protein was not altered. (b) Quantitative analysis of EGFR, Akt, P-Akt, and P21. Data are mean ± SEM of three independent experiments (*p < 0.05).

Knockdown of S100A11 suppresses renal carcinoma xenograft tumorigenesis in vivo

To determine whether knockdown of S100A11 exerts anti-tumor activity on renal carcinoma cells in vivo, we evaluated its effect in an intracranial nude mouse model. As shown in Figure 5(a) and (b), 20 days after renal carcinoma transplantation, the transplanted renal carcinoma in knockdown of S100A11 mice was visibly smaller than those in the vehicle-treated mice.

Figure 5.

Knockdown of S100A11 suppresses renal carcinoma xenograft tumorigenesis in vivo. (a and b) 786-O cells were transfected with S100A11 or control shRNA and subcutaneously injected into nude mice. After 20 days, mice were sacrificed and tumors were collected to assay the tumor diameter. Representative tumors were isolated from the control and knockdown of S100A11 groups (*p < 0.05). (c and d) Cell proliferation of the subcutaneous tumor was assessed with anti-Ki67 immunostaining. Quantitative analyses of the percentages of positive cells in the knockdown of S100A11 group normalized to the control group (*p < 0.05).

To further evaluate the effects of S100A11 on tumor growth, Ki67 was used to show cell proliferation in xenografts. The percentage of Ki67-positive cells in tumors was decreased by 61.03% in the knockdown of S100A11 group (Figure 5(c) and (d)). These data suggest that knockdown of S100A11 can inhibit tumor cell proliferation in vivo.

Discussion

S100A11, a S100 protein family member, is widely expressed in human tissues. ¹⁸ Overexpression of S100A11 has been reported in variety of human cancers, ¹⁹ and elevated S100A11 expression is associated with tumor progression.^10,20,21 Gabril et al. ²² reported that S100A11 overexpression is associated with higher grade and more severe stages of clear cell renal cell carcinoma (ccRCC) and speculated that S100A11 may be a prognostic marker for ccRCC. However, the biological significance of S100A11 in renal carcinoma development and progression is unclear. To address this, we studied S100A11 in renal carcinoma cells via downregulating endogenous expression of S100A11.

Previous studies suggested that S100A11 promoted proliferation of lung and ovarian cancer cells,^23,24 but decreased expression of S100A11 is reported to occur in bladder cancer, and downregulation of S100A11 is associated with bladder cancer progression and poor survival. ²⁵ So, whether S100A11 acts as an oncogene or a tumor suppressor in renal carcinoma is unclear. Our data indicate that S100A11 knockdown inhibited proliferation and anchorage-independent growth of 786-O and ACHN cells, suggesting that S100A11 contributes to renal cancer growth.

EMT is critical for invasion and metastasis of many solid carcinomas, ²⁶ and E-cadherin is an EMT cancer marker. ²⁷ Liu et al.’s ²⁴ group reported that knockdown of S100A11 inhibited invasion and migration of ovarian cancer cells by increasing expression of E-cadherin and decreasing expression of Snail. In agreement with previous work, we observed that knockdown of S100A11 decreased invasiveness and migration of renal carcinoma cells by upregulation of E-cadherin.

Several distinct S100 proteins are involved in cell growth, influencing cell cycle regulators such as p53, p21, and cyclin-dependent kinases (CDKs). ²⁸ p21, an important CDK inhibitor, arrested cells in G1 and G2 phases of the cell cycle. ²⁹ Foertsch et al. ³⁰ reported that S100A11 regulated stability of p21 in human keratinocyte HaCaT cells. In this study, we report that knockdown of S100A11 reduced expression of p21 in renal carcinoma cells.

Aberrant activation of the EGFR/Akt signaling pathway occurs in numerous solid tumors, including colorectal, breast, and ovarian cancers as well as gliomas, melanoma, and hepatocellular carcinoma, and it is linked to a poor prognosis. ³¹ Wang et al. ²¹ reported that S100A11 is a migration-related protein and downregulated expression of EGFR. Foertsch’s group suggested that S100A11 reduced phosphorylation of Akt and had no effect on total expression of Akt. Here, we observed that knockdown of S100A11 reduced activation of Akt and reduced expression of EGFR.

In summary, S100A11 knockdown inhibited proliferation, migration, and invasion of renal carcinoma cells, and this may be mediated by the EGFR/AKT signaling pathway and E-cadherin. Although S100A11 is implicated in several cancers, more study is warranted to explain mechanisms by which S100A11 is differentially regulated and how S100A11 can be a promising target for treating renal carcinoma.