Carbonic Anhydrase IX Promotes Human Cervical Cancer Cell Motility by Regulating PFKFB4 Expression

Abstract

Carbonic anhydrase IX (CAIX) is a hypoxia-induced protein that is highly expressed in numerous human cancers. However, the molecular mechanisms involved in CAIX and human cervical cancer metastasis remain poorly understood. In this study, CAIX overexpression in SiHa cells increased cell migration and epithelial-to-mesenchymal transition (EMT). Silencing CAIX in the Caski cell line decreased the motility of cells and EMT. Furthermore, the RNA-sequencing analysis identified a target gene, bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB4), which is influenced by CAIX overexpression and knockdown. A positive correlation was found between CAIX expression and PFKFB4 levels in the cervical cancer of the TCGA database. Mechanistically, CAIX overexpression activated the phosphorylation of extracellular signal-regulated kinases (ERKs) to induce EMT and promote cell migration. In clinical results, human cervical cancer patients with CAIX^high/PFKFB4^high expression in the late stage had higher rates of lymph node metastasis and the shortest survival time. Our study found that CAIX overexpression increases PFKFB4 expression and EMT, promoting cervical cancer cell migration. CAIX could contribute to cervical cancer cell metastasis and its inhibition could be a cervical cancer treatment strategy.

Keywords: CAIX; PFKFB4; cervical cancer; metastasis.

Figure 1

CAIX overexpression and knockdown in SiHa and Caski cell lines, respectively. (A) Endogenous CAIX protein levels were detected using Western blot analysis in human cervical cancer cell lines. (B) SiHa cells expressing low CAIX were transfected with a vector control (pcDNA 3.0) or CAIX-expressed vector (pcDNA-CAIX) for generating stably transfected clones (SiHa/pcDNA and SiHa/CAIX) after G418 selection. Caski cells expressing high CAIX were transiently transfected with scramble siRNA or CAIX siRNA. CAIX protein levels were detected through Western blot. (C) Growth curves of SiHa and Caski cells with or without CAIX expression was examined using the MTT assay. (D) Cell cycle profiles of transfected SiHa and Caski cells were determined using flow cytometry.

Figure 2

CAIX regulated cell migration and epithelial–mesenchymal transition in SiHa and Caski cells. (A) After CAIX overexpression or CAIX-silencing, cell motility was assessed by the wound healing assay and (B) Boyden chamber assay. Scale bar, 100 μm. (C) EMT proteins were detected using immunofluorescence staining. Scale bar, 50 μm. (D) The protein levels of E-cadherin and vimentin were detected by using Western blot assay. (E) The mRNA levels of E-cadherin and vimentin were detected by using real-time PCR. (*, p < 0.05).

Figure 3

PFKFB4 is involved in CAIX-regulated cervical cancer migration. (A) Summary of mRNA array for CAIX overexpression in the SiHa cell line or CAIX-silencing in the Caski cell line according to log2 fold change. (B,C) The protein level and RNA level were detected by real-time PCR and Western blot. (D,E) After PFKFB4 overexpression in the SiHa cells or silencing in the Caski cells, Western blot and Boyden chamber assay were used for analysis. Scale bar, 100 μm. (F,G) After PFKFB4 knockdown in SiHa with stable CAIX overexpression, Boyden chamber assay and Western blot were used for analysis. Scale bar, 100 μm. (H) PFKFB4 knockdown in Caski cells and analysis by Western blot. (I) An association between CAIX and PFKFB4 mRNA levels in the TCGA database. (*, p < 0.05, compared to pcDNA or Scramble siRNA; #, p < 0.05, compared to CAIX).

Figure 4

The MEK/Raf/ERK signaling pathways are crucial for CAIX-induced cell migration and EMT. (A) The levels of total and phosphorylated MEK, Raf, and ERK1/2 in SiHa and Caski cells with or without CAIX expression were determined using Western blot analyses. b-Actin was used as a loading control. (B) After knockdown PFKFB4 expression, total and phosphorylated ERK1/2 expression was determined using Western blot analyses. (C,D) Boyden chamber assay and Western blot was used to analyze the migration and protein level of PD98059 treating in SiHa cells with CAIX overexpression. Scale bar, 100 μm. (*, p < 0.05, compared to pcDNA or Scramble siRNA; #, p < 0.05, compared to CAIX).

Figure 5

Clinical late-stage and lymph node metastasis are with CAIX^high/PFKFB4^high in cervical cancer patients. (A) The association of the combined expression of CAIX and PFKFB4 with the clinical stage (I–IV) or (B) lymph node metastasis (N0–N1) in patients with cervical cancer patients. Grouped by CAIX^high/PFKFB4^high, CAIX^high/PFKFB4^low or CAIX^low/PFKFB4^high, and CAIX^low/PFKFB4^low. (C) Kaplan–Meier curves for overall cervical cancer patient survival, grouped by CAIX^high/PFKFB4^high, CAIX^high/PFKFB4^low or CAIX^low/PFKFB4^high, and CAIX^low/PFKFB4^low. (D) Schematic summary of the CAIX-PFKFB4-EMT signaling pathway.