C4.4A is associated with tumor budding and epithelial-mesenchymal transition of colorectal cancer

Abstract

C4.4A is a glycolipid-anchored membrane protein expressed in several human malignancies. The aim of this study was to explore the association between C4.4A expression at the invasion front of colorectal cancer (CRC) and tumor budding, a putative hallmark of cell invasion of CRC. Advanced CRCs (T2-4, n = 126) had a budding count of 3.66 ± 5.66, which was significantly higher than that of T1 early CRCs (1.75 ± 2.78, n = 87). C4.4A-positive CRC specimens showed a larger budding cell number than C4.4A-negative CRC specimens in T1 CRCs, and especially advanced CRCs (9.45 ± 5.83 vs 1.60 ± 3.93). Furthermore, we found a correlation between the percentage of C4.4A-positive cases and budding count in advanced CRC. Multivariate analysis for patients' survival showed that C4.4A was superior to tumor budding as a prognostic factor. With siRNA treatment, C4.4A levels were associated with cell invasion, but not with proliferation, in HCT116 and DLD1 cell lines. An immunohistochemical study in a subset of CRCs showed no relationship between C4.4A and Ki-67 proliferation marker. In vitro assays using HCT116 indicated that C4.4A levels correlated well with epithelial-mesenchymal transition (EMT) with regard to cell morphology and alterations of EMT markers including E-cadherin, vimentin, and partially N-cadherin. We also found that C4.4A expression was significantly associated with loss of E-cadherin and gain of β-catenin in clinical CRC tissue samples. These findings suggest that a tight association between C4.4A and tumor budding may, in part, be due to C4.4A promoting EMT at the invasive front of CRC.

Figure 1

Tumor budding in colorectal cancer (CRC). (A) Tumor budding (arrows) at the invasive front of specimens from early T1 CRC (H&E). (B) Tumor budding (arrows) at the invasive front of specimens from advanced T2–4 CRC (H&E). (C) The budding count of advanced CRC was significantly greater than for T1 CRC (P = 0.004). (D) The number of T1 CRC cases when stratified by budding count: 0, n = 44; 1–4, n = 31; 5–9, n = 9; ≥10, n = 3. (E) The number of advanced CRC cases when stratified by budding count: 0, n = 72; 1–4, n = 18; 5–9, n = 14; ≥10, n = 22. Magnification, (A) ×200; (B) ×200.

Figure 2

Expression of C4.4A and budding count in colorectal cancer (CRC). Strong C4.4A expression was present on the plasma membrane at the invasive front, whereas C4.4A was weakly expressed in the cytoplasm in the superficial or intermediate portion of these specimens. (A) Positive pattern of C4.4A staining at the invasive front of specimens from T1 CRC. (B) Positive pattern of C4.4A staining at the invasive front of specimens from advanced T2–4 CRC. (C,D) The budding count of T1 and advanced CRC with C4.4A positivity is significantly greater than for C4.4A negativity (P = 0.013 and P < 0.0001, respectively). (E) The C4.4A‐positive ratio for budding‐negative was 4.2%, and the ratio for a budding count ≥10 was 77.3%. The C4.4A‐positive ratio significantly correlated with the number of buddings (P < 0.001, Spearman's rank correlation coefficient). (A) Magnification, (I) ×12.5; (II) ×200; (III) ×200. (B) Magnification, (I) ×40; (II) ×200; (III) ×200.

Figure 3

In vitro growth and invasion by C4.4A levels. (A) The C4.4A expression study indicated that HCT116 and LoVo cells had higher levels of expression compared to other colorectal cancer cell lines such as Caco2 and DLD1, by RT‐PCR. (B) Knockdown assessment of C4.4A by siRNA in the HCT116 cell line. (B‐1) HCT116 cells were transfected for 24 h with siRNA using Lipofectamine RNAiMAX. Confirmation of reduced C4.4A mRNA levels was made using RT‐PCR. (B‐2) Proliferation assay showed growth of the HCT116 cell line. There were no significant differences between Neg.Cont. (transfection control of scramble sequence) and siRNAs I, II, and III. (B‐3,4) Invasion assay revealed that siRNAs I, II, and III were significantly less invasive than Neg.Cont. (B‐3) Magnification, ×200. Values are the mean ± SD of three independent experiments. (C) C4.4A transfection assessment using a plasmid in the DLD1 cell line. (C‐1) DLD1 cells were transfected for 24 h with plasmid using Lipofectamine 2000. Confirmation of C4.4A mRNA levels was by RT‐PCR. (C‐2) Proliferation assay showed growth of the plasmid‐containing DLD1 cell line. There were no significant differences between empty vector transfection control (Mock) and C4.4A plasmid‐transfected cells (C4.4A). (C‐3) Invasion assay showed that C4.4A‐transfected cells increased invasion compared to Mock cell lines. Magnification, ×200. Values are the mean ± SD of four independent experiments. N.S., not significant; Parental, a control without transfection.

Figure 4

In vitro studies on epithelial–mesenchymal transition of HCT116 colon cancer cells. (A) Morphological changes of HCT116 cells when treated with siRNA II. The cells were transfected for 48 h with C4.4A siRNA II using Lipofectamine RNAiMAX. Control HCT116 cells showed a fibroblast‐like morphology, but after C4.4A inhibition, they adopted an epithelial cell‐like morphology. Representative photographs at a high density culture and at a low density culture are shown. Magnification, ×100. (B) Cells were collected 24 h after transfection with negative control siRNA or C4.4A siRNA II. Quantitative RT‐PCR showed the mRNA levels of each culture. Values are the mean ± SD of three independent experiments. *Statistical significance. Neg.Cont., transfection control of scramble sequence; siRNA, C4.4A siRNA. (C) C4.4A plasmid and empty vector (pCMV6‐XL5) were introduced into HCT116 cells. Forty‐eight hours after transfection, cells were collected and subjected to RT‐PCR assays. Values are the mean ± SD of three independent experiments. C4.4A, C4.4A plasmid‐transfected cells; Mock, empty vector transfection control.

Figure 5

Immunohistochemistry for C4.4A, E‐cadherin and β‐catenin in clinical CRC samples. Both the superficial portion (A,C,E) and the invasive front (B,D,F) of the cancer body are shown. At the invasive front, the C4.4A protein was translocated onto the plasma membrane (A), E‐cadherin expression was largely reduced (D), and nuclear β‐catenin increased with cytoplasmic accumulation (F). Magnification, ×100.