Thioredoxin-like protein 2 is overexpressed in colon cancer and promotes cancer cell metastasis by interaction with ran

Abstract

Aims: Our previous work identified thioredoxin-like protein 2 (Txl-2) as the target of the monoclonal antibody MC3 associated with colon cancer, but its underlying mechanisms remain poorly understood. Txl-2, a novel thioredoxin (Trx) and nucleoside diphosphate kinase family member, is alternatively spliced and gives rise to three different Txl-2 isoforms. In this study, Txl-2 expression in colon cancer, differential functions for Txl-2 isoforms in cell invasion and metastasis, and the downstream signaling were investigated.

Results: Txl-2 expression was elevated in colon cancer tissues compared to normal colonic tissues, with a high correlation between the histological grade and prognosis. Knockdown of Txl-2 expression significantly inhibited cancer cell motility, and the invasive and metastatic abilities of colon cancer cells. Interestingly, Txl-2 isoforms showed differential effects on cancer cell invasion and metastasis. Cell invasion and metastasis were significantly promoted by Txl-2b but inhibited by Txl-2c, while no obvious effect was observed for Txl-2a. Furthermore, a direct interaction was identified between Txl-2b and Ran, a Ras-related protein, by yeast two-hybrid assay and coimmunoprecipitation. PI3K pathway was found to be a major pathway mediating Txl-2b induced tumor invasion and metastasis.

Innovation: The current study provides a novel biomarker and target molecule for the diagnosis and treatment of colon cancer and provides a novel paradigm to understand how alternative splicing functions in human cancer.

Conclusion: Our findings demonstrate an elevated Txl-2 expression in colon cancer and that Txl-2b promotes cell invasion and metastasis through interaction with Ran and PI3K signaling pathway.

FIG. 1.

Txl-2 expression in colon cancer tissues and its association with patients' survival. (A) Results from immunohistochemical staining. Upper row: well differentiated (left panel), moderately differentiated (middle panel), and poorly differentiated colon cancer tissues (right panel); lower row: moderately differentiated cancer tissues with omental invasion (left panel), metastatic site in lymph node (middle panel), and normal colonic epithelium (right panel). Brown color represents a positive staining for thioredoxin-like protein 2 (Txl-2). (B) Positive expression rates of the three Txl-2 splicing variants from tumor (n=50) and nontumor (n=50) samples as determined by reverse transcription polymerase chain reaction (RT-PCR). *p<0.05, **p<0.01. (C) Kaplan–Meier postoperative survival curve by Txl-2 expression. (I) Txl-2 negative expression group; (II) Txl-2 weak positive expression group; (III) Txl-2 moderate positive expression group; (IV) Txl-2 strong positive expression group.

FIG. 2.

Knockdown of Txl-2 inhibits motility, invasion, and in vivo metastatic properties of colon cancer cells. (A) Txl-2 was knocked down in SW620 Cells with two different Txl-2 siRNA expression vectors and Txl-2 downregulation was evaluated by Western blot. The sum of the densitometric units of all the three bands was counted when the positive Txl-2 expression was quantified. (B) Scratch wounds were made in monolayer cultures of cells after plating (left panel). A slower closure of the wound in the SW620-Txl-2si culture from 1 to 48 h based on the wound width is shown in the graph (right), as compared to the controls. The wound margins are indicated by arrows. (C) Tumor cell invasion capacity was measured with Transwell chambers. Representative image fields of invasive cells on the membrane are shown. Data are represented as normalized invasion (invasion index) relative to the control cells. (D) The ability of stably transfected cells to grow in an anchorage independent environment was assessed by the soft agar assay. The numbers of colonies formed on day 5 were determined. All above images shown are representative of three independent experiments. (E) Metastatic potential in vivo (n=8). H&E staining of lungs and livers was performed in samples from mice that received intravenous tail injections of SW620-Txl-2si1 and scrambled control cells, respectively, with metastatic loci marked by arrows. Number of metastatic loci in lung and liver were counted (middle). (F) Kaplan–Meier survival curves show that knock down of Txl-2 markedly prolonged the survival of the mice bearing SW620 tumors (n=10). *p<0.05, **p<0.01. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Txl-2 splicing variants produce differential effects on cell invasion and metastasis. (A) Schematic representation of the three Txl-2 splicing variants (Txl-2a, b, and c). (B) LoVo cells stably transfected with pEGFP-N3-Txl-2a, b, and c. Txl-2 isoform-EGFP fusion proteins in LoVo cells were detected with the GFP antibody (lane 1 to 3). Lane 4 is the empty vector transfected cells producing only GFP as the negative control. The accompanying image shows the green fluorescence from LoVo cells transfected with the pEGFP-N3-Txl-2b construct. Overexpression of the three Txl-2 isoform-EGFP fusion proteins in LoVo cells resulted in different effects evaluated by in vitro invasion assay (C) and soft agar colonization (D). The results were the mean±SEM from three independent experiments. (E) in vivo tail vein metastasis assay. Metastatic loci are denoted by arrows. (F) Kaplan–Meier survival curves show that overexpression of Txl-2b-EGFP markedly shortened survival of the mice bearing LoVo tumor (n=10). *p<0.05, **p<0.01 relative to vector control transfected cells.

FIG. 4.

The Trx domain is essential for inducing invasion and metastasis of colon cancer cells. (A) Schematic representation of Txl-2b isoforms harboring mutations in either the Trx domain active site WCGPC or the NDPk domain NAVH active site. Numbers below the wild type Txl-2b indicate the amino acid residues. (B) LoVo cells transfected with the different Txl-2b-EGFP mutant variants were subjected to an in vitro invasion assay. Relative invasion index represents fold change of the number of migrated cells relative to the cells transfected by wild-type Txl-2b. (C) Numbers of soft agar colony forming units were also counted. The results were the mean±SEM from three independent experiments. *p<0.05, **p<0.01. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Txl-2b interacts with the small GTPase Ran. (A) Interactions between Txl-2 isoforms and Ran protein were analyzed by the yeast two-hybrid assay. Coding sequences of three Txl-2 splicing variants or Txl-2b lacking RCC1 signature Txl-2b^ΔRCC1 were cloned into pGBKT7, and Ran into pACT2, and then cotransformed into AH109 yeast stain. A positive clone indicates an interaction between Txl-2b and Ran similar to that of the positive control (pGBKT7-53+pGADT7-T), whereas no interaction was detected between Txl-2b^ΔRCC1 and Ran and the negative control (pACT2/Ran+pGBKT7). (B) Flag-tagged Txl-2 isoforms or Txl-2b^ΔRCC1 were cotransfected with HA-tagged Ran into 293T cells. The lysate was subjected to co-IP with anti-HA antibody and probed with anti-Flag antibody, or co-IP with anti-Flag and probed with anti-HA. Interaction was detected only between Txl-2b with Ran but not for other combinations. (C) Interaction between Txl-2b and Ran in SW620 cells was further tested by coimmunoprecipitation using anti-Txl-2 and anti-Ran antibodies. (D–E) Colocalization (yellow) between Txl-2b and Ran was examined by double immune-fluorescence staining for Txl-2 (green) and Ran (red) in colon cancer cells (D) and colon cancer tissues (E).

FIG. 6.

Phospho-Akt mediates Txl-2b-Ran-matrix metalloproteinase (MMP) signaling induced tumor cell invasion. (A) Knockdown of Txl-2 expression significantly decreased the expression of MMP-2, MMP-9, and Phospho-Akt (Ser 473). (B) Overexpression of Txl-2b-EGFP fusion protein selectively upregulated MMP-2, MMP-9, and Phospho-Akt levels, whereas deletion of the RCC1 signature in Txl-2b precluded such upregulation. β-actin served as a control. (C) MMP-2/9 gelatinase activity in cancer cells with depletion of Txl-2 or overexpression of the different Txl-2 isoforms. MMP-2/9 activity is depicted as relative to that of SW620 or LoVo mock transfected cells. The results are the mean±SEM from three independent experiments. *p<0.05, **p<0.01. (D) Induced cell invasion by overexpression of Txl-2b-EGFP fusion protein was significantly blocked by siRNA-Ran, LY294002 (PI3 Kinase inhibitor), and neutralizing antibodies for MMP-2 and MMP-9, but not U0126 (MEK1/2 inhibitor) and negative controls (Control oligo, DMSO and IgG). Deletion of RCC1 signature also abolished the Txl-2b induced cell invasion. The results are the mean±SEM from three independent experiments. *p<0.05, **p<0.01. (E) MMP-9, Phospho-Akt (Ser 473), and total Akt levels were examined by Western blot in LoVo cells overexpressing Txl-2b-EGFP after treatment with siRan, LY294002, anti-MMP-2, and anti-MMP-9.