Differential expression of colon cancer associated transcript1 (CCAT1) along the colonic adenoma-carcinoma sequence

Abstract

Background: The transition from normal epithelium to adenoma and, to invasive carcinoma in the human colon is associated with acquired molecular events taking 5-10 years for malignant transformation. We discovered CCAT1, a non-coding RNA over-expressed in colon cancer (CC), but not in normal tissues, thereby making it a potential disease-specific biomarker. We aimed to define and validate CCAT1 as a CC-specific biomarker, and to study CCAT1 expression across the adenoma-carcinoma sequence of CC tumorigenesis.

Methods: Tissue samples were obtained from patients undergoing resection for colonic adenoma(s) or carcinoma. Normal colonic tissue (n = 10), adenomatous polyps (n = 18), primary tumor tissue (n = 22), normal mucosa adjacent to primary tumor (n = 16), and lymph node(s) (n = 20), liver (n = 8), and peritoneal metastases (n = 19) were studied. RNA was extracted from all tissue samples, and CCAT1 expression was analyzed using quantitative real time-PCR (qRT-PCR) with confirmatory in-situ hybridization (ISH).

Results: Borderline expression of CCAT1 was identified in normal tissue obtained from patients with benign conditions [mean Relative Quantity (RQ) = 5.9]. Significant relative CCAT1 up-regulation was observed in adenomatous polyps (RQ = 178.6 ± 157.0; p = 0.0012); primary tumor tissue (RQ = 64.9 ± 56.9; p = 0.0048); normal mucosa adjacent to primary tumor (RQ = 17.7 ± 21.5; p = 0.09); lymph node, liver and peritoneal metastases (RQ = 11,414.5 ± 12,672.9; 119.2 ± 138.9; 816.3 ± 2,736.1; p = 0.0001, respectively). qRT-PCR results were confirmed by ISH, demonstrating significant correlation between CCAT1 up-regulation measured using these two methods.

Conclusion: CCAT1 is up-regulated across the colon adenoma-carcinoma sequence. This up-regulation is evident in pre-malignant conditions and through all disease stages, including advanced metastatic disease suggesting a role in both tumorigenesis and the metastatic process.

Figure 1

CCAT1 expression analyzed by in-situ hybiridization.In situ hybrdization of CCAT1 was analyzed in normal colonic tissue (a), normal mucosa adjacent to the primary tumor site (b) and in primary adenocarcinoma of the colon (c). Samples a and b were obtained from the same patient (T877). The probe sequence and topographic location within the CCAT1 gene are outlined in the lower part of the figure.

Figure 2

A logarithmic scale of relative quantity (RQ) of CCAT1 expression in the adenoma-carcinoma sequence. The log RQ of CCAT1 expression is shown for every tissue type: inflamed colonic tissue, normal colon adjacent to the tumor site (Normal Adj.), adenomatous polyps, primary tumor tissue, lymph node metastasis (LN-Mx), liver metastasis (Liver-Mx), and peritoneal metastasis (Peritoneal-Mx). Mean RQ and standard deviation are shown in the lower part of the figure. Mean CCAT1 expression was compared to normal colon. * p < 0.09, ** p = 0.012, ***p = 0.0048, ****p < 0.0001.

Figure 3

A logarithmic scale of relative quantity (RQ) of CCAT1 expression in lymph nodes. The log RQ of CCAT1 expression is shown for benign lymph nodes (white rectangle), lymph nodes without metastasis by histopatholgical examination (gray rectangle), and for lymph nodes with metastasis from the same colon cancer patients (black rectangle). The colon cancer cell line HT29 was used as positive control and a commercially available normal lymphatic tissue was used as a calibrator (LN_NN-Ambion).