Transcription factor PDX-1 in human colorectal adenocarcinoma: A potential tumor marker?

Abstract

AIM: To examine the expression of pancreatic duodenal homeobox-1 (PDX-1) transcription factor in human colorectal cancer.

METHODS: RT-PCR, Western blotting, and immuno-histochemistry were performed to determine the expression pattern of transcription factor PDX-1 in primary colorectal tumor, hepatic metastasis, and benign colon tissue from a single patient.

RESULTS: The highest PDX-1 transcription levels were detected in the metastasis material. Lower levels of PDX-1 were found to be present in the primary tumor, while normal colon tissue failed to express detectable levels of PDX-1. Western blot data revealed a PDX-1 expression pattern identical to that of mRNA expression. Immunohistochemistry confirmed high metastasis PDX-1 expression, lower levels in the primary tumor, and the presence of only traces of PDX-1 in normal colon tissue.

CONCLUSION: These data argue for further evaluation of PDX-1 as a biomarker for colorectal cancer.

Key Words: Colorectal cancer; Pancreatic duodenal homeobox-1; Tumor marker; Transcription factor; Diagnostics

INTRODUCTION

Figure 1 Western blotting results for PDX-1 in the primary tumor, benign colon tissue, and omental metastasis.

Figure 2 RT-PCR results for PDX-1 in the primary tumor, omental adenocarcinoma, and benign colon tissue.

Figure 3 Quantitative RT-PCR results for PDX-1 in the primary tumor, omental metastasis, and benign colon tissue.

Figure 4 HE staining and immunohistochemistry for PDX-1 in the primary tumor, omental metastasis, and benign colon tissue.

In 2008, colorectal cancer is projected to be the third leading cause of cancer-related mortality in USA with an estimated 148 000 new cases and 50 000 deaths[1]. At least 40% of patients with colorectal cancer will experience metastases at some time during their illness[2]. Early detection of disease can improve prognosis, and survival varies significantly between patients with early-stage tumors and patients with metastases[3,4]. A number of studies have shown decreased mortality in populations undergoing colorectal cancer screening[5-8]. There is an increasing demand for colon cancer tumor markers for risk assessment and early diagnosis[9,10].

Pancreatic duodenal homeobox 1 (PDX-1) is a transcription factor with a critical role in pancreatic development[11]. PDX-1 regulates pancreatic cell proliferation and differentiation, and increased expression of this transcription factor has been described in human pancreatic adenocarcinoma and cell lines[12,13]. We recently found increased PDX-1 expression in benign tissues and malignant tumors from patients with pancreas, breast, colon, prostate, and renal cancers[14]. This indicates a possible role of PDX-1 as a tumor marker in patients with these malignancies.

In this report, levels of PDX-1 expression were quantified in a primary colorectal tumor, a metastasis, and in benign tissue from a single patient. Of particular interest were the expression pattern of PDX-1 and its potential use as a tumor marker in colorectal cancer.

MATERIALS AND METHODS

Samples

A 46-year-old male patient presented with a right-sided colorectal adenocarcinoma metastatic to the peritoneum and greater omentum. The patient underwent chemotherapy with capecitabine and oxaliplatin from August 2003 to February 2004 and right hemicolectomy in June 2004 for tumor-related bowel obstruction. Samples from the primary cecal tumor, omental metastases, and macroscopically normal colon mucosa from the distal end of the right hemicolectomy specimen (transverse colon) were collected during surgery (n = 16).

Immunohistochemistry

Tissues were fixed in 4% paraformaldehyde overnight at 4°C. Tissue processing, section preparation, and H&E staining were performed as described previously[14].

Western blot analysis

Western blotting was performed as described previously[15].

RNA preparations and RT-PCR

All samples were snap-frozen in liquid nitrogen. RNA was prepared according to procedures described in TRIzol Reagent manual (Cat. NO. 15596-026/-018). RT reactions were carried out according to the protocol of SuperScript III First-Strand Synthesis System (Invitrogen Cat. No. 18080-051). PCR products were loaded on a 1.5% agarose gel and visualized and quantified by ethidium bromide staining using an UVP imaging system (UVP, Upland, CA).

RESULTS

As shown in Figure 1 (Western blot), high PDX-1 protein levels were found in the metastasis and the benign colon mucosa distant from the tumor. RT-PCR results in Figures 2 and 3 show that the highest PDX-1 mRNA levels were detected in the metastasis. Significant but lower levels were present in the primary tumor, while normal colon tissue had close to undetectable levels of PDX-1 mRNA. Immunohistochemistry (Figure 4) confirmed the high PDX-1 expression in the metastasis, the lower levels in the primary tumor, and the traces of PDX-1 in normal tissue.

DISCUSSION

PDX-1 regulates pancreatic cell proliferation and differentiation[16-19]. Its aberrant expression in a number of human malignant tumors suggests a potential as a molecular marker. Among these malignancies, colorectal cancer is a common and frequently fatal disease for which screening programs are already being used in the United States[20]. Colonoscopy, the most accurate screening method, can cause significant patient discomfort and has risks of perforation and bleeding[21]. Perhaps for these reasons, participation in colorectal cancer screening programs is low[22]. Hence, safer and more acceptable screening methods are needed.

An important finding of this study is that malignant tissue was found to have significantly higher PDX-1 expression than normal colon mucosa outside the tumor. We have previously shown PDX-1 expression in 10 colon cancer specimens to be significantly elevated in both the nucleus and cytoplasm of malignant cells, compared to lower levels found in benign tissues[14]. In that study, six samples of colon tissue from colon cancer patients taken from sites outside the primary tumor were examined and found to express increased PDX-1 levels, although lower than that of tumors. In contrast, our current study showed colon mucosa distant from the primary tumor had nearly undetectable PDX-1 expression. These differences could be due to variations in the benign tissue sample distance from the primary tumor. Unfortunately, this was not recorded in our previous study and hence an accurate comparison is not possible.

Another significant observation is that, despite high levels of mRNA, PDX-1 protein levels are low in the primary tumor. This is consistent with posttranscriptional control of PDX-1 expression, which has been shown to occur in the pancreas[18]. In contrast, metastatic tissues retain high levels of both mRNA and protein expression. Although Western blotting showed lower levels of protein in the primary tumor compared to benign mucosa, immunohistochemistry did confirm high expression of PDX-1 protein in tumor cells. Hence, PDX-1 protein is present in tumor cells and normal colonocytes, which are excreted in feces. This is a prerequisite for fecal screening for colorectal cancer. Interestingly, others have shown an upregulation of PDX-1 expression in colonic hyperplastic polyps and adenomas, the latter being precursors of adenocarcinomas[23].

Although the etiology of aberrant PDX-1 expression by colorectal cancer cells is unknown, a clue for this comes from its association with caudal homeobox 2 (Cdx-2). The genes for PDX-1 and Cdx-2 are closely linked on chromosome 5[24]. Cdx-2 is expressed in colon epithelium during embryonic development and has a central role in the differentiation of midgut endoderm[24]. It encodes for a transcription factor that is expressed in the proximal colon[25]. Cdx-2 expression is significantly reduced in colorectal adenocarcinoma proximal to the splenic flexure[26] and during the later stages of colorectal carcinogenesis[27]. In vitro studies show that PDX-1 physically interacts with and inhibits transcriptional activation by Cdx-2[28].

In this report, we have found expression of PDX-1 in a colorectal adenocarcinoma, its metastases, and macroscopically normal colonic mucosa from the same patient. This indicates a potential for use of this transcription factor as a molecular marker for colorectal cancer. Levels of PDX-1 in primary tumors and metastases from a large number of patients with and without colorectal cancer would need to be measured to confirm these observations. In addition, the levels of PDX-1 in stool samples from these patients need to be determined. This case report is an initial observation that PDX-1 expression could be indicative for colorectal carcinoma. A prospective study would be required to further evaluate its impact.

COMMENTS

Background

Colon cancer is a major cause of cancer-related morbidity and mortality and new diagnostic markers could improve the results of screening. Pancreatic duodenal homeobox-1 (PDX-1) is a transcription factor that regulates differentiation and proliferation. Increased PDX-1 levels have been found in colorectal adenocarcinoma compared to normal colon mucosa from a single patient.

Research frontiers

New molecular markers that could improve the accuracy of colorectal cancer screening are being sought. Development of molecular markers aims at developing non-invasive screening methods for colorectal cancer.

Innovations and breakthroughs

Detection of colorectal cancer-specific mutations in stool has been examined but is laborious and expensive. New molecular markers that will improve the efficiency and accuracy of non-invasive screening are needed.

Applications

Demonstrating overexpression of PDX-1 in the colon of patients with colorectal cancer is the first step in evaluating this molecule as a marker for colorectal cancer. If this observation is confirmed in a large sample of colorectal cancer patients, PDX-1 could prove valuable as a colorectal cancer marker.

Terminology

PDX-1 is a transcription factor essential for normal pancreatic organogenesis. Aberrant PDX-1 expression by a number of malignant tumors has been described.

Peer review

This study is very interesting. It suggests the possibility of PDX-1 as a biomarker for early diagnosis of colorectal cancer, so further study is needed to evaluate this potential.