Increased hepcidin expression in colorectal carcinogenesis

Abstract

Aim: To investigate whether the iron stores regulator hepcidin is implicated in colon cancer-associated anaemia and whether it might have a role in colorectal carcinogenesis.

Methods: Mass spectrometry (MALDI-TOF MS and SELDI-TOF MS) was employed to measure hepcidin in urine collected from 56 patients with colorectal cancer. Quantitative Real Time RT-PCR was utilised to determine hepcidin mRNA expression in colorectal cancer tissue. Hepcidin cellular localisation was determined using immunohistochemistry.

Results: We demonstrate that whilst urinary hepcidin expression was not correlated with anaemia it was positively associated with increasing T-stage of colorectal cancer (P<0.05). Furthermore, we report that hepcidin mRNA is expressed in 34% of colorectal cancer tissue specimens and was correlated with ferroportin repression. This was supported by hepcidin immunoreactivity in colorectal cancer tissue.

Conclusion: We demonstrate that systemic hepcidin expression is unlikely to be the cause of the systemic anaemia associated with colorectal cancer. However, we demonstrate for the first time that hepcidin is expressed by colorectal cancer tissue and that this may represent a novel oncogenic signalling mechanism.

Figures

Figure 1

Validation of hepcidin expression in urine by MALDI-TOF MS. A: (a) A human urine specimen showing the two dominant forms of hepcidin; hepcidin 20 (m/z 2195.3) and hepcidin 25 (m/z 2793.8). In addition the degradation product of hepcidin 25 hepcidin 22 could also be detected by MALDI-TOF MS (m/z 2440.5); (b) A human urine specimen completely devoid of hepcidin which when spiked with synthetic hepcidin 25 clearly shows a detectable peak at 2795.4 (c); B: Synthetic hepcidin was spiked into a low hepcidin containing urine sample at concentrations between 0-80 ng/mL and analysed by MALDI-TOF MS followed by analysis of the hepcidin 25 peak intensity.

Figure 2

Hepcidin expression is associated with T-stage of colorectal cancer. Expression of hepcidin 20 (m/z 2195) and 25 (m/z 2793) was determined in urine samples of 56 colorectal cancer patients by MALDI-TOF MS, before and after desalting with desalting beads (Desalting MALDI-TOF MS), and SELDI-TOF MS and analysed with respect to T-stage. Expression of hepcidin 20 was not altered with T-stage by any of the three techniques. However, all three techniques demonstrated a significant increase in hepcidin 25 expression in T4 cancers compared to T1. Statistical significance compared to T1 (aP < 0.05); Statistical significance compared to preceding T-stage (cP < 0.05). Error bars denote 2 SEM.

Figure 3

Hepcidin mRNA expression in colorectal cancer tissue. Using Real Time RT-PCR on 34 cancer specimens (C) each with adjacent normal uninvolved mucosa (N) we demonstrate that hepcidin mRNA expression could be detected in 34% of colorectal cancer tissue specimens (10 out of 34). mRNA expression is presented as 1/dCT. Positive control included human liver. Negative control included omission of cDNA.

Figure 4

Cellular localization of hepcidin in colorectal cancer tissue. A: To determine hepcidin cellular localization in colorectal tissue immunohistochemistry was performed with a hepcidin specific antibody (Abcam 31877). (Aa) Human Liver; (Ab) Human Liver, incubated with both hepcidin antibody and immunizing hepcidin peptide; (Ac) Normal colon; (Ad) Normal colon with crypts in cross section; (Ae) Colorectal cancer (x 20); (Af) Colorectal cancer (x 40); (Ag) colorectal cancer (x 20); (Ah) Colorectal cancer (x 40). Boxes denote area subsequently magnified; B: Immunocapture of urinary hepcidin. (Ba) A human urine sample containing hepcidin 20 (m/z 2193.6), 22 (m/z 2438.2) and 25 (m/z 2792.0) was subject to immunocapture with either (Bb) protein G sepharose or with (Bc) protein G-sepharose and a polyclonal anti hepcidin antibody. Immunocaptured proteins were then eluted from the protein G sepharose and analysed by MALDI-TOF MS.