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Year : 2011  |  Volume : 54  |  Issue : 2  |  Page : 273-278
Expression of erythropoietin and its receptor increases in colonic neoplastic progression: The role of hypoxia in tumorigenesis

1 Department of Pathology, Lafayette General Medical Center, Lafayette, LA, USA
2 Department of Pathology, Comenius University School of Medicine, Bratislava, Slovakia
3 Institute of Medical Biology and Genetics, Comenius University School of Medicine, Bratislava, Slovakia
4 Department of Anatomic Pathology and Women's Oncology, Moffitt Cancer Center, Tampa, FL, USA
5 Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, PA, USA

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Date of Web Publication27-May-2011


Background: Tissue hypoxia is a characteristic patho-physiologic property of colorectal cancer. This process may also add to a therapeutic problem of solid tumor resistance to chemo- and radiation therapy. Erythropoietin (Epo) expression is induced by tissue hypoxia. Acting via its receptor (EpoR), Epo inhibits apoptosis of erythroid cells and has been shown to rescue neurons from hypoxic damage. Increased Epo and EpoR expression has been recently described in human breast, renal and cervical carcinoma. Given the characteristic tumor diathesis present in majority of colorectal cancers, we examined whether Epo signaling may play a role in colonic neoplastic progression. Materials and Methods: Expression of Epo and EpoR was examined using immunohistochemistry in 24 cases of primary colorectal and metastatic adenocarcinomas versus adenomas and normal colonic mucosa. Immunohistochemical stains were evaluated semiquantitatively based on a four-tiered scale. Based on the combination of extent and intensity of immunoreactivity, an immunostaining score (0-300) was determined for each sample. Expression of Epo and EpoR protein and mRNA was examined using Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively, in both normal colonic tissue and carcinoma specimens in five cases. Results: Epo expression was sequentially increased in normal colonic mucosa (8.3 ± 5.6, mean ± SEM), adenoma (26.4 ± 9.1), primary carcinoma (96.1 ± 12.8) and metastatic carcinoma (122 ± 51.3). EpoR expression was also sequentially increased in normal colonic mucosa (22.3 ± 11.8), adenoma (108.7 ± 24.2), primary carcinoma (178.7 ± 16.6) and metastatic carcinoma (220 ± 58.3) (P< 0.05 for all results). Epo and EpoR showed enhanced expression in the areas adjacent to ischemia/necrosis. Western blot and RT-PCR analysis revealed increased EpoR protein and mRNA levels in carcinoma compared to normal mucosal colon specimens. Focal stromal Epo and EpoR immunoreactivity was present in 10 and 12 cases, respectively. Conclusions: The uniform increase in the expression of Epo and EpoR along the colonic neoplastic sequence and further increase in ischemic/necrotic areas indicates that the Epo signaling pathway is an important component in colon carcinogenesis including possible epithelial-stromal interactions.

Keywords: Colon cancer progression, erythropoietin receptor, erythropoietin, hypoxia

How to cite this article:
Gombos Z, Danihel L, Repiska V, Acs G, Furth E. Expression of erythropoietin and its receptor increases in colonic neoplastic progression: The role of hypoxia in tumorigenesis. Indian J Pathol Microbiol 2011;54:273-8

How to cite this URL:
Gombos Z, Danihel L, Repiska V, Acs G, Furth E. Expression of erythropoietin and its receptor increases in colonic neoplastic progression: The role of hypoxia in tumorigenesis. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Aug 4];54:273-8. Available from: http://www.ijpmonline.org/text.asp?2011/54/2/273/81591

   Introduction Top

Hypoxic regions are typically present in solid tumors. Tumor hypoxia results from a variety of factors including deteriorating diffusion geometry, disturbed microcirculation and tumor related anemia. Tumor cell proliferation also results in increased oxygen consumption. The low level of oxygen in human cancers has been documented with micro-electrodes [1] and is considered to be a therapeutic problem, as it makes solid tumors resistant to radiation and chemotherapy.

Hypoxia induces the expression of a number of genes that enable cells to adapt to this stress factor; some of these genes are thought to play a role in carcinogenesis and tumor progression. Several intracellular factors are involved in the metabolic adaptation of the cell to hypoxia, such as glycolytic enzymes, glucose transporters and transcription factors such as hypoxia-inducible factor-1 (HIF-1). An immediate consequence of hypoxia is the stabilization of HIF-1, which in turn stimulates transcription of numerous genes important for tumor cell survival and tumor progression. [2] HIF-1 is a heterodimer composed of one of three alpha subunits (HIF-1α, HIF-2α and HIF-3α) and one of the HIF-1β subunits . Thus, there are three HIF-1 molecular isoforms; only HIF-1α plays a general role in hypoxic signaling. [3] HIF-1α is constitutively expressed, but under aerobic conditions it is rapidly degraded by the ubiquitin-26S proteasome pathway so that HIF-1α levels are almost non-detectable. [4] The increased activity of HIF-1 under hypoxia is achieved by stabilization of the alpha subunit. [5]

Growth factors, such as vascular endothelial growth factor (VEGF), act locally to ensure the survival of the tumor by increasing neovascularization. [6] Although angiogenesis is stimulated within tumors, the resulting microvasculature is functionally and structurally abnormal and tumor cell survival is dependent on metabolic adaptation to hypoxia.

Finally, Epo helps in the adaptation of the whole organism to hypoxia by increasing red blood cell production. However, despite these protective mechanisms, hypoxia may irreversibly damage cells inducing cell death. [7]

Epo is a 34-kDa glycoprotein hormone physiologically produced in the kidney and fetal liver. It acts via EpoR to induce differentiation, stimulate growth and prevent apoptosis of red blood cell precursors, activating Bcl-2 and Bcl-XL. [8] EpoR is a member of the cytokine receptor family. It has no intrinsic kinase activity; it binds and activates intracellular tyrosine kinase to elicit its mitogenic signal. [8],[9] In erythroid cells, binding of Epo to EpoR triggers the signaling pathway that includes multimerization of the receptor upon ligand binding, activation of Mitogen-activated protein kinase (MAPK) cascade, and phosphorylation and activation of Stat5. [10] Epo has been shown to be a cytoprotective agent as it was found to be a potent inhibitor of neuronal apoptosis induced by ischemia in vitro and in vivo. [11] Epo gene expression is primarily modulated by tissue hypoxia mediated by HIF-1. [8],[12],[13]

Recently, the physiological production of Epo in organs other than the kidney and fetal liver has been reported, including astrocytes, breast epithelial cells and human female reproductive organs, including the uterus. [14],[15],[16] EpoR is also expressed by a variety of cell types, such as endothelial cells, [17] neurons [18] and mammary epithelial cells, [15],[19] suggesting a wider biologic role for Epo signaling unrelated to erythropoiesis. Additionally, this pathway may be involved in neoplastic processes as several carcinoma cells were shown to express high levels of EpoR/Epo mRNA and protein, and exposure to exogenous Epo (rHuEpo) can activate EpoR signaling pathways and stimulate tyrosine phosphorylation, DNA synthesis and proliferation in some tumor cells. [19],[20],[21]

The goal of our study was to examine the role of hypoxia and the Epo pathway as angiogenic modulators in colon cancer neoplastic progression.

   Materials and Methods Top

Cell Culture and Treatments

Caco3, HCD116 and DLD-1 human colonic adenocarcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to ATCC directions. Forty-eight hours before hypoxic treatment, the cells were switched to serum-free medium. Hypoxic treatment of cells was performed in an enclosed chamber (Billups-Rothenberg Inc., Del Mar, CA, USA) flushed with a premixed gas mixture (1% O 2 , 5% CO 2 , 94% N 2 ) for 24 hours. The O 2 concentration in the culture medium was confirmed to be 1% using an oxygen-sensitive electrode (World Precision Instruments, Sarasota, FL, USA), and this oxygen concentration was maintained throughout the course of the experiments.

Clinical Samples

Study protocols involving human material were approved by the University of Pennsylvania Institutional Review Board. Fresh tissue samples from five exophytic colonic adenocarcinomas and adjacent benign mucosa were obtained and snap frozen. The presence of carcinoma was subsequently determined on hematoxylin and eosin (H&E) stained slides on sections taken adjacent to the specimens. Tissue samples were stored at -80°C until used for protein extraction and RNA isolation.

Fifty-three cases of colorectal adenocarcinoma were selected from the Surgical Pathology files of the University of Pennsylvania Medical Center. H&E stained slides of all cases were reviewed and the diagnoses confirmed with the assignment of TNM status.


Immunohistochemical assays were performed on formalin-fixed, paraffin-embedded sections representing benign, adenomatous, carcinoma and metastatic carcinoma specimens. Sections of 5 μm thickness were cut and deparaffinized in xylene and rehydrated in graded alcohols. Slides were steamed in 0.01 M sodium citrate buffer (pH 7.0; Biomeda Corp., Foster City, CA, USA). Endogenous peroxidase was blocked by 3% hydrogen peroxide in methanol for 20 min. Endogenous biotin was blocked by DAKO's Biotin blocking system (DAKO Corp., Carpinteria, CA, USA) according to the manufacturer's specifications. Slides were incubated with antibodies against Epo [rabbit polyclonal, H-162 (1:200 dilution); Santa Cruz Biotechnologies, Santa Cruz, CA, USA] and EpoR [rabbit polyclonal, C-20 (1:400 dilution); Santa Cruz Biotechnologies] overnight at 4°C. For both Epo and EpoR, the Dako EnVision+ System HRP was used. A negative control was obtained in each case by omitting the primary antibody. Slides of human fetal liver were used as positive controls. The specificity of the immunostain was further confirmed using A2780 ovarian carcinoma xenografts, in which the expression of EpoR was downregulated using specific shRNA vectors. Compared to parent A2780 cell xenografts, which showed strong diffuse EpoR expression [Figure 1], xenografts of cells transfected with EpoR shRNA showed significantly reduced immunostaining [Figure 2].
Figure 1: EpoR expression in A2789 ovarian carcinoma xenografts (IHC, ×400)

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Figure 2: EpoR expression in A2789 ovarian carcinoma xenografts transfected with EpoR shRNA (IHC, ×400)

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Interpretation of Immunohistochemical stains

Immunohistochemical stains were interpreted semiquantitatively by assessing the intensity and extent of staining using a four-tiered scale. [22] The percentage of weakly, moderately, and strongly staining cells was determined and a staining score was calculated as follows: score (maximum 300) = 1 × the percentage of weakly staining cells + 2 × the percentage of moderately staining cells + 3 × the percentage of strongly staining cells. The presence or absence of stromal staining was noted independently from this scoring system.

Quantitative Real-Time RT-PCR assay

RNA was extracted from fresh, snap-frozen tissue samples and colonic adenocarcinoma cell lines using the RNeasy Mini kit (Quiagen, Valencia, CA, USA). For reverse transcriptase-polymerase chain reaction (RT-PCR), 1 μg of total RNA per sample was reverse transcribed to cDNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR was performed using the iCycler Real-Time Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Amplification of specific PCR products was performed in a total reaction volume of 25 μl containing 5 μl cDNA template, sense and antisense primers, dual labeled fluorigenic internal probe and 1X IQ Supermix reagents (Bio-Rad Laboratories). Dual labeled nonextendable probes labeled with hexachlorofluorescein (HEX) at the 5′ end and with Black Hole Quencher -1 (BHQ) at the 3′ end were used for detection of Epo and EpoR. For the endogenous control beta-actin, the probe was labeled with 6-carboxyfluorescein (6FAM) at the 5′ end and with BHQ at the 3′ end. Primers and probes for Epo (sense: 5′-TGGAAGAGGATGGAGGTCGG-3′; antisense: 5′-GCTGGGAAGAGTTGACCAACAG-3′; probe, 5′-HEX-CCGCAGGACAGCTTCCGACAGCAG-BHQ-3′), EpoR (sense, 5′-CCTGACGCTCTCCCTCATCC-3′; antisense, 5′-GCCTTCAAACTCGCTCTCTGG-3′; probe, 5′-HEX-TCCTGGTGCTGCTGACCGTGCTCG-BHQ-3′) were designed using the Beacon Designer software (version 2.1, Premier Biosoft International, Palo Alto, CA, USA). Primers and probes for Epo, EpoR and beta-actin were designed to span an intron, when possible, to avoid amplification of genomic DNA. Amplifications were performed at 95°C for 3 min and for 40 cycles of 30 seconds at 95°C and 30 seconds at 60°C. Change in gene expression relative to the beta-actin endogenous control was determined by the following formula: Fold change = 2 - Δ(ΔCT), where ΔCT = CTtarget - CTbeta-actin RNA, and Δ(ΔCT) = ΔCTtreated - ΔCTcontrol (CT, threshold cycle).

Western Blotting

Protein extracts from biopsy samples and human adenocarcinoma cell lysates were normalized for protein. Fifty microgram proteins from each sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. Proteins were detected using rabbit polyclonal antibodies to Epo (H-162, 1:500 dilution, Santa Cruz Biotechnology, Inc.) and EpoR (C-20, 1:1,500 dilution, Santa Cruz Biotechnology, Inc.). As a loading control, a mouse monoclonal antibody to β-actin (clone AC-74, 1:10,000 dilution, Sigma, St. Louis, MO, USA) was used. Membranes were incubated with the primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical Analysis

The Wilcoxon signed rank test was used for the comparison of median Epo and EpoR immunohistochemical expression levels in benign, adenomatous and cancerous (including metastatic) tissue.

   Results Top

Hypoxia-induced Expression of Epo and EpoR in Human Colonic Adenocarcinoma Cell Lines and Human Colonic Adenocarcinomas

Using real-time RT-PCR, Caco-2, HCT-116 and DLD-1 human colonic adenocarcinoma cell lines cultured under normoxia (21% O 2 ) or hypoxia (1% O 2 ) and human colonic adenocarcinomas and adjacent normal mucosa were analyzed for Epo and EpoR mRNA expression. Abundant expression of Epo and EpoR mRNA was detected in all cell lines regardless of normoxic or hypoxic conditions. On comparing human carcinoma tissue with normal mucosal samples, we found a significant increase of EpoR mRNA expression in four of the five cases compared to adjacent normal tissue [Figure 3]. No difference was found in Epo mRNA expression when carcinoma tissue with normal mucosa was compared.
Figure 3: EpoR mRNA expression levels comparing normal (N) and carcinoma (T) mucosal scrapings

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Western blot analysis using Epo and EpoR antibodies revealed the presence of Epo and EpoR protein in all three carcinoma cell lines. Hypoxia induced no increase in Epo expression in any of the cell lines, whereas the HCT-116 cell line showed markedly increased EpoR expression under hypoxic conditions (band at ~66 kDa corresponding to the EpoR protein) [Figure 4].
Figure 4: Western blot for EpoR, colon carcinoma cell lines

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We examined the expression of EpoR protein using Western blotting in clinical samples of colonic adenocarcinoma and adjacent normal mucosal samples. All five carcinoma samples examined showed increased expression of a specific immunoreactive band at 66 kDa [Figure 5] in carcinoma specimens.
Figure 5: Western blot for EpoR, mucosal tissue (N - normal, T - carcinoma)

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To further investigate the potential role of the EpoR signaling pathway in colonic adenocarcinoma, we examined the expression of Epo and EpoR by immunohistochemistry in a series of clinical samples of normal colonic mucosa, adenoma and adenocarcinoma (including metastatic adenocarcinomas into lymph nodes or liver).

Cytoplasmic granular immunostaining for Epo was present in all cases of adenoma and adenocarcinoma but only focally in few cases of normal colonic mucosa. Epo immunostaining was significantly increased along the progression of adenoma-adenocarcinoma sequence [Figure 6] and [Figure 8] Furthermore, the Epo immunostaining showed increased expression pattern along the leading edge of the tumor. There was no significant difference in Epo immunoexpression when carcinoma samples were compared based on their grade and stage (data not shown). In 10 samples, we found focal stromal Epo immunoreactivity.
Figure 6: Epo immunoexpression in benign, adenomatous, cancer and metastatic cancer tissue

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Cytoplasmic and/or membrane EpoR immunostaining was seen in all cases of adenoma and adenocarcinoma in contrast to normal colonic mucosa, which showed only focal EpoR expression in rare cases. EpoR staining, similar to Epo staining, was significantly increased along the progression of adenoma-adenocarcinoma-metastatic adenocarcinoma sequence [Figure 7] and [Figure 8]. Similar to Epo, there was no significant difference in EpoR immunoexpression when carcinoma samples were compared based on their grade and stage (data not shown). In 12 samples, we found focal stromal EpoR immunoexpression.
Figure 7: Epo immunoexpression in benign, adenomatous, cancer and metastatic cancer tissue

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Figure 8: Immunohistochemical detection of Epo (a, c, e, g) and EpoR (b, d, f, h) in normal colonic mucosa (a, b, x100), adenoma (c, d, x200), invasive carcinoma (e, f, x200) and metastatic carcinoma (g, h, x200)

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   Discussion Top

It is well known that some solid tumors such as renal cell carcinoma, Wilms' tumor, cerebellar hemangioblastoma and others can be associated with paraneoplastic polycythemia. Interestingly, this phenomenon occurs in tumors arising in anatomical sites in which Epo is normally expressed in low levels. Prior studies have demonstrated that cultured human breast, cervix and endometrial carcinoma cells as well as the corresponding human carcinoma tissues express high levels of Epo and EpoR protein and mRNA. [19] Epo was shown to stimulate breast cancer cell proliferation and enhance the resistance of cervical cancer cells to chemotherapeutic drugs. Evidence suggests that increased Epo and EpoR expression paralleled with increased serum Epo levels are not merely the consequence of the hypoxic adaptation of the constantly proliferating cancer. Nevertheless, hypoxia is invariably present in solid tumors [23] and through HIF-1 activation induces a variety of gene responses to allow cells to adapt to this hypoxic stress and ultimately progress.

One of the key adaptations for tumor survival is the capability of neoangiogenesis. It is a well-orchestrated process that plays an important role in colon carcinoma progression. [24] Induction of VEGF is an essential step in tumor angiogenesis, and HIF-1 is known to be a key mediator in this process. [25],[26] Although colon carcinoma usually follows one of the two classical progression pathways including microsatellite instability and chromosomal instability, recent findings suggest that genomic instability may also be found in microsatellite instable colon cancers. [27] Several findings showed that there are important cross-talks not only between the two major progression pathways but also between the genomic instability pathway and angiogenesis, and VEGF may be induced through both HIF-dependent and HIF-independent pathways. [27]

In the current study, we demonstrated the presence of hypoxia-inducible Epo signaling, suggesting a role in the progression of human colonic adenocarcinomas. We have shown that there is abundant Epo and EpoR expression in all examined human adenocarcinoma cell lines regardless of normoxic or hypoxic conditions. When we examined human carcinoma and normal mucosal samples, we found that the level of EpoR mRNA was significantly increased in carcinoma samples, with no corresponding change in Epo mRNA level. There was no significant increase of Epo protein in carcinoma cell lines. However, we found increased EpoR protein expression in HCT-116 carcinoma cell line after hypoxic pretreatment. This cell line is characterized by endogenous beta catenin activation, and hypoxia via Epo signaling seems to play an important role in further activation. We found uniformly increased immunoexpression of EpoR protein in clinical carcinoma samples when compared to adjacent normal colonic mucosa by Western blot analysis.

Immunohistochemical analysis performed on clinical samples revealed that both Epo and EpoR expression were increased along the colonic adenocarcinoma progression. Focal stromal immunoexpression of Epo and EpoR was detected in several cases. Both Epo and EpoR showed enhanced immunoexpression along the leading edge of the adenocarcinoma specimens, which were shown to represent the most hypoxic areas in tumors. [26]

rHuEpo is frequently used in current clinical practice to treat or prevent anemia in patients receiving chemotherapy for various cancers, including colon cancer. However, numerous trials of Epo in cancer patients suggested a negative effect of rHuEpo treatment on patient survival. There was a significantly higher mortality rate in cancer patients treated with Epo as opposed to patients taking placebo. [28],[29] The authors suggest that precaution should be taken when using Epo in patients with EpoR-expressing tumors. The colon cancer cell lines and tissues we examined expressed detectable EpoR. Therefore, our findings might also have important clinical implications.

Epo was found to be a potent ischemia-induced angiogenic factor that acts independent of VEGF under non-malignant conditions. [30] Further findings suggest that rHuEpo administration may be an independent risk factor for retinopathy of prematurity due to its angiogenicity. [31] However, further clinical studies are necessary to elucidate these effects of Epo in both malignant and non-malignant conditions.

In summary, we have demonstrated that the Epo signaling pathway is an important component in colon carcinogenesis including possible epithelial-stromal interactions.

   References Top

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Correspondence Address:
Zoltan Gombos
Department of Pathology, Lafayette General Medical Center, 1214 Coolidge Blvd, Lafayette, LA 70503
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.81591

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

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