Erythropoietin signaling promotes invasiveness of human head and neck squamous cell carcinoma

Abstract

Erythropoietin (Epo) is used for managing anemia in cancer patients. However, recent studies have raised concerns for this practice. We investigated the expression and function of Epo and the erythropoietin receptor (EpoR) in tumor biopsies and cell lines from human head and neck cancer. Epo responsiveness of the cell lines was assessed by Epoetin-alpha-induced tyrosine phosphorylation of the Janus kinase 2 (JAK2) protein kinase. Transmigration assays across Matrigel-coated filters were used to examine the effects of Epoetin-alpha on cell invasiveness. In 32 biopsies, we observed a significant association between disease progression and expression of Epo and its receptor, EpoR. Expression was highest in malignant cells, particularly within hypoxic and infiltrating tumor regions. Although both Epo and EpoR were expressed in human head and neck carcinoma cell lines, only EpoR was upregulated by hypoxia. Epoetin-alpha treatment induced prominent JAK2 phosphorylation and enhanced cell invasion. Inhibition of JAK2 phosphorylation reduced both basal and Epo-induced invasiveness. Our findings support a role for autocrine or paracrine Epo signaling in the malignant progression and local invasiveness of head and neck cancer. This mechanism may also be activated by recombinant Epo therapy and could potentially produce detrimental effects in rhEpo-treated cancer patients.

Figure 1

EpoR and Epo immunohistochemistry in HNSCC. (a) Top row: A prominent increase in EpoR staining (brown color) is seen in biopsies with dysplastic (left panel) and invasive carcinoma cells (middle panel) as well as in tumoral vasculature (right panel). Middle row: EpoR immunoreactivity in normal epithelium (left panel), dysplasic epithelium (middle panel), and invasive carcinoma (right panel). Bottom row: Epo immunoreactivity in normal epithelium (left panel), perinecrotic tumor region (middle panel), and invasive carcinoma (right panel). (b) EpoR and Epo expression in lymph node metastasis. EpoR staining is seen in metastatic cancer cells (M) but not in normal lymphocytes (L). Epo staining is most prominent in the malignant cells bordering necrotic regions (N). (c) Correlation of EpoR and Epo immunoreactivity with malignant progression. P values of EpoR staining were calculated for benign and dysplasia (**P < .01), benign and carcinoma (***P < .001), and dysplasia and carcinoma (P > .05). P values of Epo staining were calculated for benign and dysplasia (***P < .001), benign and carcinoma (***P < .001), and dysplasia and carcinoma (*P < .05). Bars indicate median immunostaining score values. NS = not significant.

Figure 2

Differential invasiveness of HNSCC cell lines correlates with higher HIF and EpoR expression. (a) Quantitative real-time PCR analysis of EpoR from O22 and 22B cell cDNA with HPRT as control gene. (b) PCR amplification of Epo from O22 and 22B cell cDNA with HPRT as control gene. (c) Epo immunocytochemistry demonstrated protein expression in normoxic 022 cells and 22B cells, Epo antibody concentration 1:200, and no primary control exhibited no staining. (d) Quantitative real-time PCR analysis of Epo and GLUT3 mRNA levels in O22 cells after 24 hours of treatment with hypoxia. The amount of each mRNA in samples was normalized to the average of HPRT1 mRNA and GUS mRNA in the same sample. (e) Quantitative real-time PCR analysis of Epo and GLUT3 mRNA levels in 22B cells cultured for 24 hours under hypoxia. The amount of each mRNA in samples was normalized to the average of HPRT1 mRNA and GUS mRNA in the same sample. (f) Differential expression of HIF-1 and EpoR expression in O22 and 22B HNSCC cells. For hypoxia treatment, cells were exposed to 1% O₂ for 24 hours. (g) HIF-1 protein levels from (f) were quantified using densitometry. Densitometry values from three independent experiments were graphed and a two-tailed Student's t test was performed to compare relative HIF-1 levels of indicated treatment groups. (*, **, ***P < .05; all treatment groups were compared to HIF-1 levels of normoxic O22 cells). (h) EpoR protein levels from (f) were quantified using densitometry. Densitometry values from three independent experiments were graphed and a two-tailed Student's t test was performed to compare EpoR levels of normoxia- versus hypoxia-treated cells (*P < .05).

Figure 3

Epo signaling mediates invasion in HNSCC cell lines. (a) 22B cells display higher invasive potential as assayed with Matrigel coated Boyden chambers for a 48-hour period under serum-free conditions (*P < .05). (b) Exogenous rhEpo promotes cell invasion of O22 and 22B cells through Matrigel-coated Boyden chambers under serum-free conditions (48 hours; *P < .05). (c) Exogenous rhEpo promotes cell invasion of hepatoma (Hep3B) and prostate (DU145) cancer cell lines through Matrigel-coated Boyden chambers under serum-free conditions (48 hours; *P < .05). (d) Exogenous rhEpo (10 U/ml) treatment enhances phosphorylation of JAK2 and this activation is blocked with AG490 (20 µM; *P < .05). (e) Epo (10 U/ml)-induced invasion in O22 cells is blocked with AG490 (20 µM) treatment (*P < .05). (f) Basal invasion of 22B cells is reduced with AG490 (20 µM) treatment only under serum-free conditions (*P < .05).