Translational up-regulation of the EGFR by tumor hypoxia provides a nonmutational explanation for its overexpression in human cancer

Abstract

Overexpression of the EGF receptor (EGFR) is a recurrent theme in human cancer and is thought to cause aggressive phenotypes and resistance to standard therapy. There has, thus, been a concerted effort in identifying EGFR gene mutations to explain misregulation of EGFR expression as well as differential sensitivity to anti-EGFR drugs. However, such genetic alterations have proven to be rare occurrences in most types of cancer, suggesting the existence of a more general physiological trigger for aberrant EGFR expression. Here, we provide evidence that overexpression of wild-type EGFR can be induced by the hypoxic microenvironment and activation of hypoxia-inducible factor 2-alpha (HIF2alpha) in the core of solid tumors. Our data suggest that hypoxia/HIF2alpha activation represents a common mechanism for EGFR overexpression by increasing EGFR mRNA translation, thereby diminishing the necessity for gene mutations. This allows for the accumulation of elevated EGFR levels, increasing its availability for the autocrine signaling required for tumor cell growth autonomy. Taken together, our findings provide a nonmutational explanation for EGFR overexpression in human tumors and highlight a role for HIF2alpha activation in the regulation of EGFR protein synthesis.

Fig. 1.

The hypoxic tumor microenvironment triggers EGFR expression. (A) Western blot analysis of total EGFR and HIFα levels in U87MG glioma cells grown as 3D spheroids for 1–3 days. Control cells were grown in 2D culture. (B) Western blot analysis of EGFR and HIFα levels in U87MG, MDA-MB-231 breast, and PC3 prostate cancer cells exposed to normoxia (21% O₂) or physiological hypoxia (1% O₂) for 24 h. (C) Western blot analysis of EGFR and HIFα levels in U87MG cells exposed to hypoxia for the indicated times. (D–F) RT-PCR analysis of EGFR and GLUT mRNA levels in cells described in A–C. Actin served as a loading control in A–F.

Fig. 2.

HIF2α activation results in induction of EGFR protein levels. (A) Western blot analysis of EGFR, HIFα, and GLUT levels in U87MG cells infected with adenovirus expressing flag-tagged GFP or a dominant-negative form of HIF (dnHIF). Cells were grown as 3D spheroids for 3 days. Control cells were grown in 2D culture for the same period. (B) Western blot analysis of EGFR and HIFα levels in U87MG and MDA-MB-231 cells infected with GFP or dnHIF for 24 h. before exposure to normoxia or hypoxia for an additional 24 h. (C and D) Western blot (C) and RT-PCR (D) analysis of EGFR levels in U87MG cells infected to express GFP or HIFα variants (HIF1α and HIF2α) for 72 h in normoxia. (E) Western blot analysis of EGFR and HIFα levels in U87MG transiently transfected with siRNA (50 nM) targeting HIF1α (siHIF1α), HIF2α (siHIF2α), or a scramble sequence (siCont) for 48 h before exposure to hypoxia for an additional 24 h. (F) Western blot analysis of EGFR and HIF2α levels in U87MG pretreated with 10 nM rapamycin (Rap), or DMSO as a vehicle control, for 1 h and then exposed to normoxia or hypoxia for an additional 16 h. The phosphorylation status of the S6 ribosomal protein (ph-S6) served as a control for drug activity. Actin served as a loading control in A–F.

Fig. 3.

HIF2α activation results in increased EGFR mRNA translation. (A) Western blot and RT-PCR analysis of EGFR protein and mRNA levels in the VHL^−/− RCC, 786-0, cell line (786-0), and 786-0 cells stably expressing wild-type VHL (786-0 + VHL). (B) Western blot (Left) and RT-PCR (Right) analysis of EGFR protein and mRNA levels in 786-0 cells infected with adenovirus expressing GFP or dnHIF for 24–96 h. (C) Western blot analysis of EGFR protein levels in 786-0 + VHL cells infected with GFP, HIF1α or HIF2α for 72 h. Actin served as a loading control in A–C. (D) EGFR radiolabel incorporation in cells pulsed for the indicated times with [³⁵S]methionine ([³⁵S]Met) and immunoprecipitated with an anti-EGFR antibody. Cells were also labeled and immunoprecipitated with a second EGFR antibody as a specificity control (Right). (E) EGFR [³⁵S]Met incorporation in 786-0 cells infected with GFP or dnHIF for 72 h and labeled for 1 h. (F) EGFR [³⁵S]Met incorporation in cells labeled for 2 h and cold-chased for indicated times. Whole-cell lysates (WCL) served as radiolabel incorporation and loading controls in D–F. (G) RT-PCR analysis of EGFR mRNA levels in polysomal fractions of 786-0 cells infected with GFP or dnHIF for 48 h and subjected to sucrose gradient fractionation. The percentage of total EGFR mRNA in each fraction is plotted.

Fig. 4.

Overexpression of EGFR results in cancer cell growth autonomy but does not affect sensitivity to anti-EGFR drugs. (A) VHL-competent RCC cells (786-0 + VHL) were infected with adenovirus expressing GFP or HIF2α or treated with 20 ng/ml recombinant TGFα (rTGFα) and then cultured in the absence or presence of serum for 72 h before labeling with BrdU. (B) BrdU incorporation in VHL-deficient 786-0 cells infected with GFP or dnHIF after addition of rTGFα for the indicated times in serum-free media. (C) Western blot analysis of EGFR levels in 786-0 cells transiently transfected with the indicated amounts of siRNA (10–100 nM) targeting the EGFR or a scramble control sequence for 72 h. Actin served as a loading control. (D) BrdU incorporation in cells described in C. Bars represent standard deviation of at least three independent experiments in A, B, and D. (E) IC₅₀ concentrations of receptor tyrosine kinase inhibitors, AG1478 and PD153035, for inhibition of EGFR kinase activity in 786-0 cells infected with GFP or dnHIF for 48 h.