Cr(VI)-containing compounds are well-established environmental carcinogens. Most mechanistic investigations of Cr(VI)-induced carcinogenesis focus on oxidative stress and various cellular responses, leading to malignant cell transformation, the first stage of metal-induced carcinogenesis. The development of malignantly transformed cells into tumors in which angiogenesis is required is the second stage. This application focuses on this second stage, in particular the role of epidermal growth factor receptor (EGFR) signaling in angiogenesis and tumorigenesis of Cr(VI)-transformed cells. Our previous studies have found that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) causes malignant cell transformation. Our preliminary studies have shown that EGFR is constitutively activated in Cr(VI)-transformed cells, in lung tissues from Cr(VI)-exposed animals, and in lung tumor tissues from non-smoking workers exposed to Cr(VI). Our preliminary studies have also shown that EGFR phosphorylates p62 at serine349 (S349), that p62 upregulates nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2), and that Nrf2 upregulates both NAD(P)H:quinone oxidoreductase 1 (NQO1) and hypoxia-inducible factor (HIF)-1α in those Cr(VI)-transformed cells, leading to elevated level of vascular endothelial growth factor (VEGF). Inhibition of EGFR by its shRNA or kinase inhibitor AK1478 suppresses HIF-1α in Cr(VI)-transformed cells. Our findings indicate that EGFR functions as an initiator in angiogenesis and causes a chain of events through constitutive activation of Nrf2 in Cr(VI)-transformed cells. The central hypothesis of this application is that in Cr(VI)-transformed cells, constitutively activated EGFR phosphorylates p62 at S349, causing upregulations of Nrf2 and NQO1, which in turn activate HIF-1α and its target protein VEGF, leading to angiogenesis and tumorigenesis. Aim 1 will demonstrate that EGFR phosphorylates p62 at S349, causing increased binding affinity between p62 and Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of Nrf2, leading to reduced binding between Keap1 and Nrf2 and subsequently increased stabilization of Nrf2. Aim 2 will demonstrate that HIF-1α is upregulated by NQO1 due to its binding to HIF-1α in completion with prolyl hydroxylase domain protein 2 (PHD2) and to cap-independent but internal ribose entry site (IRES)-independent translation. We will also demonstrate the elevated protein levels of p-EGFR, p62, p-p62, Nrf2, NQO1, HIF-1α, and VEGF in human lung tumor tissues from Cr(VI)-exposed workers. Aim 3 will demonstrate that activation of EGFR/HIF-1α/VEGF causes angiogenesis and promotes tumorigenesis in vivo using animal models of intranasal exposure to Cr(VI), Matrigel plug assay, and orthotopic lung cancer.

PROJECT NARRATIVE The International Agency for Research on Cancer (IARC) classifies Cr(VI)-containing compounds as Group 1 human carcinogens. The long term goal of this application is to understand the mechanism of Cr(VI)- carcinogenesis and its prevention. This application focuses on role of epidermal growth factor receptor (EGFR) signaling in angiogenesis and tumorigenesis of Cr(VI)-transformed cells.