The major goal of this project is to study the molecular mechanisms that underlie the critical steps of bladder cancer (BC) progression: invasion and metastasis. Specifically, we will focus on how autophagy-related gene 7 (ATG7)-mediated autophagy signaling drives BC cell invasion in vitro and metastasis in vivo. During the last funding period, we found that basal-subtype muscle-invasive bladder cancer (MIBC) in mice induced by bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) markedly overexpress ATG7 and long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1), and have significantly upregulated autophagy. In stark contrast, knockin mice lacking the RING domain of XIAP, which are completely resistant to BBN-induced basal MIBCs, have markedly reduced autophagy. We also found that, during BBN-mediated bladder tumorigenesis, the RING domain of XIAP is essential for SNHG1 overexpression, and that ectopic expression of SNHG1 in vitro induces autophagy and promotes BC cell invasion accompanied by upregulated ATG7, MMP2 and MMP9. Furthermore, we showed that knockdown of ATG7 strongly inhibits autophagy, abolishes BC cell invasion and reduces the expression of basal MIBC marker KRT14. These data reveal a heretofore unknown role of autophagy in basal MIBC formation. Based on these data, we hypothesize that the upregulation of SNHG1 and ATG7 by the RING domain of XIAP, and the autophagic signaling that these molecules trigger play critical roles in the genesis and progression of basal MIBC. We will test this hypothesis in three Specific Aims.
Aim 1 will define the regulatory circuitry in the SNHG1/ATG7/autophagy signaling axis that is operative in basal MIBC in vitro.
Aim 2 will determine the biological effects of the SNHG1/ATG7/autophagy signaling on BC cell invasion in vitro and tumorigenesis and metastasis in vivo.
Aim 3 will test the hypotheses that overexpression of SNHG1 in basal urothelial cells of transgenic mice promotes basal MIBC formation, and that ablation of ATG7 in these cells of knockout mice renders mice resistant to basal MIBC formation and progression. These complementary approaches will provide definitive evidence regarding the in vivo roles of SNHG1 and ATG7 in the formation and progression of basal MIBC. While invasion and metastasis are the main reasons of the high mortality caused by MIBC, very little is known about the principal molecules or pathways that drive these crucially important biological processes. Our proposed studies that are highly focused on an important, but poorly understood signaling pathway comprising SNHG1/ATG7/autophagy should yield critical information on not only the underlying mechanisms, but also novel prognostic biomarkers to differentiate MIBC subtypes and new druggable targets to treat this aggressive form of BC.
PROJECT 3 NARRATIVE The main reason why bladder cancer is deadly is because of its tendency to invade and metastasize. The principal objective of this component project is to determine the role of a new signaling pathway in the formation and progression of a bladder cancer variant called basal-subtype muscle-invasive bladder cancer. It is expected that our studies will lead to new biomarkers for bladder cancer stratification and new druggable targets for effective treatment. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page
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