PROJECT SUMMARY SATB2 expression is critical for metal carcinogenesis. SATB2 is the only common gene that is upregulated in BEAS2B cells transformed by various carcinogenic metals and knock-down of SATB2 in normal BEAS2B cells prevents metal carcinogenesis. Knockdown SATB2 in arsenic (As) or nickel (Ni) transformed cells, results in the loss in hallmarks of transformation including anchorage independent growth, enhanced cell invasion and migration. The regulation of SATB2 involves miR-31 and miR23a. RUNX2 transcription factor down regulates the miRNAs that inhibit SATB2 expression and increases in RUNX2 brought about by exposure to Ni and As indirectly induces SATB2 by attenuating expression of these translation inhibitory miRs. The mechanisms for the increase expression of RUNX2 induced following exposure to As and Ni, and in As or Ni transformed cells will be studied. RUNX2 activation appears to be a critical upstream event that increases SATB2 protein which maintains cancer hallmarks. RUNX2 will be knocked out in normal BEAS2B to study its role in SATB2 expression and for cell transformation induced by Ni and As. RUNX2 and antogomers of miR-31 and miRNA 23a will be overexpressed and the incidence of BEAS2B transformation assessed. These later experiments will address how these miRNA block the translation of SATB2 mRNA. We will utilize a chemical inhibitor of RUNX2 (A1-10- 104) to study whether cell transformation induced by Ni and As and, cells transformed by overexpress RUNX2 is suppressed. A1-10-104 will also be used to study its effect on xenograph tumor formation in nude mice. We will use RUNX2 Chip-Seq to identify downstream targets in cells treated with As and Ni or transformed by these metals, as well as in cells engineered to overexpress RUNX2. SATB2 Chip-Seq will investigate how this transcription factor maintains cancer hallmarks. We will investigate the RUNX2/miRNA/SATB2 axis in samples of human lung cancers and surrounding normal tissue. We will study the mechanisms of how Ni and As exposure induce BEAS2B cell transformation, increase and maintain high levels of RUNX2 mRNA and protein as well as maintaining the post-translational modifications that are required to activate RUNX2. The mechanisms of how RUNX2 downregulates miRNA-31 and 23a will be investigated. We will study whether administration of miR- mimetics (miRNA-31or 23a) in lentivirus vectors, can shrink orthotopic or xenograph tumors induced by BEAS2B cells transformed with As, Ni or with overexpressed SATB2, compared to A549 lung cancer cells that have low levels of SATB2.

NARRATIVE This grant investigates the mechanism by which a gene that should only be expressed during embryonic development becomes expressed in adult cells, and studies how the expression of this embryonic gene can lead to cancer.