Over-expression of anti-apoptotic proteins is a prevalent mechanism of dysregulation in cancer that plays an essential role in tumorigenesis and confers resistance to conventional cancer therapies. The ability to induce apoptosis in cancer cells is therefore necessary for tumor sensitivity to treatment. Bcl-2 is the prototype anti apoptotic protein that has been shown to be over-expressed in many human cancers. However, recent evidence suggests that Bcl-2 can act as a pro-apoptotic protein under certain circumstances. Nuclear receptor Nur77 was shown to induce cancer cell apoptosis through interaction with Bcl-2. In response to apoptotic stimuli, Nur77 localizes to the mitochondria where it interacts with Bcl-2, causing Bcl-2 to expose its BH3 domain, the "killer" domain of the Bcl-2 protein family. Over-expression of Nur77 with wild-type Bcl-2, but not a BH3 Bcl-2 mutant, can lead to apoptosis in a human embryonic kidney cell line 293T. Similarly, co-expression of Nur77 with Bcl-2 homologs Bcl-B and Bcl2A1 causes increased apoptosis. Furthermore, addition of a peptide containing the Nur77 Bcl-2 interacting region, conjugated to a cell penetrating peptide, can induce death in several human cancer cell lines. In primary normal cells, our lab showed that the mechanism of Bcl-2 BH3 exposure by Nur77, as well as its highly homologous family member Nor-1, can be found in immature T cells (thymocytes) when they are induced to die through their T-cell receptor complex. Nur77/Nor-1 mitochondrial translocation is dependent on Protein Kinase C (PKC) signaling and inhibition of nuclear export of Nur77/Nor-1 significantly rescues thymocytes from apoptosis. This physiological example of employment of this apoptotic mechanism thus provides a unique system for the examination of the in vivo significance and molecular mechanism of this pathway and how the Bcl-2 pro-apoptotic function may be used to kill primary tumor cells in vivo.
In Aim 1, we will identify mutations that can prevent or constitutively promote mitochondrial localization of Nur77 and Nor-1. PKC's role in regulating translocation through the mutated region and the importance of translocation for induction of thymocyte and cancer cell death will be assessed. A Nor-1 mutant constitutively localized to the mitochondria will be expressed in a T cell-specific transgenic mouse model to assess the in vivo significance of this pro-apoptotic pathway and in Bcl-2/Bcl2A1 high- or low-expressing cancer cell lines to assess its apoptotic function in relation to Bcl-2/Bcl2A1 expression.
In Aim 2, we will explore the therapeutic potential of Nur77 and Nor-1 peptides with cell penetrating ability to target Bcl-2 and Bcl2A1 in vitro and in vivo. The abilityof Nur77 and Nor-1 peptides to induce apoptosis in a Bcl2A1-dependent manner will be assessed in human cancer cell lines. The mechanism of Bcl2A1's pro-apoptotic function will be delineated by interaction studies in primary thymocytes. The capacity of Nur77 and Nor-1 peptides to induce apoptosis of cells expressing Bcl-2 versus Bcl2A1 will be tested in human cancer cell lines and primary normal and malignant T cells in vitro and in xenograft and allograft models in vivo.
Cancer results from genetic alterations that lead to unrestrained cell growth and resistance to cell death. Bcl-2, an import factor used by cancer cells to resist death, can be converted into a cancer cell killing protein through interaction with Nur77 and Nor-1. With better understanding of this mechanism, cancer therapeutics can be developed to promote Bcl-2 conversion to a killer and restoration of cell death pathways, increasing the sensitivity of tumors to conventional treatments.
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|Burger, Megan L; Xue, Ling; Sun, Yuefang et al. (2014) Premalignant PTEN-deficient thymocytes activate microRNAs miR-146a and miR-146b as a cellular defense against malignant transformation. Blood 123:4089-100|