The ING1b protein is a type II tumor suppressor and a stoichiometric member of HDAC-containing protein complexes. Primarily by altering chromatin structure, ING1b contributes to regulation of gene expression, senescence and apoptosis. Mislocalization and decreased levels of ING1b are commonly observed in human tumors and cancer cell lines. Multiple independent studies in varied cell types report that ING1b overexpression promotes apoptosis. Since the inactivation of apoptosis pathways is frequent in cancer cells, modulating ING1b expression in tumors may serve as a viable approach for cancer therapy. We have defined the minimal ING1b region that confers apoptotic function as estimated by the levels of PARP cleavage, FACS analysis and using an Annexin V binding assay. We have established that ING1b-derived peptides containing its third alpha helix (A3H) and NLS/NTS domains are able to induce apoptosis at levels comparable to those of full length ING1b. The A3H-NLS/NTS peptide exhibited strong nucleolar localization characteristic of full length ING1b. Cells overexpressing the full length ING1b and A3H-NLS/NTS peptide showed similar changes in cell morphology characteristic of apoptosis and exhibited increased levels of PARP cleavage. While the A3H region that includes the N-terminal part of the highly conserved Lamin Interacting Domain (LID) was necessary but not sufficient, the NLS/NTS domain that mediates nuclear and nucleolar localization of ING1b was required and partially sufficient for induction of apoptosis. Adenoviral-mediated expression of A3H-NLS/NTS peptide in cells of osteosarcoma, glioblastoma and breast cancer origins resulted in strong reduction of cell viability. Depending on the cell line, treating cells with 45 MOI of virus expressing A3H-NLS/NTS peptide resulted in a 60 - 85% decrease in cancer cell survival compared to cells treated with the control construct (Ad-GFP), and the highly transformed triple negative tumorigenic MDA-MB-468 breast cancer line was sensitive to the peptide when infected with 5 MOI of A3H-NLS/NTS adenovirus. These pro-apoptotic effects were found to be dose and time dependent. Furthermore, using p53 wild-type, p53 mutant and p53-null cancer cell lines we demonstrated that the effects of A3H-NLS/NTS expression on cell survival and apoptosis were independent of p53-status. The evaluation of the synergy between the A3H-NLS/NTS peptide and common chemotherapeutic agents is currently ongoing. Our long-term goal is to develop ING1b-based therapeutics that can be used as an adjuvant therapy in combination with the existing breast cancer treatments.
Citation Format: Oleksandr Boyko, Karl Riabowol. Expression of ING1b-derived peptide inhibits viability of breast cancer cells and promotes apoptosis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-05-02.