Abstract P2-09-20: 2’(3’)-O-(4-benzoylbenzoyl)adenosine 5’-triphosphate (BzATP) is a potential drug for triple negative breast cancer: Autophagy and apoptosis inducer

Triple-negative breast cancer (TNBC) is among the most aggressive and therapy resistant type of breast cancer. New therapeutic strategies and new treatments are intensively sought for TNBC. Here, we report the potential of 2’(3’)-O-(4-Benzoylbenzoyl)adenosine 5’-triphosphate (BzATP), which is ATP analog, as a potential drug for TNBC. Role of extracellular nucleotides as an extracellular signaling molecule and the antitumor effects of ATP have been reported for a few decades, however, the signaling pathways that link from the purinergic receptors to the reported effects including reduced tumor xenografts volume in mice and tumor growth inhibition in vitro is not yet well established. We chose two pairs of TNBC cell lines with matching molecular subtype; one is basal A subtype and another one is basal B subtype. In each pair, one was derived from Caucasian patient and another one was derived from African American patient. This strategy allows us to cover a wide range of cell lines and also to see whether there is any disparity of BzATP treatment between ethnicity. Expression of P2Y receptors, which are one of three families of extracellular receptors for purine and pyrimidine nucleotides, in not only two pairs of TNBC in this study, but also other six breast cancer cell lines, showed five common P2Y receptors including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. Growth inhibition assay was performed by treating TNBC with agonists including ATP, BzATP, UTP, and NAD from 1 μM to the highest dose at 1000 μM, which is still lower than reported dose in in vivo studies in mice. ATP, BzATP, and NAD, but not UTP, at 100 μM and higher doses inhibited growth of TNBC regardless of ethnicity. Notably, BzATP inhibited growth of all four TNBC cell lines in a greater degree than ATP did. Among five common receptors, P2Y2, P2Y4, and P2Y6, which has UTP or UDP as an agonist, are improbable to be a candidate receptor since UTP failed to inhibit TNBC growth. P2Y11, which has ATP, BzATP, and NAD as an agonist is a first candidate receptor since P2Y1 prefers ADP to ATP. Interestingly, growth inhibition on TNBC was observed along with a morphology change including reduced cell volume and an increase in process number after 24 hours of BzATP treatment at concentration that causes 50% growth reduction (GI50), which is 320 μM, 200 μM, 250 μM, and 200 μM for HCC1143, HCC70, HCC38, and HCC1500 cell lines respectively. Although BzATP also reduced a growth rate of non-malignant human epithelium cell line (MCF10A), no aforementioned morphology change was observed on MCF10A. In addition, BzATP at GI50 induced autophagy through expression of microtubule-associated protein 1 light chain 3 (LC3) and elevated a number of autophagic vesicles. Surprisingly, autophagy-related (Atg) proteins including Atg5, Atg7, and Atg3 did not show an upregulated level. In conjunction with autophagy, BzATP at GI50 also arrested cell cycle of TNBC at G2/M phase. Further, BzATP at GI50 elevated apoptotic marker, cleaved caspase-3 and cleaved PARP, and ER stress marker, CHOP, in TNBC but not in MCF10A. BzATP specifically induced apoptosis without causing necrosis as no elevated released lactate dehydrogenase (LDH) level was detected. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-20.