Abstract 2280: Role of MAP1LC3 in 15d-PGJ2 induced non-apoptotic and non-autophagic cell death in therapy-resistant triple negative breast tumors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: In spite of significant progress in breast cancer research, hormonal therapy resistant triple negative breast cancers (TNBC) still pose a greater therapeutic challenge due to lack of a suitable therapeutic target and their resistance to apoptosis induced by chemotherapy. Therefore, alternative approaches are required to induce cell death in these apoptosis-resistant breast cancers. We previously showed that certain sulfhydryl reactive compounds, containing α,β-unsaturated ketones, such as 15d-PGJ2 induced non-apoptotic and non-autophagic cell death in apoptosis resistant and rapidly growing cancer cells through induction of microtubule-associated protein 1 light chain 3 (MAP1LC3) and sequestosome1 (p62) proteins. Purpose: In the present study, our objective is to address the role of MAP1LC3 in 15d-PGJ2 induced non-apoptotic and non-autophagic cell death of breast cancer cells. Methods: Stable knockdown of endogenous MAP1LC3 expression was achieved by shRNA expression vectors. Cell culture, cell proliferation, soft-agar colony forming assays and immunoblotting were performed using standard methods. Nude mice tumor models were used for in vivo studies. Results: First, we showed the efficacy of 15d-PGJ2 in reducing the breast tumor burden using xenograft mice models of MDA-MB-231 cells by i.p. into tumor bearing mice. We observed significant reduction of tumor growth with high levels of p27, PTEN and less pAkt compared to vehicle treated mice. Most importantly, tumors treated with 15d-PGJ2 showed extensive cytoplasmic vacuoles with remarkable increase in endoplasmic reticulum (ER) stress markers such as Bip, CHOP along with MAP1LC3 and p62 proteins as well as higher molecular weight ubiquitinated proteins. Notably, in normal human mammary epithelial cells (HMEC) 15d-PGJ2 failed to induce cell death as well as MAP1LC3 protein. Likewise, MAP1LC3 knockdown (LC3KD) in MDA-MB-231 breast cancer cells also prevented 15d-PGJ2 induced cell death with decreased accumulation of ER stress markers such as Bip and CHOP, p62 and ubiquitinated proteins. In addition, MAP1LC3 deficiency also resulted in changes in MDA-MB-231 cell morphology from an elongated mesenchymal shape to epithelial cobblestone appearance with distinct slower growth rate. Moreover, LC3KD cells failed to form colonies in soft agar, showed reduced migration and invasion in transit well chamber assays, and expressed high levels of p21, p27 and PTEN with decreased pAkt levels. Finally, MAP1LC3-deficient cells showed marked reduction in tumor growth compared to parental MDA-MB-231 cells in xenograft model using nude mice. Conclusions: Hence our results suggest that the induction of MAP1LC3 in response to treatment with sulfhydryl reactive β, α-unsaturated carbonyl compounds is an essential feature in inducing a novel form of cell death in therapy resistant breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2280. doi:1538-7445.AM2012-2280