Abstract 12: Bclxl is a key regulator of mitochondria-induced apoptosis in ovarian cancer stem cells
Authors: Alvero Ayesha B. , Pitruzello Mary , Montagna Michele , Lima Eydis , Mor Gil 
Published: August 2015

Background:

Resistance to mitochondria-initiated apoptosis is a hallmark of chemoresistant cancer stem cells. As such, in epithelial ovarian cancer (EOC), EOC stem cells exhibit cross- resistance to a broad range of chemotherapy agents irrespective of their upstream targets. Members of the Bcl2 family of proteins function as rheostats of mitochondrial stability. Pro-apoptotic members Bak and Bax can form high molecular weight oligomers that allow the release of cytochrome c to the cytoplasm and consequently activating the caspases. In turn, pro-survival members such Bclxl and Bcl2 bind Bak and Bax thus preventing their oligomerization and maintaining mitochondrial integrity. We hypothesize that a distinct and targetable subset of Bcl2 proteins may be regulating the chemoresistant EOC stem cells.

Methods:

Whole cell lysates and cytoplasmic/mitochondrial fractions were collected from pure clones of chemoresistant CD44+/MyD88+ and chemosensitive CD44-/MyD88- EOC cells. Expression of proteins in the Bcl2 family was determined by western blot and net intensity of regions of interest (ROI) was determined to provide quantitative measure. Mitochondrial membrane potential (MMP) was measured using Mitotracker Red, caspase activity was quantified by Celltiter GloTM, and Bclxl expression was knocked-down in the EOC stem cells using siRNA.

Results:

We observed a differential expression profile of Bcl2 family members between the chemoresistant CD44+/MyD88+ and chemosensitive CD44-/MyD88- EOC cells. Using whole cell lysates, chemoresistant CD44+/MyD88+ EOC stem cells surprisingly express 3-fold and 5-fold more of the pro-death proteins Bak and Bax, respectively compared to the chemosensitive CD44-/MyD88- EOC cells. In addition, the EOC stem cells express 6 -fold more Bclxl but 5-fold less Bcl2 than CD44-/MyD88- EOC cells. At the level of the mitochondria, the significantly higher levels of Bak, Bax, and Bclxl are maintained in the EOC stem cells. Interestingly, as little as 14% knock-down of Bclxl in the EOC stem cells was able to induce loss of MMP, 3-fold increase in caspase-9 and 17-fold increase in caspase 3/7.

Conclusion:

Ovarian cancer stem cells have a unique mitochondrial phenotype and Bclxl is a key regulator of its stability and may play a role in resistance to chemotherapy-induced apoptosis. Targeting Bclxl may be an approach to complement current standard of care in ovarian cancer. The demonstration that apoptosis can be fully induced with minimal loss of Bclxl suggests an acceptable therapeutic window for Bclxl inhibitors.

Citation Format: Ayesha B. Alvero, Mary Pitruzello, Michele Montagna, Eydis Lima, Gil Mor. Bclxl is a key regulator of mitochondria-induced apoptosis in ovarian cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 12. doi:10.1158/1538-7445.AM2015-12