8/12/2019 Breast cancer resistance research abstract

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A NEW MECHANISM FOR ESTROGEN-STARVATION RESISTANCE IN BREAST CANCER

Amy S. Lee, Yong Fu, and Minal Patel

University of Southern California

Background and Objectives: The recent development of aromatase inhibitors as effective treatment for

estrogen-receptor positive breast cancer represents a major advance in improving breast cancer therapy.

However, cancer cells often acquire adaptations that result in resistance. Estrogen-starvation inducedapoptosis of breast cancer cells requires BIK, an apoptotic BH3-only protein located primarily at the

endoplasmic reticulum (ER). In search of novel partners that interact with BIK at the ER, we discovered

that glucose-regulated protein GRP78/BiP, which is upregulated in about 70% of breast tumors, forms

complexes with endogenous BIK. GRP78 overexpression decreases apoptosis induced by ER-targetedBIK in human cell cultures. For estrogen-dependent MCF-7/BUS breast cancer cells, overexpression of

GRP78 inhibits estrogen-starvation-induced Bax activation, mitochondrial permeability transition, and

subsequent apoptosis. Furthermore, knockdown of endogenous GRP78 by siRNA sensitizes MCF-7/BUScells to estrogen-starvation-induced apoptosis. This project aims to test whether: (1) suppression of

GRP78 will re-sensitize estrogen-receptor positive breast cancer cells that have acquired resistance to

estrogen-starvation therapy; and (2) GRP78 inhibits the proapoptotic activity of BIK through interferenceof its cooperation with other members of the BH3-protein family.

Methods: Aim 1 is to isolate a panel of human breast cancer cells that are resistant to estrogen-starvation-induced apoptosis. MCF-7/BUS cells were cultured in estrogen-starvation medium (DMEM supplemented

with 5% charcoal-dextran stripped fetal calf serum) for 5 to 10 days so that about 10% of cells survived.The surviving MCF-7/BUS cells were cultured in regular DMEM with 5% fetal calf serum for 2 to 3 days

to allow expansion of remaining cells. This selection process was repeated 6 times to obtain resistant

clones that could grow and divide in the estrogen-starvation medium. The resistant clones of MCF-7/BUSwere maintained in this medium for at least 2 months to stabilize resistant properties before being analyzed

for ER chaperone levels and ER stress markers. Our plan is to introduce siRNA targeted against GRP78

and/or other induced chaperones into the resistant cells and test whether lowering their levels will re-sensitize the resistance cells to estrogen-starvation-induced apoptosis. In Aim 2, we will determine the

interactive domains between GRP78 and BIK. In Aim 3, we will examine the mechanisms whereby

GRP78 inhibits estrogen-starvation-induced cell death. This will include testing: (a) whether GRP78suppresses the cooperative mechanism between NOXA and BIK, both of which are pro-apoptotic;

(b) whether GRP78 releases the pro-survival BCL-2 from BIK; and (c) whether GRP78 suppresses caspase

activation by estrogen starvation.

Results: We have obtained 14 resistant clones and observed prominent elevation of ER chaperones in 9 ofthe clones and will proceed with the proposed experiments.

Conclusion: Our preliminary results suggest that induction of the cytoprotective ER chaperone is an

adaptive mechanism to allow breast cancer cells to be resistant to estrogen-starvation. If this is validated,

drugs that can suppress ER chaperones in breast tumors will sensitize the cancer cells to aromataseinhibitor therapy.

This work was supported by the U.S. Army Medical Research and Materiel Command under W81XWH-07-1-0480.