P4-01-03: Establishment and Characterization of an Endocrine Resistance Model In Vitro and In Vivo by Inducible PTEN Knockdown.
Authors: Fu X , Shea M , Biswal NC , Mitchell T , Giuliano M , Healy NA , Meerbrey KL , Joshi A , Westbrook T , Hilsenbeck SG , Osborne CK , Schiff R 
Published: June 2012

Background

Growth factor receptor and estrogen receptor (ER) are two major driving pathways for initiating and sustaining breast cancer (BC) development and progression. We have previously shown that an inverse correlation exists between the PI3K pathway and ER expression/activity in luminal type BCs. High PI3K activation signaling correlates to the luminal B subtype of BC with low ER expression/activity. However, the involvement of the PI3K pathway tumor suppressor PTEN in resistance to endocrine therapy is less clear. Here we attempt to develop an experimental system to better understand the role of PTEN in this resistance.

Materials and Methods: Two luminal BC cell lines, MCF7L and ZR75-B, were stably infected with a lentivirus pINDUCER (Meerbrey et al., PNAS, 2011), containing Tet-on responsive shPTEN, turboRFP (tRFP) as an inducible tag, and enhanced GFP (eGFP) as a constitutive expressed tag for positive cells selection. Immunoblotting of PTEN, phosphorylated (p) Akt, pMAPK, pc-Jun, ER, and ER's downstream gene products (PR, IGF-1R) was performed on cells after 6 days of doxycycline (Dox) incubation. After pre-starvation for 5 days in estrogen deprivation (ED) conditions, the cells -/+ Dox were subjected to induction with estrogen (E2) or to endocrine treatment [continued ED, tamoxifen (Tam, 10–7M), or fulvestrant (Ful, 10–7M)] in 96-well plate format for 9 days. In situ cell cytometry (Celigo, Cyntellect Inc., San Diego, CA) was applied to count the cell number by scanning the same 96-well plate every other day. Ovariectomized nude mice bearing MCF7L-shPTEN xenografts, established in the presence of E2 supplementation, were randomized to minus and plus Dox groups, with each treatment group continuing E2 or endocrine therapies (ED, Tam, or Ful). The in vivo GFP/RFP imaging was performed with a home-built cooled and image intensified CCD camera system.

Results: Immunoblot analysis showed a striking loss of PTEN, and significant upregulation of pAkt, as well as pMAPK and pc-Jun in cells with +Dox. In contrast, levels of ER, PR and IGF1R were reduced in cells with +Dox compared to -Dox. In all -Dox groups, cell growth was significantly reduced in endocrine treated groups compared to the E2 group, whereas +Dox rescued the endocrine treatment growth suppression, especially in ED and Tam groups. In vivo induction of PTEN shRNA expression was confirmed by RFP imaging after feeding the mice with +Dox water. MCF7L-shPTEN xenografts randomized to +Dox under ED conditions continued to grow over 4 weeks, in contrast to the marked regression of control tumors (−Dox) under this endocrine regimen (p&lt;0.05). Comparison between plus and minus Dox groups under E2, Tam, or Ful treatment is ongoing.

Discussion: These data further support the existence of crosstalk between PI3K and ER pathways in luminal type BC. Decreasing PTEN levels by shRNA renders the luminal type BC cells de novo resistant to endocrine therapy both in vitro and in vivo. The pINDUCER PTEN knockdown system combined with live animal imaging offers successful real-time, noninvasive tracking of endocrine sensitivity by controllably manipulating the level of target gene. Combination therapies to overcome endocrine resistance under PTEN knockdown conditions are currently underway.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-03.