Acquisition of estrogen independence induces TOB1-related mechanisms supporting breast cancer cell proliferation

Abstract

Resistance to therapies targeting the estrogen pathway remains a challenge in the treatment of estrogen-receptor positive breast cancer. To address this challenge, a systems biology approach was used. A library of siRNAs targeting an estrogen receptor- and aromatase-centered network identified 46 genes that are dispensable in estrogen-dependent MCF7 cells, but are selectively required for the survival of estrogen-independent MCF7-derived cells, and multiple additional estrogen-independent breast cancer cell lines. Integration of this information identified a tumor suppressor gene TOB1 as a critical determinant of estrogen-independent estrogen receptor-positive breast cell survival. Depletion of TOB1 selectively promoted G1 phase arrest and sensitivity to AKT and mTOR inhibitors in estrogen-independent cells but not estrogen-dependent cells. Phosphoproteomic profiles from reverse phase protein array analysis supported by mRNA profiling identified a significant signaling network reprogramming by TOB1 that differed in estrogen-sensitive and estrogen-resistant cell lines. These data support a novel function for TOB1 in mediating survival of estrogen-independent breast cancers. These studies also provide evidence for combining TOB1 inhibition and AKT/mTOR inhibition as a therapeutic strategy, with potential translational significance for the management of patients with estrogen receptor-positive breast cancers.

TOB1 Deficiency Enhances the Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Tendon-Bone Healing in a Rat Rotator Cuff Repair Model

“…Our study found that knockdown of TOB1 enhanced the proliferative activity of MSCs, which is consistent with results in mouse embryonic stem cells [26]. However, a recent study found that TOB1 knockdown inhibited growth of estrogen-independent estrogen receptor positive breast cancer cells [27]. The reported variability in TOB1 function may be due to distinctive regulation of survival and cell cycle signaling in different cell types.…”

Protein network construction using reverse phase protein array data

“…In addition, it is unclear how well WGCNA performs under small sample size restrictions. To explore the mechanisms of resistance to therapies targeting estrogen pathways in the treatment of estrogen receptor (ER) positive breast cancer, we previously performed a systemic biological screening of a library of siRNAs targeting an ER- and aromatase-centered network [15]. We identified 46 genes that are dispensable in the estrogen-dependent MCF7 cell lines, but are selectively required for the survival of estrogen-independent MCF7-derived cell lines (LCC1 and LCC9).…”

RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells

“…SiRNAs against those genes were distributed into 11 x 96-well plates. Two siRNAs were selected for each gene and mixed in one well (Zhang et al ., 2016). The advantage of our method provides high-throughput screening by using automatic machines (Cybio, Combi-nL or Wellmate dispenser) to dispense liquid to speed the screening process.…”

“…Different types of cancer cell lines had been used in RNAi screening with our methods (Astsaturov et al ., 2010; Murray et al ., 2014; Zhang et al ., 2016), such as estrogen positive breast cancer MCF7, estrogen-independent MCF7 (LCC1 and LCC9), triple negative breast cancer MDA-MB-231, epidermoid cancer A431 and human fibroblast HFF1 cells etc . For each cell line, the optimal transfection reagent has to be determined before RNAi library screening.…”

“…Z′-score is taken as a quantitative parameter to control the experiment quality for various cell lines and corresponding transfection reagents. In this assay, we utilize ER-network RNAi screening in MCF7 cells as an example to describe the protocol (Zhang et al ., 2016). It also fits other cell lines or other gene network RNAi library with minor modification, such as type of transfection reagent, cell plating density, Cell Titer blue incubation time or RNAi library scale (total number of siRNA library plates), which will be noted.…”