No abstract

Cancer tumor cell dormancy is a significant clinical problem in breast cancer (BrCa). We used a 3D in vitro model of the endosteal bone niche (EN), consisting of endothelial, bone marrow stromal cells and fetal osteoblasts in a 3D collagen matrix (GELFOAM™), to identify genes required for dormancy. Human triple-negative MDA-MB-231 BrCa cells, but not the bone-tropic metastatic variant, BoM1833, established dormancy in 3D-EN cultures in a p38-MAPK-dependent manner, whereas both cell types proliferated on 2D plastic or in 3D collagen alone. “Dormancy-reactivation suppressor genes” (DRSG) were identified using a genomic shRNA screen in MDA-MB-231 cells for gene knockdowns that induced proliferation in the 3D-EN. DSRG candidates enriched for genes controlling stem cell biology, neurogenesis, MYC targets, ribosomal structure and translational control. Several potential DRSG were confirmed using independent shRNAs, including BHLHE41, HBP1 and WNT3. Overexpression of the WNT3/a antagonists, secreted frizzled-related protein 2 or 4 (SFRP2/4), induced MDA-MB-231 proliferation in the EN. In contrast, overexpression of SFRP3, known not to antagonize WNT3/a, did not induce proliferation. Decreased WNT3 or BHLHE41 expression was found in clinical BrCa metastases compared to primary-site lesions, and the loss of WNT3 or BHLHE41 or gain of SFRP1, 2 and 4 in the context of TP53 loss/mutation correlated with decreased progression-free and overall survival.

<div>Abstract<p>Tumor cell dormancy is a significant clinical problem in breast cancer. We used a three-dimensional (3D) <i>in vitro</i> model of the endosteal bone niche (EN), consisting of endothelial, bone marrow stromal cells, and fetal osteoblasts in a 3D collagen matrix (GELFOAM), to identify genes required for dormancy. Human triple-negative MDA-MB-231 breast cancer cells, but not the bone-tropic metastatic variant, BoM1833, established dormancy in 3D-EN cultures in a p38-MAPK–dependent manner, whereas both cell types proliferated on two-dimensional (2D) plastic or in 3D collagen alone. “Dormancy-reactivation suppressor genes” (DRSG) were identified using a genomic short hairpin RNA (shRNA) screen in MDA-MB-231 cells for gene knockdowns that induced proliferation in the 3D-EN. DRSG candidates enriched for genes controlling stem cell biology, neurogenesis, MYC targets, ribosomal structure, and translational control. Several potential DRSG were confirmed using independent shRNAs, including <i>BHLHE41, HBP1</i>, and <i>WNT3</i>. Overexpression of the WNT3/a antagonists secreted frizzled-related protein 2 or 4 (SFRP2/4) and induced MDA-MB-231 proliferation in the EN. In contrast, overexpression of SFRP3, known not to antagonize WNT3/a, did not induce proliferation. Decreased <i>WNT3</i> or <i>BHLHE41</i> expression was found in clinical breast cancer metastases compared with primary-site lesions, and the loss of <i>WNT3</i> or <i>BHLHE41</i> or gain of <i>SFRP1, 2,</i> and <i>4</i> in the context of <i>TP53</i> loss/mutation correlated with decreased progression-free and overall survival.</p>Implications:<p>These data describe several novel, potentially targetable pathways controlling breast cancer dormancy in the EN.</p></div>

<p>pGIPZ Lentiviral shRNAmir-GFP Human shRNA clones</p>

<p>DSRG candidates from modules 2 and 3</p>