RB1 loss induces quiescent state through downregulation of RAS signaling in mammary epithelial cells

Abstract

While loss of function (LOF) of retinoblastoma 1 (RB1) tumor suppressor is known to drive initiation of small-cell lung cancer and retinoblastoma, RB1 mutation is rarely observed in breast cancers at their initiation. In this study, we investigated the impact on untransformed mammary epithelial cells given by RB1 LOF. Depletion of RB1 in anon-tumorigenic MCF10A cells induced reversible growth arrest (quiescence) featured by downregulation of multiple cyclins and MYC, upregulation of p27^KIP1, and lack of expression of markers which indicate cellular senescence or epithelial-mesenchymal transition (EMT). We observed a similar phenomenon in human mammary epithelial cells (HMEC) as well. Additionally, we found that RB1 depletion attenuated the activity of RAS and the downstream MAPK pathway in an RBL2/p130-dependent manner. The expression of farnesyltransferase β, which is essential for RAS maturation, was found to be downregulated following RB1 depletion also in an RBL2/p130-dependent manner. These findings unveiled an unexpected mechanism whereby normal mammary epithelial cells resist to tumor initiation upon RB1 LOF.

Keywords: MYC; RAS; RB1; RBL2/p130; quiescence.

FIGURE 1

Retinoblastoma 1 (RB1) loss induces MCF10A cells’ growth arrest. (A) Phase contrast image of MCF10A cells at 72 h after transduction. Scale bar: 200 μm. (B) Representative image of colony formation assay for Scr, shRB1#1, or shRB1#2 in MCF10A. (C) Quantitative analysis of colony number (N = 3). (D) Quantitative analysis of colony area (N = 3). (E) Immunoblotting of indicated proteins in Scr, shRB1#1, and shRB1#2 MCF10A cells. (F) Immunocytochemistry of Ki67 in MCF10A cells at 72 h after transduction. (G) Immunofluorescence staining of Ki67 in MCF10A cells and quantitative analysis of Ki67 mean fluorescence intensity (MFI) (N = 3). *p < 0.05, ***p < 0.001.

FIGURE 2

Retinoblastoma 1 (RB1) deletion induces c‐Myc downregulation. (A) Immunoblotting of indicated proteins in Scr, shRB1#1, and shRB1#2 MCF10A cells. (B) Immunoblotting of total c‐Myc, phospho‐c‐Myc (Serine 62), and phospho‐c‐Myc (threonine 58) proteins. (C) Immunoblotting of indicated proteins in Scr, shRB1#1, and shRB1#2 MCF10A cells treated with vehicle or proteasomal inhibitor MG‐132. Cells were treated with vehicle or MG132 for 5 h. (D) Quantitative analysis of c‐Myc relative protein level (N = 3). *p < 0.05, ****p < 0.0001.

FIGURE 3

c‐Myc overexpression overcomes cell growth arrest. (A) Representative image of colony formation assay for MCF10A cells. c‐Myc was induced by 1 μg/mL Dox for 72 h in MCF10A. Then, cells were transduced with RBsh lentivirus and subjected to selection by puromycin (N = 3). (B) Quantitative analysis of colony number (N = 3). (C) Quantitative analysis of colony area (N = 3). (D) Immunoblotting of indicated proteins in MCF10A cells. *p < 0.05, **p < 0.01.

FIGURE 4

Overexpression of active RAS restores c‐Myc levels and induces transformation. (A) Pull‐down assay of RAS protein and GTP protein. Pan‐RAS protein was analyzed by immunoblotting. α‐tubulin was used as a loading control. (B) Quantitative analysis of RAS activity (RAS‐GTP/Pan‐RAS, N = 3). (C) Immunoblotting of indicated proteins in MCF10A cells using CRISPR. (D) Immunoblotting of indicated protein in MCF10A cells after induction of gRNA for retinoblastoma 1 (RB1) and RBL2. (E) Immunoblotting of indicated proteins in MCF10A cells. Cells were transduced with lentivirus harboring tetracycline‐inducible mutant RAS and subjected to selection with 200 μg/mL hygromycin under DOX (−) condition. Then, cells were transduced with RBsh lentivirus and subjected to selection with puromycin. After selection, cells were treated with 1 μg/mL doxycycline for 24 h and analyzed by immunoblotting. (F) Phase contrast image of MCF10A cells in sphere assay. Scale bar: 200 μm. (G) Quantitative analysis of sphere number (sphere size >25,000 μm², N = 3). *p < 0.05, ***p < 0.001, ****p < 0.0001.