Outgrowth of single oncogene-expressing cells from suppressive epithelial environments

Abstract

Tumorigenesis is a clonal evolution process that is initiated from single cells within otherwise histologically normal tissue. It is unclear how single, sporadic mutant cells that have sustained oncogenic alterations evolve within a tightly regulated tissue environment. Here we investigated the effects of inducing oncogene expression in single cells in organotypic mammary acini as a model to elucidate the processes by which oncogenic alterations initiate clonal progression from organized epithelial environments. Sporadic cells induced to overexpress oncogenes that specifically perturb cell-cycle checkpoints (for example, E7 from human papilloma virus 16, and cyclin D1), deregulate Myc transcription or activate AKT signalling remained quiescent within growth-arrested acini. By contrast, single cells that overexpress ERBB2 initiated a cellular cascade involving cell translocation from the epithelial layer, as well as luminal outgrowth that is characteristic of neoplastic progression in early-stage epithelial tumours. In addition, ERBB2-mediated cell translocation to the lumen was found to depend on extracellular-regulated kinase and matrix metalloproteinase activities, and genetic alterations that perturb local cell-matrix adhesion drove cell translocation. We also provide evidence that luminal cell translocation may drive clonal selection by promoting either the death or the expansion of quiescent oncogene-expressing cells, depending on whether the pre-existing alterations allow anchorage-independent survival and growth. Our data show that the initial outgrowth of single oncogene-expressing cells from organized epithelial structures is a highly regulated process, and we propose that a cell translocation mechanism allows sporadic mutant cells to evade suppressive micro-environments and elicits clonal selection for survival and proliferative expansion outside the native niches of these cells.

Figure 1. Single-cell induction of oncogenic alternation in mammary acinar culture

a) Representative images of Day16 MCF10A acini. Left: Low magnification phase contrast image. Scale bar, 50mm. Middle and right: Representative acini immunostained with laminin-γ2 (LAMC2, red) and GM130 (green). Scale bars, 10µm. b) Schematic of single-cell lentiviral infection and doxycycline (Dox)-induction of oncogenes and reporters (green) in growth-arrested, polarized acini. c) Representative images and d) quantification of clonal expansion eight days after induction. e) Representative images of 3D confocal reconstructions of infected acini. f) Acini with expanded ErbB2-clones in lumen were immunostained for GFP (ErbB2- cells), laminin-γ2 (LAMC2), α₆-integrin (ITGA6), E-cadherin (Ecad), or ErbB2. Nuclei were counterstained with DAPI (blue). Nuclei of GFP-expressing clones are outlined in dotted white lines in some cases to aid visualization. Raw data are shown in Supplementary Table 1. Error bars represent standard deviations of four experiments. Asterisks denote statistically significant change calculated using two-tailed t-tests. Scale bars, 10µm.

Figure 2. Mechanisms of luminal cell translocation

a) Confocal sections from time series of acini (red nuclei) containing single ErbB2- or GFP-cell (green) captured starting from approximately 24 hours after induction (0h). b) Representative images and quantification of c) translocation and d) single ErbB2-cells in lumen of Day16 acini treated with aphidicolin (10µM) or DMSO for eight days. e) Representatives images and f) quantification of ErbB2-cells translocation in 3D acini treated with MEK inhibitor (PD325901, 1µM), PI3K/mTor inhibitor (LY294002, 20µM), or DMSO for eight days. g) Representative images and h) quantifications of luminal translocation of MEK2DD- or myrAKT1-cells from growth-arrested acini. Nuclei of GFP-expressing clones are outlined in dotted white lines to aid visualization. Raw data are shown in Supplementary Table 2. Error bars represent standard deviations of four experiments. Asterisks denote statistically significant change calculated using two-tailed t-tests. Scale bars, 10µm.

Figure 3. Luminal translocation and clonal outgrowth from suppressive epithelial environment

a) Representative images, and quantification of b) luminal translocation and c) single-cell in lumen eight days after induction of Ets1 and MT1-MMP expression in 3D acini. d) Representative images of ErbB2 cells/clones and quantification of e) translocation and f) single-cells in epithelial layer of acini treated with GM6001, Batimastat, MMP inhibitor III, or DMSO for eight days. g) Representative images and h) quantification of translocation of talin-1 knockdown cells in 3D acini. Western blot indicates efficient talin-1 knockdown. Similar results were obtained by a different talin-1 knockdown construct (data not shown). Raw data are shown in Supplementary Table 3. Error bars represent standard deviations of three-four experiments. Asterisks denote statistically significant change calculated using two-tailed t-tests. Scale bars, 10µm.

Figure 4. Cell translocation provokes clonal selection or outgrowth of quiescent mutant cells

a–c) Single cells within Day16 MCF10A/pLT-MT1-MMP-iGSP or MCF10A/pLT-iGSP (IRES-GFP) acini were infected with pLT-Myc-iCSA, pLT-myrAKT1-iCSA or pLT-iCSA (IRES-mCherry) to inducibly drive oncogenes and reporters co-expression. b) Representative images and c) quantifications of Myc/mCherry, myrAKT1/mCherry, or mCherry cells/clones co-expressing MT1-MMP/GFP or GFP control (yellow) 12 days after doxycycline induction. Nuclei (DAPI, blue) of MT1-MMP/myrAKT1 clones are outlined with white dotted line. Quantification of translocated cells/clones for c) proliferation and d) apoptosis. e) Acini with Myc/MT1-MMP, Ets1-, and MT1-MMP-expressing cells were immunostained with antibody against cleaved Caspase-3 eight days after induction. f–g) Single cells within pre-formed, Day16 MCF10A/pTRIPZ-p120KD or MCF10A/pTRIPZ-NS (non-silencing) acini were infected with pLT-Myc-iG, pLT-myrAKT-iG, pLT-ErbB2-iG or pLT-iG. Acini were induced with doxycycline to drive expression of p120KD or NS shRNA in all cells (red) with co-expression of Myc, myrAKT1, ErB2, or GFP in single cells (green). Acini were induced with doxycycline for 48 hours to express p120KD or NS shRNA before infection with pLT-ErbB2-iG. f) Representative images and g) quantification of expanded clones in epithelial layer eight days after induction. Raw data are shown in Supplementary Table 4. Error bars represent standard deviations of four experiments. Asterisks denote statistically significant change calculated using two-tailed t-tests. Scale bars, 10µm.