Induction of the RNA regulator LIN28A is required for the growth and pathogenesis of RESTless breast tumors - PubMed (original) (raw)

Induction of the RNA regulator LIN28A is required for the growth and pathogenesis of RESTless breast tumors

Kearney T W Gunsalus et al. Cancer Res. 2012.

Abstract

The transcription factor RE1 silencing transcription factor (REST) is lost in approximately 20% of breast cancers. Although it is known that these RESTless tumors are highly aggressive and include all tumor subtypes, the underlying tumorigenic mechanisms remain unknown. In this study, we show that loss of REST results in upregulation of LIN28A, a known promoter of tumor development, in breast cancer cell lines and human breast tumors. We found that LIN28A was a direct transcriptional target of REST in cancer cells and that loss of REST resulted in increased LIN28A expression and enhanced tumor growth both in vitro and in vivo, effects that were dependent on heightened LIN28A expression. Tumors lacking REST expression were locally invasive, consistent with the increased lymph node involvement observed in human RESTless tumors. Clinically, human RESTless breast tumors also displayed significantly enhanced LIN28A expression when compared with non-RESTless tumors. Our findings therefore show a critical role for the REST-LIN28A axis in tumor aggression and suggest a causative relationship between REST loss and tumorigenicity in vivo.

©2012 AACR.

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Figures

Figure 1

Figure 1

REST knockdown enhances tumorigenicity of cells in culture. A–B, Western blot showing REST knockdown in MCF7 and MDA-MB-231 cells. C–E, Plating efficiency of RESTlow and RESTnorm MCF7, MCF10A and MDA-MB-231 cells in a clonogenic assay (p≤0.050). F–G, Soft agar colony formation assay comparing anchorage-independent growth of RESTlow versus RESTnorm MCF7 and MCF10A cells (p≤0.050).

Figure 2

Figure 2

REST knockdown increases the aggressiveness of MCF7 tumor growth in nude mouse xenografts. A, 106 control (RESTnorm) or REST knockdown (RESTlow) MCF7 cells were injected subcutaneously into the mammary fat pads of female athymic nude mice. Tumor incidence was monitored weekly, and is significantly higher for RESTlow versus RESTnorm cells (p=0.0022). B, Tumor burden in the mammary fat pads is significantly larger in RESTlow versus RESTnorm tumors (p<0.0001). C, 106 control (RESTnorm) or REST knockdown (RESTlow) MCF7 cells were injected subcutaneously into the flanks of female athymic nude mice. Tumor incidence upon injection into flank is significantly higher for RESTlow versus RESTnorm MCF7 cells (p=0.0382). D, Tumor burden in flanks is significantly larger in RESTlow versus RESTnorm tumors (p=0.0016). E–G, Bright field photomicrographs of H&E stained sections of RESTlow tumors. Representative images show the histopathology (E) and local invasion (F) observed in these tumors; arrows in (F) indicate muscle fibers incorporated into the tumor. G, RESTlow tumor and adjacent mammary fat pad; arrows indicate lympho-vascular invasion.

Figure 3

Figure 3

REST is a direct transcriptional repressor of LIN28A in breast cancer cells. A, LIN28A mRNA in RESTlow and RESTnorm T47D cells was analyzed by qPCR and normalized to actin (p=0.05). B, Western blot for LIN28A and β-actin (loading control) in RESTlow and RESTnorm T47D cells. C, LIN28A mRNA in RESTlow and RESTnorm MCF7 cells was analyzed by qPCR and normalized to actin (p=0.0053). D, Immunofluorescent staining for LIN28A in RESTnorm and RESTlow MCF7 cells. E, Chromatin immunoprecipitations (ChIP) in MCF7 cells with anti-REST antibody and IgG (sham). DNA analyzed by qRT-PCR with primers in the LIN28 and BDNF (positive control) promoter regions; negative control (NC) lacks RE1 site. F, ChIP in MCF7 cells using anti-REST or anti-G9a antibody, and IgG (sham). DNA was analyzed via qRT-PCR using primers up- and downstream of TSS. G–I, Schematic representation of the LIN28 promoter region showing placement of primers used to clone the LIN28 promoter region including (+RE1) or excluding (−RE1) the REST binding site into a pGL3 luciferase reporter plasmid, which was transfected into RESTnorm and RESTlow MCF7 (F) and HEK (G) cells. Luciferase expression was normalized to renilla. J, LIN28A immature and processed mRNA in RESTlow versus RESTnorm tumors measured by qPCR (p≤0.04). Housekeeping genes showed no significant difference.

Figure 4

Figure 4

LIN28A is necessary and sufficient for RESTlow phenotypes in vitro. A, Immunoblot for LIN28A or β-actin (loading control) in control (LIN28Anorm) or LIN28A knockdown (LIN28Alow) MCF7 cells. B, Soft agar colony formation of RESTnorm and RESTlow MCF7 cells with and without anti-LIN28 shRNA (LIN28low) (p=0.0383). C–D, Growth of RESTnorm and RESTlow MDA-MB-231 cells with and without anti-LIN28 shRNA (LIN28low) in high (C, 10%) or low (D, 2.5%) serum (72h, p≤0.05). E–G, Lentiviral delivery of a LIN28A expression construct was used to generate LIN28 over-expressing MCF7 cells; expression was verified by western blot (E). Clonogenicity (F) and soft agar colony formation (G) were measured (p≤0.05).

Figure 5

Figure 5

LIN28A contributes to the tumorigenicity of RESTlow MCF7 cells in mice. 106 RESTlow/LIN28Anorm or RESTlow/LIN28Alow MCF7 cells were injected subcutaneously into the mammary fat pads of athymic nude mice, and tumor incidence and growth were monitored weekly. A–B Tumor incidence (A, p=0.020) and burden (B, p=0.0002) are decreased upon LIN28A knockdown.

Figure 6

Figure 6

LIN28A mRNA is increased in human RESTless tumors. A–B, Box and whisker plot of fold median LIN28A mRNA expression in RESTless and REST-containing (“RESTfl”) breast tumors from datasets GSE2034 (A, p<0.0001) and GSE2990 (B, p=0.0050). Lines of the box represent 75th, 50th and 25th percentiles; whiskers represent 90th and 10th percentile of LIN28A expression in each group.

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