REST knockdown enhances tumorigenic phenotypes in vitro
Recently we demonstrated that REST is lost in 20% of human breast cancers, and that these RESTless tumors are highly aggressive(1
). Here, we demonstrate that REST loss drives tumor aggression. We used lentiviral delivery of shRNAs to generate stable control (RESTnorm
) or REST knockdown cell lines (RESTlow
cells; the term “RESTless” refers only to human tumors). We knocked down REST in a non-transformed breast cell line (MCF10A), and in estrogen receptor positive (ERα+) and triple negative (ER-/PR-/Her2-) breast cancer cell lines (MCF7 and MDA-MB-231, respectively); REST knockdown was confirmed by western blot ( and previously shown(1
REST knockdown enhances tumorigenicity of cells in culture.
We first tested the tumorigenicity of RESTlow
breast cells using a clonogenic assay(23
), which estimates the ability of individual cells to form colonies. In all three cell lines tested, REST knockdown significantly increased plating efficiency ().
Using an ERα+ (MCF7) and an ER negative (ER-; MCF10A) cell line, we repeated the clonogenicity assay using a soft agar protocol. In both cell lines, REST knockdown significantly increased (>3-fold) the number of colonies observed ().
REST knockdown increases tumor growth in mice
We performed xenograft experiments to test the in vivo tumorigenicity of RESTnorm and RESTlow MCF7 cells. Work that will be described elsewhere suggested that RESTlow MCF7 cells would show enhanced growth in vivo in the absence of estrogen supplementation; RESTnorm or RESTlow MCF7 cells were injected subcutaneously into the flanks or mammary fat pads of intact female athymic Foxn1nu mice, and tumor growth was monitored. 200 days post-injection, 30% (8/28) of RESTlow mammary fat pad injection sites developed tumors (1980 mm3 tumor volume), compared with 0% (0/28, 0 mm3 tumor volume) of RESTnorm injections (). The tumor take rate was also significantly greater for RESTlow versus RESTnorm cells injected subcutaneously into the flanks of the mice, with 34.4% (11/32) of RESTlow injection sites giving rise to tumors, compared to 12.5% (4/32) of RESTnorm sites (). The total tumor burden for flank tumors was significantly greater for RESTlow than RESTnorm tumors, at 4314mm3 and 1017mm3, respectively (). REST knockdown therefore results in a significant increase in tumorigenicity of MCF7 cells at both orthotopic and non-orthotopic sites.
REST knockdown increases the aggressiveness of MCF7 tumor growth in nude mouse xenografts.
Histopathological examination of RESTlow tumors showed that they were highly anaplastic, with enlarged nuclei, prominent nucleoli, many convoluted nuclear envelopes and a high mitotic rate (). RESTlow tumors contain ~1.5-fold more cells undergoing mitosis than RESTnorm tumors; however, due to the small number of RESTnorm tumors available for analysis (n=3), this difference did not reach statistical significance (p=0.1). 62.5% (5/8) of RESTlow flank tumors examined show localized invasion into adjacent muscle (), and one showed lympho-vascular invasion ().
REST regulates LIN28A expression in breast cancer cells
We hypothesized that one or more of the genes that become simultaneously de-repressed upon REST knockdown(1
) must contribute to RESTless breast tumor aggression. One such gene was LIN28A; given that LIN28A was up-regulated in RESTless cells in vitro
and in vivo
, was a REST target(6
), and was previously implicated in breast cancer progression(19
), we sought to determine whether LIN28A was an effector of the RESTless phenotype. We confirmed previously published microarray data showing that LIN28A mRNA was elevated in the breast cancer cell line T47D upon REST knockdown(1
) by quantitative RT-PCR (); this increase in mRNA correlated with increased LIN28A protein (). LIN28A mRNA and protein are similarly up-regulated upon REST knockdown in MCF7 cells (). REST has been shown to regulate LIN28 in embryonic stem and neural progenitor cells(14
), and the LIN28A promoter region contains an evolutionarily-conserved REST binding site. To determine whether LIN28A is a direct target of transcriptional regulation by REST in our tumor model (MCF7 cells), we performed chromatin immunoprecipitation with an anti-REST antibody and interrogated for the LIN28A RE1 sequence (); a region of DNA that does not contain an RE1 site serves as a negative control (NC). To confirm that REST binding to the putative RE1 site was ablated by REST knockdown, this ChIP was performed in both RESTnorm
MCF7 cells. We find robust enrichment of REST at the LIN28A RE1 site in RESTnorm
MCF7 cells; indeed, REST enrichment at the LIN28A RE1 is twice that at the BDNF RE1, a well-characterized REST binding site(25
). As expected, upon REST knockdown, REST binding enrichment is lost at both the BDNF and LIN28 RE1 sites. To demonstrate the specificity of REST binding and co-repressor recruitment to the LIN28 RE1 site we performed a ChIP using an anti-REST antibody or anti-G9a (a REST co-repressor(12
)) antibody, and sham IgG; the ChIP DNA was analyzed via qRT-PCR using primers spaced every 2kb from the LIN28A transcriptional start site (TSS) to 12kb upstream, and primers 8kb downstream of the TSS. REST and G9a enrichment is found exclusively at the LIN28A RE1 site and not elsewhere in the vicinity of the LIN28A gene ().
REST is a direct transcriptional repressor of LIN28A in breast cancer cells.
To determine whether REST directly represses transcription by binding to the LIN28A RE1 site, we transfected a luciferase reporter containing ~2kb of the LIN28A promoter region including (+RE1) or excluding (−RE1) the REST binding site into RESTnorm and RESTlow MCF7 and HEK cells. As predicted, luciferase activity was decreased by greater than 50% in the presence (+RE1) compared to the absence (−RE1) of the RE1 site in RESTnorm cells; this repression was lost in RESTlow cells (). This repression therefore requires both REST and the REST binding site. These data indicate that REST directly represses transcription from the LIN28A promoter region by binding to the RE1 site.
To test whether the regulation of LIN28A by REST observed in vitro was also present in vivo, we measured LIN28A RNA levels in RESTnorm and RESTlow MCF7 tumor tissue. Both LIN28A mRNA, as measured by two different primer pairs (pair 1 and pair 2) and LIN28A immature RNA (pre-mRNA), measured using primers in the second intron, are significantly increased in RESTlow versus RESTnorm MCF7 tumors (). None of the housekeeping genes analyzed show significant differences in expression between RESTnorm and RESTlow tumors.
RESTlow in vitro phenotypes are LIN28A-dependent
To evaluate the contribution of LIN28A to the RESTlow phenotype, RESTnorm and RESTlow MCF7 cells were transduced with a lentiviral construct expressing an anti-LIN28A shRNA (LIN28Alow) or control shRNA (LIN28Anorm), and LIN28A knockdown was verified (); the anti-LIN28A shRNA used does not knock down LIN28B (data not shown). We then performed a soft agar colony formation assay and found that the increase in colony formation observed upon REST knockdown is ablated by concurrent knockdown of LIN28A ().
LIN28A is necessary and sufficient for RESTlow phenotypes in vitro.
We then examined the effects of REST and LIN28A on the growth rate of estrogen receptor negative MDA-MB-231 cells. When cells were grown in high serum (10% FBS), there was no significant difference in growth rate between RESTlow
cells; however, LIN28A knockdown significantly retarded the growth of RESTlow
but not RESTnorm
cells (), suggesting that the RESTlow
cells have acquired a dependence upon LIN28A that is not present in RESTnorm
cells. Because MDA-MB-231 cells proliferate at near-maximal rate in the presence of 10% FBS, we then performed the growth assay using low-serum (2.5%) conditions previously shown to unmask growth phenotypes in MDA-MB-231 cells(21
). Under low-serum conditions, RESTlow
cells show increased proliferation relative to RESTnorm
cells, but lose their growth advantage upon LIN28A knockdown ().
LIN28A is sufficient to recapitulate REST knockdown phenotypes in MCF7 cells
To determine whether LIN28A over-expression is a functional effector of the RESTlow phenotypes, LIN28A was stably over-expressed in MCF7 cells (). LIN28A over-expression caused a ~2× increase in plating efficiency and colonies grown in soft agar (). We therefore conclude that in MCF7 cells, LIN28A over-expression is sufficient to induce the cell culture phenotype of RESTlow cells.
LIN28A contributes to RESTless tumor formation
To determine whether the up-regulation of LIN28A observed in RESTlow cells contributes to the tumorigenicity observed in vivo, we compared the tumorigenicity of RESTlow cells with and without LIN28A expression. RESTlowLIN28Anorm or RESTlowLIN28Alow MCF7 cells were injected subcutaneously into the mammary fat pads of athymic nude mice as for . After 100 days, 50% (6/12) of RESTlowLIN28Anorm mammary fat pad injections had given rise to tumors, compared with only 8.3% (1/12) of fat pads injected with RESTlowLIN28Alow cells (). The tumor burden in the mammary fat pads was also significantly decreased when LIN28A was knocked down, with a total tumor volume of 345mm3 for RESTlowLIN28Anorm compared to only 56mm3 for RESTlowLIN28Alow tumors ().
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. (more ...)
LIN28A expression is increased in human RESTless breast tumors
Given the dependence of tumor growth on LIN28A in RESTlow
cells in xenograft assays, we assessed LIN28A levels in human RESTless tumors. We analyzed publicly-available cDNA microarray data from two breast tumor cohorts, GSE2034(28
) and GSE2990(30
) and classified ER+ tumors as RESTless or REST-expressing (RESTfl) using the 24-gene signature method previously described(1
) (Supplemental Fig 1A
). In these two independent breast tumor cohorts, LIN28A expression was significantly higher in RESTless versus RESTfl tumors (). The list of tumors in each dataset defined as RESTless or RESTfl is provided (Supplemental Table 1
). Our results strongly argue that LIN28A over-expression contributes to the previously described aggression of RESTless tumors(1
LIN28A mRNA is increased in human RESTless tumors.