Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells

Abstract

Long Interspersed Elements (L1 elements) are biologically active retrotransposons that are capable of autonomous replication using their own reverse transcriptase (RT) enzyme. Expression of the normally repressed RT has been implicated in cancer cell growth. However, at present, little is known about the expression of L1-encoded RT activity or the molecular changes that are associated with RT activity in the development of breast cancer. Here, we report that RT activity is widespread in breast cancer cells. The expression of RT protein decreased markedly in breast cancer cells after treatment with the antiretroviral drug, efavirenz. While the majority of cells showed a significant reduction in proliferation, inhibition of RT was also accompanied by cell-specific differences in morphology. MCF7 cells displayed elongated microtubule extensions that adhered tightly to their substrate, while a large fraction of the T47D cells that we studied formed long filopodia projections. These morphological changes were reversible upon cessation of RT inhibition, confirming their dependence on RT activity. We also carried out gene expression profiling with microarrays and determined the genes that were differentially expressed during the process of cellular differentiation. Genes involved in proliferation, cell migration, and invasive activity were repressed in RT-inhibited cells. Concomitantly, genes involved in cell projection, formation of vacuolar membranes, and cell-to-cell junctions were significantly upregulated in RT-inhibited cells. qRT-PCR examination of the mRNA expression of these genes in additional cell lines yielded close correlation between their differential expression and the degree of cellular differentiation. Our study demonstrates that the inhibition of L1-encoded RT can reduce the rate of proliferation and promote differentiation of breast cancer cells. Together, these results provide a direct functional link between the expression of L1 retrotransposons and the development of breast cancer.

Fig. 1

L1-encoded RT activity in breast cancer cells. a Endogenous RT activity was detected after incubation of synthetic MS2 phage RNA with cell extracts from a panel of breast cancer cells. Control reactions were set up by omitting cell extract (negative control) or adding cell extracts from NTera.2D1 human embryonic carcinoma cells (positive control). The PCR product of 110 bp (corresponding to the reverse-transcribed MS2 cDNA) is shown. Marker, 1 kb-plus DNA marker. b The L1-encoded ORF2p, which contains the RT enzyme, was detected by western blotting of whole-cell lysates from normal and a panel of breast cancer cells. NTera.2D1 were used as positive controls. For protein normalization, α-tubulin was used as a loading control. c The L1 ORF2 mRNAs derived from L1 expression were quantified by qRT-PCR with primer specific for the ORF2 sequence. The data are shown as fold change in each breast cancer panel cells compared to normal MCF10A after normalization to the HPRT1 housekeeping gene. Each point represents the average of three independent experiments, with each experiment performed in triplicate. Error bars indicate SD (n = 3)

Fig. 2

RT inhibition promotes differentiation in breast cancer cell lines. a Western blots of L1-encoded RT protein in normal (MCF10A) and breast cancer (T47D) cells after treatment with 15 and 45 μM efavirenz for 72 h. The analyses of the parental cells (control) and cells treated with DMSO (mock) were performed in parallel. α-tubulin was used as an internal control. b Quantitative real-time RT-PCR analysis of endogenous L1 mRNAs in T47D cells that were treated with 15 and 45 μM efavirenz or DMSO for 72 h. The MCF10A cell line was used as a negative control. The data are shown as the relative fold changes of ORF1 and ORF2 mRNAs with respect to the control HPRT1. The error bars indicate SD (n = 3). Unpaired t test, * p > 0.05 and ** p < 0.001. c Morphological differentiation of normal MCF10A and breast cancer T47D and MDA-MB-231 cells after treatment with 15 μM efavirenz or DMSO (control) for 72 h. Representative panels from phase-contrast microscopy are shown. Bar 50 μM. d Phase-contrast microscopy of weakly-and moderately-invasive breast cancer cells. Cells exposed to DMSO (control) or efavirenz were examined in vivo after 72 h of culture to assess morphological changes. The arrows indicate the appearance of filopodial projections on the cell edges. e Quantitative morphological changes. The percentages of morphological changes in drug-treated cells were calculated by comparison to mock DMSO-treated cells or parental cells in 100 cells from ten randomly selected fields. Each point represents an average from two independent experiments. P values were calculated by unpaired t test in comparisons to mock-treated and parental cells. Error bars indicate SD

Fig. 3

RT inhibition leads to distinct changes in the surface architecture of cells. a Scanning electron microscopy images illustrates the formation of filopedial projections in DMSO-treated control and RT-inhibited T47D cells. b Immunofluorescence of T47D and MCF7 cells in the absence (control) or presence of RT inhibition (efavirenz). Cells stained with anti-tubulin (green) and DAPI staining of nucleus (blue) are shown. Bar represents 100 μM

Fig. 4

RT inhibition reduces proliferation in breast cancer cells. a 1 × 10³ cells were seeded in 96-well plates, cultured with or without efavirenz at the concentration of 15 μm for 72 h and color formation measured at absorbance 492 nm after 2, 6, and 20 h incubation with XTT. Analysis of parental cells was performed in parallel. The reference wavelength was measured at the absorbance 690 nm. The staining intensity represents the ratio of absorbance at 492 and 690 nm. Error bars indicate SD of three independent assays. b An equivalent number of cells were cultured in the presence of efavirenz for 72 h and the cells then counted and replated in drug-free medium for 48 h. Proliferation in response to drug-free medium was measured using an ELISA reader after staining with XTT. The (*) and (**) symbols denote a significant difference compared to drug-treated cells, with a p value < 0.05 and <0.001, respectively

Fig. 5

RT inhibition modulates gene expression. a Heatmap of log2 expression values of genes in the four most significant gene annotation clusters identified by the DAVID analysis from lists of genes that were 2-fold up- or downregulated and with p value for differential expression ≤ 0.05 after Benjamini–Hochberg correction for multiple testing. The color scale corresponds to the degree of fold change. Green upregulated genes; red downregulated genes, black no change. b Confirmation of differentially expressed genes from the microarray expression profiling experiments. Relative levels of differentially expressed genes were quantified by qRT-PCR with primers specific for their sequences. The data are shown as the fold change compared to mock-treated cells after normalization to the HPRT1 housekeeping gene. Data are expressed as mean ± SD (n = 4)