Enhanced RNA polymerase III-dependent transcription is required for oncogenic transformation

Abstract

RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5 S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. Although it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that Rat1a fibroblasts undergoing oncogenic transformation by the TATA-binding protein or c-Myc display enhanced RNA pol III transcription. Decreased expression of the RNA pol III-specific transcription factor Brf1 prevented this increase in RNA pol III transcription. Although the overall proliferation rates of these cells remained unchanged, the ability of cells to grow in an anchorage-independent manner and form tumors in mice was markedly reduced. Although overexpression of Brf1 modestly stimulated RNA pol III transcription, expression of a phosphomimic, Brf1-T145D, more significantly induced transcription. However, these increases in transcription were not sufficient to promote cellular transformation. Together, these results demonstrate that enhanced RNA pol III transcription is essential for anchorage-independent growth and tumorigenesis and that these events can be uncoupled from effects on anchorage-dependent proliferation.

FIGURE 1.

RNA pol III transcription is increased by enhanced TBP expression. A, stable cell lines expressing increased TBP or mutant TBP proteins. Protein lysates from Rat1a cells stably transfected with empty vector or expression plasmids encoding HA-tagged wild-type or mutant (L185E and K249E) human (H) TBP cDNAs were subjected to immunoblot analysis using antibody against HA, TBP, or β-actin. B, RNA pol III gene activity is increased in cell lines expressing increased TBP but not TBP-L185E or TBP-K249E. RT-qPCR was performed using RNA isolated from stable cell lines in A with primers for pre-tRNA^Leu, 7SL RNA, and GAPDH. Values are the means ± S.E. (n ≥ 3). C, RNA pol III transcription is enhanced in tumors derived from stable cell lines expressing increased TBP or TBP-L185E. RT-qPCR was performed as described for B using RNA isolated from tumors from mice injected with the vector, TBP, and TBP-L185E cell lines. D, relative levels of TBP, Brf1, and Bdp1 mRNAs in tumors derived from stable cell lines. RT-qPCR using primers for murine TBP, rat Brf1, rat Bdp1, HA-tagged human TBP, and GAPDH was performed with RNA from tumors in C. -Fold changes were calculated based on GAPDH-normalized transcript levels in the tumors derived from the vector cell line (rat TBP, rat Brf1, and rat Bdp1) or TBP cell line (HA-tagged human TBP). Values are the means ± S.E. (n ≥ 4) from tumors from two different mice (C and D). E, tumor cells expressing TBP-L185E recultured in vitro display reduced RNA pol III transcription capacity. Stable cell lines were established from tumors from mice injected with the vector, TBP, and TBP-L185E cell lines. Following propagation in selection medium, total RNA was isolated, and RT-qPCR was performed as described for B. Values are the means ± S.E. (n ≥ 4) from two independently derived cell lines.

FIGURE 2.

Tumor formation is enhanced by increased TBP or mutant TBP expression. A, proliferation rates are not altered by changes in RNA pol III transcription. Stable cell lines on duplicate plates were trypsinized and counted daily. A representative plot of three independent experiments is shown. B, enhanced expression of TBP, but not mutant TBP proteins, promotes anchorage-independent growth. Stable cell lines in A or cells expressing c-Myc were analyzed for growth in soft agar. Values are the means ± S.E. (n ≥ 3). C, tumor formation in mice with cells overexpressing TBP or mutant TBP proteins. Cell lines expressing TBP, TBP-L185E, or TBP-K249E were injected subcutaneously into the groins of nude mice (six mice/group). Representative results from two independent experiments are shown.

FIGURE 3.

Decreased Brf1 expression inhibits TBP-mediated increase in RNA pol III transcription and anchorage-independent growth. A, Brf1 levels in stable cell lines expressing Brf1 shRNA. Protein lysates from vector and TBP stable lines expressing mismatch (mmRNA) or Brf1 shRNA were subjected to immunoblot analysis with anti-Brf1 or anti-β-actin antibodies. B, decreased Brf1 expression abrogates TBP-mediated increases in RNA pol III transcription. RT-qPCR was performed on RNA from infected stable cell lines in A. Values are the means ± S.E. (n ≥ 3). C, accumulation rate of stable lines expressing Brf1 shRNA. Infected stable cell lines in A on duplicate plates were trypsinized and counted daily. A representative plot of three independent experiments is shown. D, down-regulating Brf1 expression reduces TBP-mediated anchorage-independent growth. Cells in A or those expressing c-Myc were analyzed for growth in soft agar. Values are the means ± S.E. (n ≥ 3).

FIGURE 4.

Enhanced RNA pol III transcription is required for TBP-mediated tumorigenesis. A, down-regulating Brf1 expression inhibits TBP-mediated tumorigenesis. Vector and TBP stable cell lines expressing mismatch RNA (mmRNA) or Brf1 shRNA were injected subcutaneously into nude mice (six mice/group), and tumor volumes were determined. B, Brf1 expression remains repressed in tumors from cells expressing Brf1 shRNA. RT-qPCR was performed on RNA from tumors derived from mice injected with stable cell lines using primers for Brf1 or GAPDH. C, RNA pol III transcription remains repressed in tumors from cells expressing Brf1 shRNA. RT-qPCR was performed on RNA from tumors in B using primers for pre-tRNA^Leu, 7SL RNA, or GAPDH. Values are the means ± S.E. (n ≥ 3) from tumors from two different mice per cell line.

FIGURE 5.

Enhanced RNA pol III transcription is not sufficient for promoting anchorage-independent growth. A, analysis of expression of TBP, Brf1, and Brf1-T145D in stable cell lines. Cells were stably transfected with empty vector or expression plasmids encoding HA-Brf1, HA-Brf1-T145D, or HA-TBP. Protein lysates were subjected to immunoblot analysis with anti-HA or anti-β-actin antibody. h, human. B, expression of Brf1-T145D induces RNA pol III-dependent transcription. RT-qPCR was performed using RNA isolated from each cell line in A and primers for pre-tRNA^Leu, 7SL RNA, tRNA^Met_i, and GAPDH. Transcript levels were normalized to GAPDH levels, and -fold change was calculated based on levels in the pCEP4 vector cell line. Values are the means ± S.E. (n ≥ 3). C, enhanced Brf1 or Brf1-T145D expression does not increase anchorage-independent growth. Stable cell lines in A or those expressing c-Myc were analyzed for growth in soft agar. Values are the means ± S.E. (n ≥ 3).

FIGURE 6.

Enhanced RNA pol III transcription is required for c-Myc-mediated transformation and tumorigenesis. A, Brf1 and TBP levels in lines expressing c-Myc and Brf1 shRNA. Vector and c-Myc stable cell lines were infected with lentivirus expressing mismatch RNA (mmRNA; –) or Brf1 shRNA (+). Protein lysates were subjected to immunoblot analysis. B, decreased Brf1 expression abrogates c-Myc-induced RNA pol III transcription. RT-qPCR was performed on RNA isolated from each cell line in A with primers for pre-tRNA^Leu, 7SL RNA, tRNA^Met_i, and GAPDH. Values are the means ± S.E. (n ≥ 3). C, decreased Brf1 expression inhibits c-Myc-mediated anchorage-independent growth. Cell lines in A were analyzed for growth in soft agar. Values are the means ± S.E. (n ≥ 3). D, decreased Brf1 expression inhibits c-Myc-mediated tumorigenesis. Stable cell lines in A were injected subcutaneously into mice (six mice/group). Tumor volumes were determined by measuring the dimensions (height × width × depth) of tumors.