Not just protein factories : role of ribosome biogenesis as an architect of epithelial-mesenchymal transition and breast cancer progression

Abstract

RNA polymerase I (Pol I)-mediated transcription of ribosomal DNA (rDNA) is considered to be the rate-limiting step in ribosome biogenesis and is a well-known hallmark of cell growth and proliferation. The process of synthesizing new ribosomes is executed by the coordination of multiple complex processes in the nucleolus. The initial step of transcribing 47S ribosomal RNA (rRNA) transcript by the Pol I complex is followed by its processing into 28S, 18S and 5.8S rRNAs. These transcripts, together with 5S rRNA transcribed by Pol III and auxiliary proteins transcribed by Pol II, proceed to form a mature ribosome after being exported into the cytoplasm. Regulation of ribosome biogenesis occurs in a cell cycle dependent manner, and actively transcribing nucleolar organizing regions (NORS) indicating active rDNA transcription have been associated with tumor proliferation and poor prognosis in cancer patients. Numerous oncogenic and tumor suppressive pathways modulate tumor growth through rDNA transcription.

We have previously shown that the tumor suppressive effects of Wnt5a is mediated though suppression of rDNA transcription by recruitment of Dishevelled 1 (DVL1) to the nucleolus and the rDNA gene cassette. In this thesis, we show that de novo ribosome biogenesis is essential for the epithelial-to-mesenchymal transition (EMT), which is indispensable for embryonic development and for the acquisition of migratory phenotype during cancer progression. The induced de novo rRNA synthesis occurring in the absence of cell proliferation is mediated by increased recruitment of Pol I complex components and EMT transcription factor Snail1 to the rDNA gene cassette. This is accompanied by the opening of the otherwise silenced rDNA operons by the release of TTF-I interacting protein 5 (TIP5), a major component of the repressive nuclear chromatin remodeling NoRC complex, from the rDNA. Pharmacological inhibition of rRNA synthesis by the small molecule CX-5461 reduced the invasive capacity of cells in vitro, which correlated with a decrease in mesenchymal proteins, together confirming an important role of de novo ribosome biogenesis in EMT. In accordance with previous literature that have shown association of the mTORC2 complex with ribosomes, expression of Rictor, a mTORC2 complex component, was found to be induced in the nucleolus during EMT. This association of Rictor was observed to be rRNA dependent. Furthermore, inhibition of ribosome biogenesis significantly reduced the nucleolar expression of Rictor. Mouse models of metastatic breast cancer showed reduced tumor volume upon treatment with CX-5461 and a significant reduction in lung metastasis was observed. Interestingly, CX-5461 treated primary tumors were also more differentiated, as they had increased expression of cytokeratin 8/18, and were also Estrogen Receptor-alpha (ERα) positive and Rictor-negative, which altogether correlates with a less aggressive phenotype in 2 the MMTV-PyMT mouse tumor model.

Further investigation into the driving mechanism of EMT by de novo ribosome biogenesis revealed pervasive changes in the translational control of gene expression program during EMT. This translational control during EMT was affected by inhibition of de novo rRNA synthesis by the Pol I assembly inhibitor, CX-5461. Though the transcriptional profiles remained the same, about 1478 genes were differentially expressed in the ribosome protected fragments during EMT. CX-5461 treatment blocked the upregulation of 185 and the downregulation of 179 translationally controlled genes. The expression of the translationally controlled genes post-TGFβ stimulation significantly overlapped with the translationally controlled genes affected by CX-5461. Interestingly, a translational reprogramming of the mTORC1 signaling cascade was observed during EMT, revealed by the downregulation of transcripts with short UTRs. This reprogramming was diminished by the inhibition of Pol I mediated rDNA transcription. These findings collectively provide compelling evidence that the EMT-associated ribosome biogenesis program, and by extension the ribosomes generated by this process, fuel the pro-migratory, pro-invasive gene expression program underpinning EMT and thus the mesenchymal phenotype. They also demonstrate compelling evidence that rRNA biogenesis plays a unique and targetable role in metastatic breast cancer development, progression and metastasis.