Investigation of the role and regulation of RNA polymerase I transcription in cancer

Abstract

Aberrant transcription driven by oncogenic genetic lesions is central for malignant transformation. Among the three major classes of transcription, transcription of ribosomal RNA (rRNA) genes by RNA polymerase I (Pol I) provides the catalytic and structural rRNAs essential for ribosome formation and function. Synthesis of rRNA by Pol I is rate-limiting for ribosome biogenesis, thus directly governs protein synthesis capacity critical for cell fate decisions including cell cycle progression, apoptosis, and differentiation. Therefore, precise regulation of Pol I transcription is crucial to maintain cellular homeostasis. Hyperactive Pol I transcription, and upregulation of its basal transcriptional machinery are common features of cancer. Mutations that result in the aberrant activity of tumor suppressors and/or oncogenes accelerate Pol I transcription, suggesting that elevated rRNA synthesis is a pre-requisite for oncogenesis. Initiation of Pol I transcription requires the assembly of class-specific basal transcription factors (bTFs) including, upstream binding factor (UBF), selectivity factor 1 [SL1, a complex of TATA-binding protein (TBP), and four TBP-associated factors (TAFs) TAF1A, TAF1B, TAF1C, and TAF1D)], and RRN3-associated Pol Iβ at the rDNA promoter into a pre-initiation complex (PIC). SL1 recognizes and binds to the rDNA promoter, stabilizes UBF binding, and recruits Pol Iβ to the transcription start site via RRN3. These protein - protein and protein - ribosomal DNA (rDNA) interactions are direct targets of pro-survival cues, and regulated by tumor suppressors such as pRb, p53, and proto-oncogenes including c-MYC, AKT and mTOR. Obviously, oncogenic aberrations perturbing this regulation enhance PIC activity on the rDNA promoter, resulting in unrestrained rRNA synthesis. Recent studies have provided crucial insights pertaining to the role of Pol I transcription in cancer. Notably, elevated Pol I transcription has been attributed to adverse prognosis, chemoresistance, and epithelial–mesenchymal transition (EMT) in cancer. Intriguingly, loss of a highly conserved m1acp3Ψ modification of 18S rRNA has been reported as a major oncogenic feature. In addition, upregulation of the expression of PIC components has been identified in multiple cancers, however, the underlying molecular mechanisms remain undetermined. Collectively, upregulation of Pol I transcription and its machinery may have compelling role(s) in tumorigenesis, beyond facilitating ribosome biogenesis, and plausibly a pre-requisite for malignant transformation. However, the direct role(s) of upregulated Pol I transcription and its transcriptional machinery contributing to oncogenesis remain undetermined. These yet to be deciphered crucial mechanisms forms the basis of this thesis work, as below. 1. What are the underlying molecular mechanisms for the upregulation of PIC components in cancer and their consequences in carcinogenesis? 2. Whether upregulated rRNA synthesis has any independent roles in malignant transformation of a cell? 3. What are the individual roles of SL1 components in Pol I transcription and their implications in cancer? Results: 1. What are the molecular mechanisms underlying the upregulation of PIC components in cancer and their consequences in carcinogenesis? To investigate the mechanism(s) underlying the upregulation of Pol I transcription in cancer, the genetic aberrations of the basal PIC components were examined using The Cancer Genome Atlas (TCGA) database. The analysis revealed that the PIC components are altered in approximately 46.9% of lung adenocarcinoma (LUAD) cases with mRNA high as major genetic alteration. Differential expression analysis of PIC components using TCGA data revealed a significant upregulation in LUAD compared to control samples. Further, the expression of PIC components and 47S rRNA in LUAD patient samples were examined and results revealed a significant upregulation compared to control. Notably, the upregulation of PIC components significantly correlated with the major hallmarks of cancer and showed similar expression trends to that of LUAD driver genes, EGFR and KRAS. Preliminary analysis ruled out epigenetic changes as a causal factor for the upregulation of PIC components and suggesting a transcriptional or post-transcriptional regulatory mechanisms. Dysregulated microRNAs (miRs) alter the target genes expression which has implications in carcinogenesis. We questioned whether the dysregulation of miRs could plausibly contribute to the upregulation of Pol I transcription in LUAD. To investigate this, we first predicted the putative miRs targeting TAF1A, TAF1B, TAF1C, RRN3, and POLR1B transcripts using miRWalk. Among all the miRs, miR-330-5p and miR-1270 were predicted to target SL1 components TAF1B and TAF1C, and TAF1A, RRN3, and POLR1B, respectively. Next, we verified the expression of these miRs in LUAD patient samples. MiR-330-5p and miR-1270 expression was significantly reduced in LUAD tumor samples compared to adjacent normal control (NAT). Further, a significant negative correlation was observed between the expression of miR-330-5p and miR1270 and their respective target genes in LUAD tumors compared to NAT. Overexpression of miR-330-5p or miR-1270 in LUAD cell lines A549 and H23 resulted in a significant downregulation of their respective target genes at mRNA and protein levels. Based on these results, using in-silico, in-vitro and in-vivo approaches, we established that the loss of miR-330-5p and miR-1270 expression contributes to the upregulation of Pol I transcription in LUAD. Constitutive overexpression of these miRs in LUAD cell lines suppressed the expression of core components of Pol I transcription and reduced global rRNA synthesis. Importantly, miR-330-5p/miR-1270-mediated repression of Pol I transcription exerted multiple tumor suppressive functions including reduced proliferation, cell cycle arrest, enhanced apoptosis, reduced migration, increased drug sensitivity, and reduced tumor burden in a mouse xenograft model. Mechanistically, the downregulation of miR-330-5p and miR-1270 is regulated by Pol I subunit-derived circular RNA circ_0055467 and DNA hypermethylation, respectively. This study uncovers a novel miR-330-5p/miR-1270 mediated posttranscriptional regulation of Pol I transcription and establish tumor suppressor properties of these miRs in LUAD. Ultimately, these crucial findings provide a rationale for the therapeutic targeting of Pol I transcriptional machinery for LUAD. 2. Whether upregulated rRNA synthesis has any independent role(s) in malignant transformation of a cell? Hyperactive Pol I transcription is a hallmark feature of cancer. To investigate the direct role(s) of Pol I transcription system in tumorigenesis, we constitutively overexpressed Pol I-specific transcription factor TAF1C to endogenously upregulate rRNA synthesis. TAF1C-mediated upregulation of Pol I transcription in non-malignant lung epithelial cells (BEAS-2B) alone increased proliferation, reduced apoptosis, promoted migration, and strikingly, caused anchorage-independent growth. Based on these proofs-ofconcept, we embarked on determining the underlying gene expression changes by whole transcriptome analysis. Computational analysis of RNA-Seq data revealed that the upregulation of Pol I transcription caused global gene expression changes (coding and non-coding) that showed functional implications in cancer hallmarks. Intriguingly, TAF1C transcriptionally upregulated c-MYC expression. These novel finding provides evidence for the functioning of Pol I machinery outside of rRNA synthesis, and lays foundation for the functional crosstalk between Pol I and Pol II transcription systems during oncogenesis. Our findings rationalize Pol I transcription as a therapeutic target for broad-spectrum of cancers. 3. What are the individual roles of SL1 components in Pol I transcription and their implications in cancer? Our findings suggested that targeting and overexpression of SL1 subunits have implications in cancer. The role of SL1 as a complex in Pol I transcription is known, however, the role of individual TAF subunits in Pol I transcription and its implications in cancer remains elusive. To understand the independent roles of SL1 subunits in-vivo, we devised an auxin-inducible CRISPR-Cas9 system to generate conditional knockouts of individual TAF subunits. Currently, we are validating the stable knockouts, and further studies will be conducted to determine the individual roles of TAFs in and out of Pol I transcription. The outcomes of this study will provide novel drug targets to inhibit Pol I transcription for cancer therapy.