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TGFβ functions as a tumor suppressor or promoter, depending on the context, making TGFβ a useful predictive biomarker. Genes related to TGFβ signaling and Aurora kinase were tested for their ability to predict the progression risk of primary prostate tumors. Using data from The Cancer Genome Atlas (TCGA), we trained an elastic-net regularized Cox regression model including a minimal set of gene expression, copy number (CN), and clinical data. A multi-step feature selection and regularization scheme was applied to minimize the number of features while maintaining predictive power. An independent hold-out cohort was used to validate the model. Expanding from prostate cancer, predictive models were similarly trained on all other eligible cancer types in TCGA. AURKA, AURKB, and KIF23 were predictive biomarkers of prostate cancer progression, and upregulation of these genes was associated with promotion of cell-cycle progression. Extending the analysis to other TCGA cancer types revealed a trend of increased predictive performance on validation data when clinical features were complemented with molecular features, with notable variation between cancer types and clinical endpoints. Our findings suggest that TGFβ signaling genes, prostate cancer related genes and Aurora kinases are strong candidates for patient-specific clinical predictions and could help guide personalized therapeutic decisions.

Transforming growth factor β (TGF-β) is a multifunctional cytokine regulating homeostasis and immune responses in adult animals and humans. Aberrant and overactive TGF-β signaling promotes cancer initiation and fibrosis through epithelial–mesenchymal transition (EMT), as well as the invasion and metastatic growth of cancer cells. TGF-β is a key factor that is active during hypoxic conditions in cancer and is thereby capable of contributing to angiogenesis in various types of cancer. Another potent role of TGF-β is suppressing immune responses in cancer patients. The strong tumor-promoting effects of TGF-β and its profibrotic effects make it a focus for the development of novel therapeutic strategies against cancer and fibrosis as well as an attractive drug target in combination with immune regulatory checkpoint inhibitors. TGF-β belongs to a family of cytokines that exert their function through signaling via serine/threonine kinase transmembrane receptors to intracellular Smad proteins via the canonical pathway and in combination with co-regulators such as the adaptor protein and E3 ubiquitin ligases TRAF4 and TRAF6 to promote non-canonical pathways. Finally, the outcome of gene transcription initiated by TGF-β is context-dependent and controlled by signals exerted by other growth factors such as EGF and Wnt. Here, we discuss the synergistic cooperation between TGF-β and hypoxia in development, fibrosis and cancer.

Background: Transforming growth factor β (TGFβ) is overexpressed in several advanced cancer types and promotes tumor progression. We have reported that the intracellular domain (ICD) of TGFβ receptor (TβR) I is cleaved by proteolytic enzymes in cancer cells, and then translocated to the nucleus in a manner dependent on the endosomal adaptor proteins APPL1/2, driving an invasiveness program. How cancer cells evade TGFβ-induced growth inhibition is unclear.

Methods: We performed microarray analysis to search for genes regulated by APPL1/2 proteins in castration-resistant prostate cancer (CRPC) cells. We investigated the role of TβRI and TRAF6 in mitosis in cancer cell lines cultured in 10% FBS in the absence of exogenous TGFβ. The molecular mechanism of the ubiquitination of AURKB by TRAF6 in mitosis and the formation of AURKB–TβRI complex in cancer cell lines and tissue microarrays was also studied.

Findings: During mitosis and cytokinesis, AURKB–TβRI complexes formed in midbodies in CRPC and KELLY neuroblastoma cells. TRAF6 induced polyubiquitination of AURKB on K85 and K87, protruding on the surface of AURKB to facilitate its activation. AURKB–TβRI complexes in patient's tumor tissue sections correlated with the malignancy of prostate cancer.

Interpretation: The AURKB–TβRI complex may become a prognostic biomarker for patients with risk of developing aggressive PC.

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ₂) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3′-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

A wide variety of factors are required for the conversion of pre-tRNA molecules into the mature tRNAs that function in translation. To identify factors influencing tRNA biogenesis, we previously performed a screen for strains carrying mutations that induce lethality when combined with a sup61-T47:2C allele, encoding a mutant form of tRNA_CGA^Ser. Analyzes of two complementation groups led to the identification of Tan1 as a protein involved in formation of the modified nucleoside N⁴-acetylcytidine (ac⁴C) in tRNA and Bud13 as a factor controlling the levels of ac⁴C by promoting TAN1 pre-mRNA splicing. Here, we describe the remaining complementation groups and show that they include strains with mutations in genes for known tRNA biogenesis factors that modify (DUS2, MOD5 and TRM1), transport (LOS1), or aminoacylate (SES1) tRNA_CGA^Ser. Other strains carried mutations in genes for factors involved in rRNA/mRNA synthesis (RPA49, RRN3 and MOT1) or magnesium uptake (ALR1). We show that mutations in not only DUS2, LOS1 and SES1 but also in RPA49, RRN3 and MOT1 cause a reduction in the levels of the altered tRNA_CGA^Ser. These results indicate that Rpa49, Rrn3 and Mot1 directly or indirectly influence tRNA_CGA^Ser biogenesis.

Messenger RNAs are transcribed and co-transcriptionally processed in the nucleus, and transported to the cytoplasm. In the cytoplasm, mRNAs serve as the template for protein synthesis and are eventually degraded. The removal of intron sequences from a precursor mRNA is termed splicing and is carried out by the dynamic spliceosome. In this thesis, I describe the regulated splicing of two transcripts in Saccharomyces cerevisiae. I also describe a study where the mechanisms that control the expression of magnesium transporters are elucidated.

The pre-mRNA retention and splicing (RES) complex is a spliceosome-associated protein complex that promotes the splicing and nuclear retention of a subset of pre-mRNAs. The RES complex consists of three subunits, Bud13p, Snu17p and Pml1p. We show that the lack of RES factors causes a decrease in the formation of N⁴-acetylcytidine (ac⁴C) in tRNAs. This phenotype is caused by inefficient splicing of the pre-mRNA of the TAN1 gene, which is required for the formation of ac⁴C in tRNAs. The RES mutants also show growth defects that are exacerbated at elevated temperatures. We show that the temperature sensitive phenotype of the bud13Δ and snu17Δ cells is caused by the inefficient splicing of the MED20 pre-mRNA. The MED20 gene encodes a subunit of the Mediator complex. Unspliced pre-mRNAs that enter the cytoplasm are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway, which targets transcripts that contain premature translation termination codons. Consistent with the nuclear retention function of the RES complex, we find that NMD inactivation in the RES mutants leads to the accumulation of both TAN1 and MED20 pre-mRNAs. We also show that the cis-acting elements that promote RES-dependent splicing are different between the TAN1 and MED20 pre-mRNAs.

The NMD pathway also targets transcripts with upstream ORFs (uORFs) for degradation. The ALR1 gene encodes the major magnesium importer in yeast, and its expression is controlled by the NMD pathway via a uORF in the 5’ untranslated region. We show that the ribosome reaches the downstream main ORF by a translation reinitiation mechanism. The NMD pathway was shown to control cellular Mg²⁺ levels by regulating the expression of the ALR1 gene. We further show that the NMD pathway targets the transcripts of the vacuolar Mg²⁺ exporter Mnr2p and the mitochondrial Mg²⁺ exporter Mme1p for degradation.

In summary, we conclude that the RES complex has a role in the splicing regulation of a subset of transcripts. We also suggest a regulatory role for the NMD pathway in maintaining the cellular Mg²⁺ concentration by controlling the expression of Mg²⁺ transporters.

The heterotrimeric pre-mRNA retention and splicing (RES) complex, consisting of Bud13p, Snu17p and Pml1p, promotes splicing and nuclear retention of a subset of intron-containing pre-mRNAs. Yeast cells deleted for individual RES genes show growth defects that are exacerbated at elevated temperatures. Although the growth phenotypes correlate to the splicing defects in the individual mutants, the underlying mechanism is unknown. Here, we show that the temperature sensitive (Ts) growth phenotype of bud13Δ and snu17Δ cells is a consequence of inefficient splicing of MED20 pre-mRNA, which codes for a subunit of the Mediator complex; a co-regulator of RNA polymerase II transcription. The MED20 pre-mRNA splicing defect is less pronounced in pml1Δ cells, explaining why they grow better than the other 2 RES mutants at elevated temperatures. Inactivation of the cytoplasmic nonsense-mediated mRNA decay (NMD) pathway in the RES mutants leads to accumulation of MED20 pre-mRNA, indicating that inefficient nuclear retention contributes to the growth defect. Further, the Ts phenotype of bud13Δ and snu17Δ cells is partially suppressed by the inactivation of NMD, showing that the growth defects are augmented by the presence of a functional NMD pathway. Collectively, our results demonstrate an important role of the RES complex in maintaining the Med20p levels.

The conserved pre-mRNA retention and splicing (RES) complex, which in yeast consists of Bud13p, Snu17p and Pml1p, is thought to promote nuclear retention of unspliced pre-mRNAs and enhance splicing of a subset of transcripts. Here, we find that the absence of Bud13p or Snu17p causes greatly reduced levels of the modified nucleoside N-4-acetylcytidine (ac(4)C) in tRNA and that a lack of Pml1p reduces ac(4)C levels at elevated temperatures. The ac(4)C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine. We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac(4)C. Analyses of cis-acting elements in TAN1 pre-mRNA showed that the intron sequence between the 5' splice site and branchpoint is necessary and sufficient to mediate RES dependency. We also show that in RES-deficient cells, the TAN1 pre-mRNA is targeted for degradation by the cytoplasmic nonsense-mediated mRNA decay pathway, indicating that poor nuclear retention may contribute to the tRNA modification defect. Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac(4)C in tRNA.