TTF1 control of LncRNA synthesis delineates a tumor suppressor pathway directly regulating the ribosomal RNA genes.

The tumor suppressor p14/19ARF regulates ribosomal RNA (rRNA) synthesis by controlling the nucleolar localization of Transcription Termination Factor 1 (TTF1). However, the role played by TTF1 in regulating the rRNA genes and in potentially controlling growth has remained unclear. We now show that TTF1 expression regulates cell growth by determining the cellular complement of ribosomes. Unexpectedly, it achieves this by acting as a "roadblock" to synthesis of the noncoding LncRNA and pRNA that we show are generated from the "Spacer Promoter" duplications present upstream of the 47S pre-rRNA promoter on the mouse and human ribosomal RNA genes. Unexpectedly, the endogenous generation of these noncoding RNAs does not induce CpG methylation or gene silencing. Rather, it acts in cis to suppress 47S preinitiation complex formation and hence de novo pre-rRNA synthesis by a mechanism reminiscent of promoter interference or occlusion. Taken together, our data delineate a pathway from p19ARF to cell growth suppression via the regulation of ribosome biogenesis by noncoding RNAs and validate a key cellular growth law in mammalian cells.

EPH receptor tyrosine kinases phosphorylate the PAR-3 scaffold protein to modulate downstream signaling networks.

EPH receptors (EPHRs) constitute the largest family among receptor tyrosine kinases in humans. They are mainly involved in short-range cell-cell communication events that regulate cell adhesion, migration, and boundary formation. However, the molecular mechanisms by which EPHRs control these processes are less understood. To address this, we unravel EPHR-associated complexes under native conditions using mass-spectrometry-based BioID proximity labeling. We obtain a composite proximity network from EPHA4, -B2, -B3, and -B4 that comprises 395 proteins, most of which were not previously linked to EPHRs. We examine the contribution of several BioID-identified candidates via loss-of-function in an EPHR-dependent cell-segregation assay. We find that the signaling scaffold PAR-3 is required for cell sorting and that EPHRs directly phosphorylate PAR-3. We also delineate a signaling complex involving the C-terminal SRC kinase (CSK), whose recruitment to PAR-3 is dependent on EPHR signals. Our work describes signaling networks by which EPHRs regulate cellular phenotypes.

Liquid-Liquid Phase Separation in Cancer Signaling, Metabolism and Anticancer Therapy.

The cancer state is thought to be maintained by genetic and epigenetic changes that drive a cancer-promoting gene expression program. However, recent results show that cellular states can be also stably maintained by the reorganization of cell structure leading to the formation of biological condensates via the process of liquid-liquid phase separation. Here, we review the data showing cancer-specific biological condensates initiated by mutant oncoproteins, RNA-binding proteins, or lincRNAs that regulate oncogenic gene expression programs and cancer metabolism. Effective anticancer drugs may specifically partition into oncogenic biological condensates (OBC).

Ribosomal DNA promoter recognition is determined in vivo by cooperation between UBTF1 and SL1 and is compromised in the UBTF-E210K neuroregression syndrome.

Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.

Cellular senescence limits translational readthrough.

The origin and evolution of cancer cells is considered to be mainly fueled by DNA mutations. Although translation errors could also expand the cellular proteome, their role in cancer biology remains poorly understood. Tumor suppressors called caretakers block cancer initiation and progression by preventing DNA mutations and/or stimulating DNA repair. If translational errors contribute to tumorigenesis, then caretaker genes should prevent such errors in normal cells in response to oncogenic stimuli. Here, we show that the process of cellular senescence induced by oncogenes, tumor suppressors or chemotherapeutic drugs is associated with a reduction in translational readthrough (TR) measured using reporters containing termination codons withing the context of both normal translation termination or programmed TR. Senescence reduced both basal TR and TR stimulated by aminoglycosides. Mechanistically, the reduction of TR during senescence is controlled by the RB tumor suppressor pathway. Cells that escape from cellular senescence either induced by oncogenes or chemotherapy have an increased TR. Also, breast cancer cells that escape from therapy-induced senescence express high levels of AGO1x, a TR isoform of AGO1 linked to breast cancer progression. We propose that senescence and the RB pathway reduce TR limiting proteome diversity and the expression of TR proteins required for cancer cell proliferation.

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.

New Insights into CDK Regulators: Novel Opportunities for Cancer Therapy.

Cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), control the transition between different phases of the cell cycle. CDK/cyclin activity is regulated by CDK inhibitors (CKIs), currently comprising the CDK-interacting protein/kinase inhibitory protein (CIP/KIP) family and the inhibitor of kinase (INK) family. Recent studies have identified a third group of CKIs, called ribosomal protein-inhibiting CDKs (RPICs). RPICs were discovered in the context of cellular senescence, a stable cell cycle arrest with tumor-suppressing abilities. RPICs accumulate in the nonribosomal fraction of senescent cells due to a decrease in rRNA biogenesis. Accordingly, RPICs are often downregulated in human cancers together with other ribosomal proteins, the tumor-suppressor functions of which are still under study. In this review, we discuss unique therapies that have been developed to target CDK activity in the context of cancer treatment or senescence-associated pathologies, providing novel tools for precision medicine.

Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men and the third cause of cancer mortality. PCa initiation and growth are driven by the androgen receptor (AR). The AR is activated by androgens such as testosterone and controls prostatic cell proliferation and survival. Here, we report an AR signaling network generated using BioID proximity labeling proteomics in androgen-dependent LAPC4 cells. We identified 31 AR-associated proteins in nonstimulated cells. Strikingly, the AR signaling network increased to 182 and 200 proteins, upon 24 h or 72 h of androgenic stimulation, respectively, for a total of 267 nonredundant AR-associated candidates. Among the latter group, we identified 213 proteins that were not previously reported in databases. Many of these new AR-associated proteins are involved in DNA metabolism, RNA processing, and RNA polymerase II transcription. Moreover, we identified 44 transcription factors, including the Kru¨ppel-like factor 4 (KLF4), which were found interacting in androgen-stimulated cells. Interestingly, KLF4 repressed the well-characterized AR-dependent transcription of the KLK3 (PSA) gene; AR and KLF4 also colocalized genome-wide. Taken together, our data report an expanded high-confidence proximity network for AR, which will be instrumental to further dissect the molecular mechanisms underlying androgen signaling in PCa cells.

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability.

In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI-Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI-Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.

NFE2L3 Controls Colon Cancer Cell Growth through Regulation of DUX4, a CDK1 Inhibitor.

Constitutive nuclear factor κB (NF-κB) activation is a hallmark of colon tumor growth. Cyclin-dependent kinases (CDKs) are critical cell-cycle regulators, and inhibition of CDK activity has been used successfully as anticancer therapy. Here, we show that the NFE2L3 transcription factor functions as a key regulator in a pathway that links NF-κB signaling to the control of CDK1 activity, thereby driving colon cancer cell proliferation. We found that NFE2L3 expression is regulated by the RELA subunit of NF-κB and that NFE2L3 levels are elevated in patients with colon adenocarcinoma when compared with normal adjacent tissue. Silencing of NFE2L3 significantly decreases colon cancer cell proliferation in vitro and tumor growth in vivo. NFE2L3 knockdown results in increased levels of double homeobox factor 4 (DUX4), which functions as a direct inhibitor of CDK1. The discovered oncogenic pathway governing cell-cycle progression may open up unique avenues for precision cancer therapy.

The Short N-Terminal Repeats of Transcription Termination Factor 1 Contain Semi-Redundant Nucleolar Localization Signals and P19-ARF Tumor Suppressor Binding Sites.

The p14/p19ARF (ARF) tumor suppressor provides an important link in the activation of p53 (TP53) by inhibiting its targeted degradation via the E3 ligases MDM2/HDM2. However, ARF also limits tumor growth by directly inhibiting ribosomal RNA synthesis and processing. Initial studies of the ARF tumor suppressor were compounded by overlap between the INK4A and ARF genes encoded by the CDKN2A locus, but mouse models of pure ARF-loss and its inactivation in human cancers identified it as a distinct tumor suppressor even in the absence of p53. We previously demonstrated that both human and mouse ARF interact with Transcription Termination Factor 1 (TTF1, TTF-I), an essential factor implicated in transcription termination and silencing of the ribosomal RNA genes. Accumulation of ARF upon oncogenic stress was shown to inhibit ribosomal RNA synthesis by depleting nucleolar TTF1. Here we have mapped the functional nucleolar localization sequences (NoLS) of mouse TTF1 and the sequences responsible for interaction with ARF. We find that both sequences lie within the 25 amino acid N-terminal repeats of TTF1. Nucleolar localization depends on semi-redundant lysine-arginine motifs in each repeat and to a minor extent on binding to target DNA sequences by the Myb homology domain of TTF1. While nucleolar localization of TTF1 predominantly correlates with its interaction with ARF, NoLS activity and ARF binding are mediated by distinct sequences within each N-terminal repeat. The data suggest that the N-terminal repeats of mouse TTF1, and by analogy those of human TTF1, cooperate to mediate both nucleolar localization and ARF binding.

Phosphorylation of SOCS1 Inhibits the SOCS1-p53 Tumor Suppressor Axis.

Expression of the suppressor of cytokine signaling-1 (SOCS1) is inactivated in hematopoietic and solid cancers by promoter methylation, miRNA-mediated silencing, and mutations. Paradoxically, SOCS1 is also overexpressed in many human cancers. We report here that the ability of SOCS1 to interact with p53 and regulate cellular senescence depends on a structural motif that includes tyrosine (Y)80 in the SH2 domain of SOCS1. Mutations in this motif are found at low frequency in some human cancers, and substitution of Y80 by a phosphomimetic residue inhibits p53-SOCS1 interaction and its functional consequences, including stimulation of p53 transcriptional activity, growth arrest, and cellular senescence. Mass spectrometry confirmed SOCS1 Y80 phosphorylation in cells, and a new mAb was generated to detect its presence in tissues by IHC. A tyrosine kinase library screen identified the SRC family as Y80-SOCS1 kinases. SRC family kinase inhibitors potentiated the SOCS1-p53 pathway and reinforced SOCS1-induced senescence. Samples from human lymphomas that often overexpress SOCS1 also displayed SRC family kinase activation, constitutive phosphorylation of SOCS1 on Y80, and SOCS1 cytoplasmic localization. Collectively, these results reveal a mechanism that inactivates the SOCS1-p53 senescence pathway and suggest that inhibition of SRC family kinases as personalized treatment in patients with lymphomas may be successful. SIGNIFICANCE: These findings show that SOCS1 phosphorylation by the SRC family inhibits its tumor-suppressive activity, indicating that patients with increased SOCS1 phosphorylation may benefit from SRC family kinase inhibitors.

Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex.

Senescence is a tumor suppressor program characterized by a stable growth arrest while maintaining cell viability. Senescence-associated ribogenesis defects (SARD) have been shown to regulate senescence through the ability of the ribosomal protein S14 (RPS14 or uS11) to bind and inhibit the cyclin-dependent kinase 4 (CDK4). Here we report another ribosomal protein that binds and inhibits CDK4 in senescent cells: L22 (RPL22 or eL22). Enforcing the expression of RPL22/eL22 is sufficient to induce an RB and p53-dependent cellular senescent phenotype in human fibroblasts. Mechanistically, RPL22/eL22 can interact with and inhibit CDK4-Cyclin D1 to decrease RB phosphorylation both in vitro and in cells. Briefly, we show that ribosome-free RPL22/eL22 causes a cell cycle arrest which could be relevant during situations of nucleolar stress such as cellular senescence or the response to cancer chemotherapy.

Ribosomal Proteins Control Tumor Suppressor Pathways in Response to Nucleolar Stress.

Ribosome biogenesis includes the making and processing of ribosomal RNAs, the biosynthesis of ribosomal proteins from their mRNAs in the cytosol and their transport to the nucleolus to assemble pre-ribosomal particles. Several stresses including cellular senescence reduce nucleolar rRNA synthesis and maturation increasing the availability of ribosome-free ribosomal proteins. Several ribosomal proteins can activate the p53 tumor suppressor pathway but cells without p53 can still arrest their proliferation in response to an imbalance between ribosomal proteins and mature rRNA production. Recent results on senescence-associated ribogenesis defects (SARD) show that the ribosomal protein S14 (RPS14 or uS11) can act as a CDK4/6 inhibitor linking ribosome biogenesis defects to the main engine of cell cycle progression. This work offers new insights into the regulation of the cell cycle and suggests novel avenues to design anticancer drugs.

SOCS1: phosphorylation, dimerization and tumor suppression.

Suppressor of cytokine signaling (SOCS) family members are upregulated following JAK-STAT pathway activation by cytokines. SOCS proteins are recognized inhibitors of cytokine signaling playing roles in cell growth and differentiation. Moreover, SOCS1 and SOCS3 have been shown to be involved in tumor suppression through their ability to interact with p53 leading to the activation of its transcriptional program and showing the implication of SOCS family members in the regulation of apoptosis, ferroptosis and senescence. More recently, we demonstrated that the SRC family of non-receptor tyrosine kinases (SFK) can phosphorylate SOCS1 leading to its homodimerization and inhibiting its interaction with p53. Then, we reactivated the SOCS1-p53 tumor suppressor axis with the SFK inhibitor dasatinib in combination with the p53 activating compound PRIMA. This work suggests new avenues for cancer treatment and leaves open several new questions that deserve to be addressed.

Senescence-associated ribosome biogenesis defects contributes to cell cycle arrest through the Rb pathway.

Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.

A CDK4/6-Dependent Epigenetic Mechanism Protects Cancer Cells from PML-induced Senescence.

Promyelocytic leukemia (PML) plays a tumor suppressive role by inducing cellular senescence in response to oncogenic stress. However, tumor cell lines fail to engage in complete senescence upon PML activation. In this study, we investigated the mechanisms underlying resistance to PML-induced senescence. Here, we report that activation of the cyclin-dependent kinases CDK4 and CDK6 are essential and sufficient to impair senescence induced by PML expression. Disrupting CDK function by RNA interference or pharmacological inhibition restored senescence in tumor cells and diminished their tumorigenic potential in mouse xenograft models. Complete senescence correlated with an increase in autophagy, repression of E2F target genes, and an gene expression signature of blocked DNA methylation. Accordingly, treatment of tumor cells with inhibitors of DNA methylation reversed resistance to PML-induced senescence. Further, CDK inhibition with palbociclib promoted autophagy-dependent degradation of the DNA methyltransferase DNMT1. Lastly, we found that CDK4 interacted with and phosphorylated DNMT1 in vitro, suggesting that CDK activity is required for its stabilization. Taken together, our findings highlight a potentially valuable feature of CDK4/6 inhibitors as epigenetic modulators to facilitate activation of senescence programs in tumor cells. Cancer Res; 76(11); 3252-64. ©2016 AACR.

Permanent farnesylation of lamin A mutants linked to progeria impairs its phosphorylation at serine 22 during interphase.

Mutants of lamin A cause diseases including the Hutchinson-Gilford progeria syndrome (HGPS) characterized by premature aging. Lamin A undergoes a series of processing reactions, including farnesylation and proteolytic cleavage of the farnesylated C-terminal domain. The role of cleavage is unknown but mutations that affect this reaction lead to progeria. Here we show that interphase serine 22 phosphorylation of endogenous mutant lamin A (progerin) is defective in cells from HGPS patients. This defect can be mimicked by expressing progerin in human cells and prevented by inhibition of farnesylation. Furthermore, serine 22 phosphorylation of non-farnesylated progerin was enhanced by a mutation that disrupts lamin A head to tail interactions. The phosphorylation of lamin A or non-farnesylated progerin was associated to the formation of spherical intranuclear lamin A droplets that accumulate protein kinases of the CDK family capable of phosphorylating lamin A at serine 22. CDK inhibitors compromised the turnover of progerin, accelerated senescence of HGPS cells and reversed the effects of FTI on progerin levels. We discuss a model of progeria where faulty serine 22 phosphorylation compromises phase separation of lamin A polymers, leading to accumulation of functionally impaired lamin A structures.

Mutant lamin A links prophase to a p53 independent senescence program.

Expression of oncogenes or short telomeres can trigger an anticancer response known as cellular senescence activating the p53 and RB tumor suppressor pathways. This mechanism is switched off in most tumor cells by mutations in p53 and RB signaling pathways. Surprisingly, p53 disabled tumor cells could be forced into senescence by expression of a mutant allele of the nuclear envelope protein lamin A. The pro-senescence lamin A mutant contains a deletion in the sequence required for processing by the protease ZMPSTE24 leading to accumulation of farnesylated lamin A in the nuclear envelope. In addition, the serine at position 22, a target for CDK1-dependent phosphorylation, was mutated to alanine, preventing CDK1-catalyzed nuclear envelope disassembly. The accumulation of this mutant lamin A compromised prophase to prometaphase transition leading to invaginations of the nuclear lamina, nuclear fragmentation and impaired chromosome condensation. Cells exited this impaired mitosis without cytokinesis and re-replicated their DNA ultimately arresting in interphase as polyploid cells with features of cellular senescence including increased expression of inflammatory gene products and a significant reduction of tumorigenicity in vivo.

Conditional inactivation of Upstream Binding Factor reveals its epigenetic functions and the existence of a somatic nucleolar precursor body.

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).