The Intrinsic Requirement for Eliminated S6K1 Activity in Connection with the ER/Hypoxia-Dependent Breast Cancer

Iouk T, Glebov Anastasia and Titorenko V

Published on: 2018-12-28

Abstract

The mammalian S6K1 kinase triggers breast cancer in the estrogen-receptor and hypoxia-dependent manner. In yeast, sterol sensing and hypoxia are transcriptionally interconnected, and we challenged yeast cells with a steroid compound (lithocholic bile acid) to obtain mutants that would have a decreased relative proliferation rate. In these mutants, we found mutations in the S6K1 homolog (SCH9) and its upstream activator gene (TOR1), suggesting that a steroid-/ hypoxia-dependent regulation of cell proliferation, which involves S6K1, is not unique to high eukaryotes. However, if in mammalian cells such regulation involves soluble nuclear receptors, in yeast it involves mitochondria which communicates the hypoxic state and sterol availability to nucleus. In presence of a steroid compound, yeast Sch9 kinase acted exactly as S6K1, by increasing metabolism and promoting cell growth. Conversely, deleterious mutations in SCH9 or its upstream regulator (TOR1) gene were consistent with a decreased competitive growth rate, increased resistance to stress and longevity.

The mammalian S6K1 kinase triggers breast cancer in the estrogen-receptor and hypoxia-dependent manner. In yeast, sterol sensing and hypoxia are transcriptionally interconnected, and we challenged yeast cells with a steroid compound (lithocholic bile acid) to obtain mutants that would have a decreased relative proliferation rate. In these mutants, we found mutations in the S6K1 homolog (SCH9) and its upstream activator gene (TOR1), suggesting that a steroid-/ hypoxia-dependent regulation of cell proliferation, which involves S6K1, is not unique to high eukaryotes. However, if in mammalian cells such regulation involves soluble nuclear receptors, in yeast it involves mitochondria which communicates the hypoxic state and sterol availability to nucleus. In presence of a steroid compound, yeast Sch9 kinase acted exactly as S6K1, by increasing metabolism and promoting cell growth. Conversely, deleterious mutations in SCH9 or its upstream regulator (TOR1) gene were consistent with a decreased competitive growth rate, increased resistance to stress and longevity.

Keywords

S6K1; Sch9; Hypoxia; Sterol sensing; Cancer; Yeast

Introduction

The 40S ribosomal S6 kinase 1 (S6K1) is a conserved serine/threonine protein kinase that belongs to the AGC family of protein kinases [1]. S6K1 is the principal kinase effector downstream of the mammalian target of rapamycin complex 1 (mTORC1) [2,3]. S6K1 is linked to an estrogen receptor (ER)-positive breast cancer, as it directly phosphorylates and activates an estrogen receptor α (ERα). Moreover, the expression of S6K1 is estrogen-dependent [4-6].

ERα cooperates with the hypoxia-inducible factor 1α (HIF-1α) in the breast-cancer cells. Some HIF-1α targets contain both hypoxia- and estrogen-response elements and they are regulated by ERα. The HIF-1α gene also contains an estrogen response element, and its expression is directly regulated by ERα [7,8]. Numerous studies suggest parallelism between S6K1 and yeast Sch9 kinase, the latter of which stimulates yeast growth in a manner that primarily involves glucose-dependent phosphorylation of translation-initiation factors [9,10]. Recent studies also implicate Sch9 in regulating the sphingolipid metabolism and synthesis of the very-long-chain fatty acids (VLCFA) [11-13]. If the role of Sch9 in the VLCFA synthesis is of a biochemical nature, its link to the sphingolipid metabolism appears to be genetic, since sch9? knockout rescues chronological longevity of the isc1? cells which lack inositol phosphosphingolipid phospholipase C. sch9? was shown to reduce the entropy-based cell death since it decreases the accumulation of ROS (reactive oxygen species), while causing positive changes in the subcellular redox homeostasis and vacuole dynamics, and decreasing protein aggregation [12,14,15]. Sch9 regulates both chronological and replicative lifespan (CLS and RLS). Negative regulation of RLS involves the Sch9-dependent phosphorylation of Rim15 kinase, which regulates the release of PP2ACdc55 phosphatase [16,17].

A distinction exists between the TOR-dependent regulation of S6K-like activity in the mammalian and yeast cells. The single mammalian mTOR complex activates S6K1 and S6K2 kinases, while two distinct TOR kinase complexes (TORC1 and TORC2) activate kinase signaling in yeast [2].  TORC1 targets Sch9, while TORC2 is primarily linked to the regulation of cell-wall integrity and endocytosis via the Pkh1 and Pkh2 kinases which have different from Sch9 phosphorylation substrate specificity [18,19].

The lithocholic bile acid (LCA) supports viability of the glucose-limited yeast [20]. Yeast do not have receptors to steroid compounds, however LCA, which induces hypoxia early in yeast growth, triggers a specific transcriptional response that involves low-oxygen sensing [21,22]. Several hypoxia-specific transcription factors were identified in yeast [23]. These factors respond to the loss of mitochondrial membrane potential (??) and also to shortage of the synthesized in mitochondria ergosterol that occurs in response to oxidative stress [24,25]. Therefore, despite being a steroid compound, lithocholic acid actually sends the sterol-starvation signal early in yeast growth, a signal that requires involvement of specific transcriptional regulators such as Sut1 [24]. Sut1 not only regulates gene expression, but it also has a role in sequestering – inside the nucleus – of several serine-threonine kinases [26]. These kinases which have an overlapping with S6K1 (Sch9) phosphorylation-substrate specificity, and because of their accumulation in the nucleus, the function of Sch9 in cytoplasm increases dramatically [11]. While such an increase is thought to support yeast growth overall, it may nevertheless decrease the Pkh1/Pkh2-signaling output and cell-wall integrity [19,27].

LCA-inducible hypoxia is temporary, and the mitochondrial function of yeast cells is improved subsequently so that, overall, the drug appears to increase and not to decrease a lifespan [20,21]. However, it decreases the competitive fitness of yeast cells, i.e. their ability to grow alongside the control cells on various carbon substrates [28,29]. The obtained in presence of LCA long-lived yeast mutants appeared to either lack the SCH9 (S6K1) function altogether, or to contain mutations in the SCH9 gene and its upstream activator TORC1 (this study). Relative ability of all mutants to proliferate (the mentioned above competitive fitness) was decreased on several growth substrates, and some mutants exhibited a decreased early-growth rate on all substrates tested. Our findings suggest that an activated in yeast sterol- and hypoxia-sensing stimulate a genetic inhibition of the Sch9(S6K1) function and that such an inhibition in turn is accompanied by a partially decreased cell proliferation. Our study implies that S6K1-dependent breast cancer that occurs in the ERα- and HIF-1α dependent manner is part of a basic evolutionary-conserved mechanism which links the principal signaling queues to cell proliferation in various types of cells.

Materials And Methodology

Media, Growth Conditions, and Chronological Lifespan

Yeast were grown in synthetic medium in presence of 0.2% glucose, as described [29,30]. For the CLS assay, cells were plated on the YEPD plates, and the colonies were scored at specific time-points (Ibid.).

Telomeres assay

As described [31]. A pCT300 probe was used to detect telomeric fragments. DIG DNA labeling and detection kit was from Roche.

cDNA synthesis, PCR, and sequence analysis

RNA was synthesized using a hot-phenol method. The cDNA synthesis kit was from Thermo Fisher Scientific. The primers for PCR analysis and sequencing services were from Bio Basic Canada.

Other

Phospho-serine antibodies were from Santa Cruz Abs.

Results

Exposure to LCA produces long-lived mutants with a decreased relative growth rate

Previously, we described a lithocholic-acid driven selection of novel yeast species which appeared to be long-lived [29] (Figure 1A-B). The longevity is often co-incident with a decreased competitive-growth rate [28,32] (Figure 1D). The decreased competitive-growth rate is determined based on ability of the mutant cells to survive in a mixed yeast culture alongside the control cells [28,29]. 

The longevity of mutants was linked to recombinant telomeres (Figure 1, B and E). If a typical digestion pattern contained the terminal fragment of 1.2 kb (Figure 1B, an arrow) and specific subset of larger bands (as in the lanes 10 and 13), the recombinant telomeres produced additional bands and they exhibited an aberrant digestion pattern indicative of homologous DNA recombination at the chromosomal ends [33,34]. In addition to the recombination, telomeric mutants are known to frequently contain genetic defects that decrease their fitness [33].

The exceptionally long-lived mutants 3, 5 and 12 are characterized by an altered age-related chronology of mitochondrial respiration, which was previously identified in the sch9? cells that lack yeast homolog of the S6K1 [21,29]. One of the mutants (mutant #3) was subjected to the DNA microarray analysis to determine that mitochondrial-protein genes were equally likely to be up-regulated or down-regulated in these cells (Figure 2A and Supplementary Table 1). The detected expression was indicative of a modified mitochondrial activity, as in the sch9? cells [35]. Overall, in the mutant we determined the repression of 106 genes (>2fold, p<0.05). However, 22 of these genes are the so-called “frequently modified ORFs” the expression of which varies in response to different factors [36].

87 genes were induced (>2fold, p<0.05), and both up regulated and down regulated genes were likely to encode mitochondrial proteins. In order to determine whether the affected genes were linked to the mutant’s longevity, we genetically reduced the respiratory function in the mutant #3. Specifically, we disrupted a COX5A gene (complex IV of the mitochondrial electron transport chain), thereby decreasing the longevity (from >2.5 down to 1.3 fold). The relative growth rate was also decreased, and no growth was detected on glycerol, which requires the unaffected mitochondrial function (data not shown). With COX5A deleted, 47 genes remained repressed (2fold, p<0.05), while 29 genes remained up regulated (Figure 2A). Among the repressed were genes that have a role in entering G0 growth arrest. Numerous mitochondrial-protein genes were also repressed, which was not surprising given the elimination of COX5A (Supplementary Table 1). Noticeable changes were detected in the expression of molecular-transport genes some of which were up regulated and some were repressed, while several metabolism-related genes were up regulated [13].

The detected changes in gene expression were more apparent in the intact long-lived mutant. Greater number of genes required to enter G0 were repressed. In contrast, numerous mitochondrial-protein genes were up regulated, and so were some metabolic genes.  The mutant exhibited the decreased expression of RGS2 (a glucose-inducible signaling gene), PHM8 (phosphate starvation and ribose salvage gene), stress response YRO2 and HSP30 genes, and the HXT2 and HXT7 hexose transporter genes. Despite the down-regulation of HXT2 and HXT7, several other transporter genes were activated. The activated mitochondrial-protein genes primarily included those responsible for the mitochondrial-genome maintenance, however the induced genes also included the HSP60 mitochondrial-transporter gene and PGS1 that has a role in the biosynthesis of cardiolipin, a specific to mitochondria cone-shaped glycerophospholipid with a role in cristae formation. The up regulated transcription-factor genes included ZNF1 responsible for the respiratory growth and gluconeogenesis, SAS4, as well as the repressor of hypoxic genes ROX1.

Since some mitochondrial protein genes were up-regulated in the mutant #3 while others were down-regulated, it appeared that the mutant was flip-flopping between mild oxidative stress and the increased respiratory function. Such a regulation would be consistent with a previously shown pattern of the LCA (longevity-drug)-dependent regulation, when mild oxidative stress is followed by the activation of respiratory function [21]. DNA microarray analysis suggested that a mutant #3 is lacking the expression of several genes that regulated thickening of cell wall. This meant that a TORC2 signaling, which is parallel to TORC1-S6K1 cascade, was down-regulated. Further analysis revealed that Sch9 (S6K1) signaling output was also missing in these cells.  

Mutations in the S6K1 homologue

Unlike other mutants, the mutant #3 showed excess protein phosphorylation (Figure 2B) that often occurs in cells with a swapped kinase activity, when the function of one kinase is substituted by another kinase with a similar substrate specificity [1,11,18]. We asked if mutant #3 lacks Sch9 activity.

The SCH9 cDNA from the mutants was sequenced to find a STOP codon in place of Asp186 in the #3 mutant. Other long-lived survivors also contained mutations in the SCH9 gene (Figure 2B). One mutant had a mutation which led to the N217Y amino-acid substitution in the regulatory C2 calcium-binding domain of Sch9 (the 652 A>T nucleotide substitution), while another mutant had a C44S amino-acid substitution, which possibly decreased the ability of Sch9 to aggregate [37]. However, with exception of the Asp186>>STOP, neither of the mentioned above mutations is known to inhibit the Sch9 function. These mutations are characterized by the ≤1.0 SIFT (Sorting Intolerant from Tolerant) score, suggesting that the arising amino amino-acid substitutions do not significantly influence the protein function [38]. The TOR1 cDNA, which encodes the upstream regulator of Sch9, was also analyzed. In the long-lived #5 mutant, TOR1 contained the W1748G amino-acid substitution in the FKBP12 rapamycin-binding domain (a.k.a. FAT domain) [39]. This domain has a role in the formation of TORC1 protein complex, binding to rapamycin (the inhibitor of Tor1), and recruiting the effector kinase, i.e. Sch9. The 1748 residue of Tor1 is evolutionary conserved, and its substitution could potentially decrease this protein’s function. Still, the mutation produced high SIFT score, and it therefore was not considered deleterious.

In addition to the tor1 mutation, the mutant #5 showed the strongly decreased expression of FKH1, one of the two FOXO transcription factors in yeast (Figure 3). In mammals, Forkhead-box O (FOXO) transcription factors suppress tumor growth and they control cell differentiation. The FOXO transcription is known to maintain cellular homeostasis during aging in the C. elegans nematode [40]. In yeast, the overexpression of FKH1 was linked to a modified sensitivity to mild oxidative stress, including nutrient-related oxidative stress [41,42]. In case of the #5 mutant, the decreased expression of FKH1 resulted in a greatly increased resistance to an oxidative stress [29]. Notably, the expression of FKH2, the FKH1 homologue, remained unchanged in this mutant (Supplementary Figure 1).

The mutant #12 had no mutations directly related to the TORC1 signaling. The initial sequencing analysis suggested presence of a STOP codon in the DBP3 rRNA processing gene, i.e. a dbp3D mutation which decreases competitive-growth rate [43,29]. However, the dbp3? mutation was not confirmed, and further analysis revealed the presence of mutations in the RPP2(POP7) gene, which encodes a subunit of the nuclear MRP RNase and P RNase [44,45] (Figure 3A). The detected in the POP7 gene mutations (32T>K and 43S>R) increase the binding to nucleic acid, thereby decreasing the release of the substrate and a nucleolytic activity. Remarkably, because of a decreased ribosomal biogenesis, this mutant’s cells were smaller in size than control (4.5+/-1.4 mm versus 5.5 mm for the ScBy4742 control), and they had a slightly decreased growth rate.

The mutant #5 had a mutation in the rapamycin-binding domain of Tor1 kinase and it also had a mutation in the UBP6 gene that potentially increased the levels of polyubiquitinated proteins inside the cell, while decreasing function of the proteasome [46] (Figure 3A). The UBP6 was not associated with the cell proliferation previously, however the loss-of-function mutation in the UBP7 gene, that also decreases protein degradation by a proteasome, is consistent with the increased survival in the context of chemically challenging environment, including the biotechnology applications [27]. The mutant #5 was highly resistant to stress, including the oxidative, high-temperature, and salt-inducible stress [29]. In part, the resistance to stress was linked to the decreased expression of FKH1 transcription factor [41,48] (Supplementary Figure 1).

Discussion and Conclusions

In this study, we characterized the subset of long-lived yeast mutants which exhibited a decreased competitive-growth rate on several growth substrates [29] (Figure 1D-E). Some of these mutants could be viewed as a prototype cells with the decreased proliferative activity but increased lifespan. The source of increased lifespan is long recombinant telomeres (Figure 1B) and the sch9? mutation in the #3 mutant (Figure 2C). The extended chromosomal ends are typically linked to increased replicative longevity, however they could also define a greater CLS, since determining one in yeast cells is usually based on ability to form colonies, i.e. on the ability of yeast cells to divide. 

Several mutants had mutations in the SCH9 gene - yeast homologue of the mammalian S6K1, and one mutant had a mutation in the TOR1 gene, which encodes the kinase-activator for Sch9 (Figure 2C). The mutants were obtained in presence of lithocholic acid (LCA) [20]. The steroid LCA (Figure 1C) has no known mutagenic effects, however it causes temporary oxidative stress, which is then followed by increased mitochondrial function [21,22]. The drug has an effect on both glucose-limited and non-glucose limited yeast, however it substantially increases age-related survival only of the glucose-limited yeast, which lack cytoplasmic glycolysis (20,49].

The mutant number #3 exhibited the increased expression of mitochondrial-protein genes, which appeared to be linked to the sch9D mutation (Figure 2A and Supplementary Table 1). The protein phosphorylation was increased in this mutant - the probable result of swapped kinase activity (Figure 2B). Elucidated in connection with a cytoplasmic synthesis of long-chain fatty acids, swapping the kinase activity is known to involve several TORC1-, TORC2- and the Cdc28-cyclin regulated kinases that have an overlapping phosphorylation-target specificity [1,11]. Cell-wall integrity is often compromised in the sch9-deficient cells, typically in the YPK2-involving manner [27]. The mutant #5, which has a mutation in a FAT-domain of Tor1, is highly resistant to stress, including the hydrogen-peroxide inducible stress [29]. The resistance to stress however was linked not only to TORC1, but also to the decreased expression of FKH1 transcription factor (Supplementary Figure 1). In the mammalian cells, the FOXO family members are known to protect against oxidative stress in a manner that involves manganese superoxide-dismutase and catalase gene expression [48,50]. The mutant #5 also contained a mutation in the UBP6, a ubiquitin-specific protease gene.

The TORC1 signaling and Sch9 function could be decreased by the means of glucose limitation (a.k.a. calorie restriction, CR). CR decreases the accumulation of ROS, and it increases chronological and replicative longevity [51,52]. CR however does not decrease growth rate, at least not in yeast cells [53]. Detailed analysis had established that a moderate CR (2-4 fold decrease of the glucose intake) has no negative effect on the ATP synthesis (Ibid). Instead, it decreases the release of acetic acid, which negatively influences mitochondrial function and which may cause an apoptosis. Instead of a calorie restriction, the sch9? knockout is frequently used to obtain a longevity phenotype [54,10]. In contrast to CR, the sch9? mutation could decrease a competitive-growth rate, while increasing sensitivity to the inhibitors of sphingolipid biosynthesis [12,13].

Despite some deleterious effects of the sch9D mutation, the micro-evolution of yeast cells challenged by the chemically induced hypoxia - and by activated sterol sensing – often involves modifications of the genetic determinant of S6K1 activity (Figure 2C). The outcome - in the form of a modified yeast transcriptome - appears to increase mitochondrial activity and longevity, while potentially decreasing the competitive-growth rate [35] (Figure 2A, Supplementary Table 1). As already mentioned, the decreased competitive-growth rate is linked to swapping a kinase activity, increased sensitivity to chemicals, and to decreased YPK2 kinase expression in the #3 mutant that appears to compromise the integrity of yeast cell wall [27]. Long recombinant telomeres (Figure 1B) could also decrease the competitive-growth rate while increasing longevity; however, in contrast to sch9D, they do not eliminate the accumulation of ROS and do not decrease an oxidative damage to cell.

Sch9 and its up-stream activator TOR1-kinase gene are responsible for the programmed yeast aging. The theories of programmed aging imply existence of the evolutionary forces that actively restrict organismal lifespan. Such a restriction accelerates death of an individual in order to benefit kin, i.e. to ensure that a fate of progeny is not jeopardized because of spontaneous errors, free radicals, glycation, and other detrimental factors accumulated by the parental organism [55,56]. An aging organism is often a threat to populations and ecosystems; therefore, mechanisms of a programmed aging were developed that target both RLS and CLS of a specie. In yeast, such a mechanism exists primarily in a form of TORC1 signaling pathway, which functions in coordination with other nutrient-sensitive pathways, including RAS/cAMP-dependent protein kinase A and AMP-dependent protein kinase (Snf1) pathways [57,58]. If Sch9 and its up-stream activator TOR1-kinase gene are responsible for a programmed aging, the non-programmed chronological aging depends on variety of genes, including the mitochondrial-protein genes, regulators of redox homeostasis, genes that regulate alkalinization of vacuole and protein sorting to endosome, tRNA methylation genes, etc. [59-61]. The deletions of many of these genes decrease CLS while also decreasing yeast growth.

The gene deletions were also identified that increase RLS but not CLS, suggesting that they work against the evolution [60,61]. Among others, the implicated knockouts include those of the retrograde-transport genes, which communicate mitochondrial malfunction to nucleus and some histone-deacetylase knockouts [51,25]. Some mutations identified in this study (Figure 3A) could be linked to the chemically-challenging growth conditions, but they have no effect on the competitive-growth rate or longevity. The ubp mutations, for example, were previously detected in yeast cells in the context of industrial applications, including the exposing yeast cells to a lignocellulosic chemical waste [27,62]. Other mutations - and the respective amino-acid substitutions in proteins of interest - were typical for the industrially used methanol-utilizing yeast such as Candida boidinii and other yeast species. Overall however, our findings suggest that in the hypoxia-challenged cells with an activated sterol sensing the selective evolutionary forces work towards the elimination of the S6K1 and its upstream regulatory activity. Indeed, out of 16 mutations detected, 5 mutations or 31% were in the SCH9 (Chr. VIII) or TOR1 (Chr. X) genes. By comparison, the mutations in all other genes appeared to be quite rare. It would be interesting to determine if the SCH9 (S6K1) locus, and perhaps the TOR1 (mTOR) as well, are somehow prone to mutagenesis in yeast and other eukaryotic cells: it seems that S6K1-like genetic barrier, which triggers aging in yeast and cancer in the mammalian cells, is relatively easy to break.

Funding

This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada and Concordia University Chair Fund.

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