Expression Of Ribosomal Protein S6 Research Articles

Effects of Fish Meal Replacement with Composite Mixture of Soybean Protein Hydrolysates and Other Plant Proteins on Growth Performance, Antioxidant Capacity, and Target of Rapamycin Pathway in Largemouth Bass

AbstractA feeding trial was conducted to evaluate the effect of fish meal replacement with a composite mixture of soybean protein hydrolysates and other plant proteins on growth performance, antioxidant capacity, and target of rapamycin pathway in Largemouth Bass Micropterus salmoides. Triplicate groups of Largemouth Bass were fed the diet formulated with 40% (FM40; control), 35% (FM35), 30% (FM30), or 25% (FM25) fish meal for 10 weeks. The results revealed that the best growth performance was achieved in the FM35 group, while a negative effect on growth was observed in the FM25 group. The feed intake of the FM30 and FM25 groups was significantly lower than the other two groups, while no significant difference was observed in feed efficiency ratio and protein efficiency ratio. No statistical difference was observed in the activity of serum alanine transaminase and aspartate transaminase with the replacement of fish meal. However, the replacement of fish meal with composite plant protein significantly reduced hepatic malondialdehyde content, and a significant improvement in total antioxidant capacity was observed in the FM35 group. Gene expression analysis revealed that the expression of amino acid transporter LAT1 was significantly elevated in the FM35 group, while no significant difference was observed in the LAT2 expression among different treatments. No significant difference was observed in the expression of target of rapamycin among treatments. However, the expression of ribosomal protein S6 in the FM35 and FM30 groups was significantly higher than in the other two groups, while the replacement of fish meal significantly decreased the expression of eukaryotic initiation factor 4EBP1. In summary, the composite mixture of soybean protein hydrolysates and other plant proteins can reduce the use of fish meal from 40% to 35%.

Glioma Cells Acquire Stem-like Characters by Extrinsic Ribosome Stimuli.

Although glioblastoma (GBM) stem-like cells (GSCs), which retain chemo-radio resistance and recurrence, are key prognostic factors in GBM patients, the molecular mechanisms of GSC development are largely unknown. Recently, several studies revealed that extrinsic ribosome incorporation into somatic cells resulted in stem cell properties and served as a key trigger and factor for the cell reprogramming process. In this study, we aimed to investigate the mechanisms underlying GSCs development by focusing on extrinsic ribosome incorporation into GBM cells. Ribosome-induced cancer cell spheroid (RICCS) formation was significantly upregulated by ribosome incorporation. RICCS showed the stem-like cell characters (number of cell spheroid, stem cell markers, and ability for trans differentiation towards adipocytes and osteocytes). In RICCS, the phosphorylation and protein expression of ribosomal protein S6 (RPS6), an intrinsic ribosomal protein, and STAT3 phosphorylation were upregulated, and involved in the regulation of cell spheroid formation. Consistent with those results, glioma-derived extrinsic ribosome also promoted GBM-RICCS formation through intrinsic RPS6 phosphorylation. Moreover, in glioma patients, RPS6 phosphorylation was dominantly observed in high-grade glioma tissues, and predominantly upregulated in GSCs niches, such as the perinecrosis niche and perivascular niche. Those results indicate the potential biological and clinical significance of extrinsic ribosomal proteins in GSC development.

CBMS-09 Intercellular communication at glioblastoma stem cell niches

Glioblastoma multiforme (GBM) contains heterogenous population of cells including a small population of GBM stem cells (GSCs), which potentially cause therapeutic resistance and tumor recurrence. GSCs harbored in special microenvironments, such as perinecrotic niche, perivascular niche, border niche. However, the mechanisms underlying the pathogenesis and maintenance of GSCs remain largely unknown. Stemness and chemo-radioresistance was promoted by not only additional mutation, but also microenvironment of GBM cells. Previously, we had reported that growth factors and cytokines secreted by oligodendrocyte lineage cells and macrophages/microglia induce stemness and chemo-radioresistance into GBM cells. Recently, Ito et al. reported that incorporation of ribosomes and ribosomal proteins into somatic cells promoted lineage trans-differentiation toward multipotency. Ribosomal proteins exist intra- and extracellularly. There is a possibility that ribosomal proteins promote stemness into cancer cells, we focused on 40S ribosomal protein S6 (RPS6), which is related to cell proliferation in lung and pancreatic cancer, but not reported in GBM. RPS6 was significantly upregulated in high-grade glioma. SiRNA-mediated RPS6 knock-down significantly suppressed the characteristics of GSCs, including their tumorsphere potential and stemness marker expression, such as Nestin and Sox2. RPS6 overexpression enhanced the tumorsphere potential of GSCs. Moreover, RPS6 expression was significantly correlated with SOX2 expression in different glioma grades. Immunohistochemistry data indicated that RPS6 was predominant detected at GSC niches, concurrently with the data from IVY GAP databases. Furthermore, RPS6 and other ribosomal proteins were upregulated in GSC-predominant areas in this database. The present results indicate that, in GSC niches, ribosomal proteins play crucial roles in the development and maintenance of GSCs and are clinically associated with chemo-radioresistance and GBM recurrence. These results suggested that intercellular communications through growth factors, cytokines, and ribosomes are regarded as new treatment targets of GBM.

Knockdown of ribosomal protein S6 suppresses proliferation, migration, and invasion in epithelial ovarian cancer

BackgroundOvarian cancer typically is diagnosed late because insensitivity and lack of specificity of current biomarkers prior to its clinical detection. Ribosomal protein S6 (RPS6) is a ribosomal protein involved in the ribosomal 40S subunit, but its biological role in epithelial ovarian cancer (EOC) is still unknown.ResultsRPS6 was elevated in EOC compared to normal ovarian tissues and adenomas. Higher expression of RPS6 predicted worse prognosis in EOC. The level of RPS6 was correlated with clinical stage, histological type and pathological grade. Knockdown of RPS6 reduced the proliferation of ovarian cancer cell lines SKOV-3 and HO8910, and inhibit the migration and invasion ability. It revealed that cells arrested at G0G1 phase after knockdown of RPS6, and the expressions of CyclinD1, Cyclin E, CDK2, CDK4, CDK6 and pRb were also reduced.ConclusionsRPS6 is involved in EOC and knockdown of RPS6 could inhibit the proliferation, invasion and migration ability of EOC in vitro by inducing G0/G1 phase arrest. RPS6 is expected to be a novel biomarker and molecular target to the EOC.

Ribosomal protein S6 promotes stem-like characters in glioma cells.

Glioblastoma multiforme (GBM), a lethal brain tumor developing in the white matter of the adult brain, contains a small population of GBM stem cells (GSCs), which potentially cause chemotherapeutic resistance and tumor recurrence. However, the mechanisms underlying the pathogenesis and maintenance of GSCs remain largely unknown. A recent study reported that incorporation of ribosomes and ribosomal proteins into somatic cells promoted lineage trans‐differentiation toward multipotency. This study aimed to investigate the mechanism underlying stemness acquisition in GBM cells by focusing on 40S ribosomal protein S6 (RPS6). RPS6 was significantly upregulated in high‐grade glioma and localized at perivascular, perinecrotic, and border niches in GBM tissues. siRNA‐mediated RPS6 knock‐down significantly suppressed the characteristics of GSCs, including their tumorsphere potential and GSC marker expression; STAT3 was downregulated in GBM cells. RPS6 overexpression enhanced the tumorsphere potential of GSCs and these effects were attenuated by STAT3 inhibitor (AG490). Moreover, RPS6 expression was significantly correlated with SOX2 expression in different glioma grades. Immunohistochemistry data herein indicated that RPS6 was predominant in GSC niches, concurrent with the data from IVY GAP databases. Furthermore, RPS6 and other ribosomal proteins were upregulated in GSC‐predominant areas in this database. The present results indicate that, in GSC niches, ribosomal proteins play crucial roles in the development and maintenance of GSCs and are clinically associated with chemoradioresistance and GBM recurrence.

Inhibition of the Spectraplakin Protein Microtubule Actin Crosslinking Factor 1 Sensitizes Glioblastomas to Radiation.

BackgroundMicrotubule actin crosslinking factor 1 (MACF1) is a spectraplakin cytoskeletal crosslinking protein whose function and role in cancer biology has lacked investigation. Recent studies have identified MACF1 as a novel target in glioblastomas expressed in tissue from tumor patient explants but not normal brain tissue and when silenced has an antitumorigenic impact on these tumors. Radiation as a single agent therapy to treat glioblastomas has been used for decades and has done little to improve survival of individuals diagnosed with this disease. However, contemporary clinical radiotherapy protocols have provided evidence that combinatorial radiotherapy approaches confer a therapeutic benefit in glioblastoma patients. In this study MACF1 was investigated as a radiosensitization target in glioblastomas.MethodsTo provide context of MACF1 in glioblastomas, The Cancer Genome Atlas expression analyses were performed in conjunction with genes associated with glioblastoma evolution, while a genetic inhibitory approach, cell migratory assays, and immunofluorescence procedures were used to evaluate responses to MACF1 suppression with radiation. Additionally, expression analyses were conducted to assess co-expression of mTOR signaling pathway regulators and MACF1 in glioblastoma patient samples.ResultsOur amalgamation approach demonstrated that negative regulation of MACF1, which was positively correlated with epidermal growth factor receptor and p70s6k expression, enhanced the sensitivity of glioblastoma cells to radiation as a consequence of reducing glioblastoma cell viability and migration. Mechanistically, the antitumorigenic effects on glioblastoma cell behaviors after radiation and impairing MACF1 function were associated with decreased expression of ribosomal protein S6, a downstream effector of p70s6k.ConclusionMACF1 represents a diagnostic marker with target specificity in glioblastomas that can enhance the efficacy of radiation while minimizing normal tissue toxicity. This approach could potentially expand combinatorial radiation strategies for glioblastoma treatments via impairment of translational regulatory processes that contribute to poor patient survival.

Modulation of oxidative-nitrosative stress and inflammatory response by rapamycin in target and distant organs in rats exposed to hindlimb ischemia-reperfusion: the role of mammalian target of rapamycin.

Mammalian target of rapamycin (mTOR) has been recognized with potential immunomodulatory properties playing an important role in various physiopathological processes including ischemia-reperfusion (I/R) injury. I/R injury stimulate reactive oxygen and nitrogen species by activating nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase, respectively. Controversial results have been obtained in different I/R models following localized I/R; however, the precise role of the mTOR signaling pathway remains undefined. The objective of the current study was to evaluate the role of the mTOR in oxidative-nitrosative stress and inflammation in hindlimb I/R-induced injury in target and remote organ injuries. In rats subjected to I/R, an increased expression of ribosomal protein S6 (rpS6), inhibitor κB (IκB)-α, nuclear factor-κB (NF-κB) p65, inducible nitric oxide synthase, cyclooxygenase 2, gp91phox, and levels of tumor necrosis factor α, nitrite, nitrotyrosine, malondialdehyde and the activities of myeloperoxidase and catalase in the tissues and (or) sera were detected. Treatment with rapamycin, a selective inhibitor of mTOR, reversed all the I/R-induced changes as manifested by its anti-inflammatory and antioxidant effects in kidney and gastrocnemius muscle of rats. Collectively, these findings suggest that rapamycin protects against I/R-induced oxidative-nitrosative stress and inflammation leading to organ injuries via suppression of mTOR/IκB-α/NF-κB signaling pathway.

Delta-6-desaturase (FADS2) inhibition and omega-3 fatty acids in skeletal muscle protein turnover

Polyunsaturated fatty acids (PUFAs) are essential dietary components. They are not only used for energy, but also act as signaling molecules. The delta-6 desaturase (D6D) enzyme, encoded by the FADS2 gene, is one of two rate limiting enzymes that convert the PUFA precursors – α-linolenic (n-3) and linoleic acid (n-6) to their respective metabolites. Alterations in the D6D enzyme activity alters fatty acid profiles and are associated with metabolic and inflammatory diseases including cardiovascular disease and type 2 diabetes. Omega-3 PUFAs, specifically its constituent fatty acids DHA and EPA, are known for their anti-inflammatory ability and are also beneficial in the prevention of skeletal muscle wasting, however the mechanism for muscle preservation is not well understood. Moreover, little is known of the effects of altering the n-6/n-3 ratio in the context of a high-fat diet, which is known to downregulate protein synthesis. Twenty C57BL6 male mice were fed a high-fat lard (HFL, 45% fat (mostly lard), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 13:1) diet for 6 weeks. Mice were then divided into 4 groups (n = 5 per group): HFL– , high-fat oil– (HFO, 45% fat (mostly Menhaden oil), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 1:3), HFL+ (HFL diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day), and HFO+ (HFO diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day). After 2 weeks on their respective diets and treatments, animals were sacrificed and gastrocnemius muscle harvested. Protein turnover signaling were analyzed via Western Blot. 4-EBP1 and ribosomal protein S6 expression were measured. A two-way ANOVA revealed no significant change in the phosphorylation of both 4EBP-1 and ribosomal protein S6 with diet or inhibitor. There was a significant reduction in STAT3 phosphorylation with the inhibition of FADS2 (p = 0.03). Additionally, we measured markers of protein degradation through levels of FOXO phosphorylation, ubiquitin, and LC3B expression; there was a trend towards increased phosphorylation of FOXO (p = 0.08) and ubiquitinated proteins (p = 0.05) with FADS2 inhibition. LC3B expression, a marker of autophagy, was significantly higher in the HFL plus FADS2 inhibition group from all other comparisons. Lastly, we analyzed activation of mitochondrial biogenesis which is closely linked with protein synthesis through PGC1-α and Cytochrome-C expression, however no significant differences were associated with either marker across all groups. Collectively, these data suggest that the protective effects of muscle mass by omega-3 fatty acids are from inhibition of protein degradation. Our aim was to determine the role of PUFA metabolites, DHA and EPA, in skeletal muscle protein turnover and assess the effects of n-3s independently. We observed that by inhibiting the FADS2 enzyme, the protective effect of n-3s on protein synthesis and proliferation was lost; concomitantly, protein degradation was increased with FADS2 inhibition regardless of diet.

Mammalian target of rapamycin inhibition attenuates myocardial ischaemia-reperfusion injury in hypertrophic heart.

Pathological cardiac hypertrophy aggravated myocardial infarction and is causally related to autophagy dysfunction and increased oxidative stress. Rapamycin is an inhibitor of serine/threonine kinase mammalian target of rapamycin (mTOR) involved in the regulation of autophagy as well as oxidative/nitrative stress. Here, we demonstrated that rapamycin ameliorates myocardial ischaemia reperfusion injury by rescuing the defective cytoprotective mechanisms in hypertrophic heart. Our results showed that chronic rapamycin treatment markedly reduced the phosphorylated mTOR and ribosomal protein S6 expression, but not Akt in both normal and aortic‐banded mice. Moreover, chronic rapamycin treatment significantly mitigated TAC‐induced autophagy dysfunction demonstrated by prompted Beclin‐1 activation, elevated LC3‐II/LC3‐I ratio and increased autophagosome abundance. Most importantly, we found that MI/R‐induced myocardial injury was markedly reduced by rapamycin treatment manifested by the inhibition of myocardial apoptosis, the reduction of myocardial infarct size and the improvement of cardiac function in hypertrophic heart. Mechanically, rapamycin reduced the MI/R‐induced iNOS/gp91phox protein expression and decreased the generation of NO and superoxide, as well as the cytotoxic peroxynitrite. Moreover, rapamycin significantly mitigated MI/R‐induced endoplasmic reticulum stress and mitochondrial impairment demonstrated by reduced Caspase‐12 activity, inhibited CHOP activation, decreased cytoplasmic Cyto‐C release and preserved intact mitochondria. In addition, inhibition of mTOR also enhanced the phosphorylated ERK and eNOS, and inactivated GSK3β, a pivotal downstream target of Akt and ERK signallings. Taken together, these results suggest that mTOR signalling protects against MI/R injury through autophagy induction and ERK‐mediated antioxidative and anti‐nitrative stress in mice with hypertrophic myocardium.

Differential Requirement of the Ribosomal Protein S6 and Ribosomal Protein S6 Kinase for Plant-Virus Accumulation and Interaction of S6 Kinase with Potyviral VPg.

Ribosomal protein S6 (RPS6) is an indispensable plant protein regulated, in part, by ribosomal protein S6 kinase (S6K) which, in turn, is a key regulator of plant responses to stresses and developmental cues. Increased expression of RPS6 was detected in Nicotiana benthamiana during infection by diverse plant viruses. Silencing of the RPS6 and S6K genes in N. benthamiana affected accumulation of Cucumber mosaic virus, Turnip mosaic virus (TuMV), and Potato virus A (PVA) in contrast to Turnip crinkle virus and Tobacco mosaic virus. In addition, the viral genome-linked protein (VPg) of TuMV and PVA interacted with S6K in plant cells, as detected by bimolecular fluorescence complementation assay. The VPg-S6K interaction was detected in cytoplasm, nucleus, and nucleolus, whereas the green fluorescent protein-tagged S6K alone showed cytoplasmic localization only. These results demonstrate that the requirement for RPS6 and S6K differs for diverse plant viruses with different translation initiation strategies and suggest that potyviral VPg-S6K interaction may affect S6K functions in both the cytoplasm and the nucleus.

Expression of Ribosomal Protein S6 (R6SP) and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1) Is Correlated in Acute Myelogenous Leukemia (AML) and Is Highly Prognostic, Especially in NPM1 and FLT3 Wildtype Patients

Background. Conceptually mRNA processing and ribosomal regulation should interact as both affect mRNA translation and protein production. We studied protein expression and functional relationships between proteins in AML using a custom made reverse phase protein array (RPPA), probed with 231 strictly validated antibodies. We found a relationship between expression of Ribosomal Protein S6 (HUGO name R6SP, a.k.a. S6RP) and Eukaryotic Translation Initiation Factor 4EBinding Protein 1, (HUGO name EIF4EBP1). R6SP, a 40S ribosomal subunit component, activated by phosphorylation, regulates cell growth via selective mRNA translation. EIF4EBP1 interacts with eIF4E to recruit the 40S ribosomal subunit, thereby affecting ribosomal assembly. When phosphorylated, in response to cellular signaling, it releases eIF4E allowing transcription.Methods. Our RPPA has protein from leukemia enriched cells from 511 newly diagnosed AML patients and was probed with 231 strictly validated antibodies, including antibodies against total RPS6 and forms phosphorylated on S235-236 and S240-244, and against total EIF4EBP1 and forms phosphorylated on T37 & 46, T70 and S65. Expression was compared to normal bone marrow derived CD34+ cells. Interaction networks with the other 224 proteins were generated from the RPPA data using glasso and supplemented by the literature of known interactions.Results. A heatmap of expression of the 3 R6SP and 4 PA2 forms was generated and hierarchical k-and means clustering performed (Fig A). Using the “Prototype Clustering ”method an optimal division into four clusters (Fig B) was determined. This includes an “All-Off” state (18%), a state characterized by weak activation of RPS6 alone (RP-Only, 36%) activation of only EIF4EBP1 (EIF4EBP1-Only, 26%) and a group where both were on simultaneously (Both-On). The RPS6 interactome (Fig B) showed the expected positive correlation with mTOR, and P70 (Hugo RPS6KB1) and a previously unknown, but very strong, negative correlation with transcription factor ZNF296. The EIF4EBP1 interactome showed the expected strong positive correlation with many signal transduction pathways (MAP2K1, MAPK14) and proliferation related proteins (pRB, EIF2AK, EIF2S1, FOXO3) and negative correlation with several transcription factors (GATA3, SPI1, CREB). Cluster membership was unassociated with most clinical features including cytogenetics, FLT3 , RAS and NPM1 mutation, excluding gender (more F in All-Off, more M in Both-On, p=0.01). EIF4EBP1 and Both-On had higher WBC (p=0.0001) and % marrow (p=0.0001) and blood blasts (0.0007) and lower platelet counts (p=0.025). Response rates did not differ, although fewer All-Off were primary refractory. Relapse was more common in EIF4EBP1-Only and Both-On clusters. Overall survival (OS) and remission duration (RemDur) (Fig C) of the EIF4EBP1-Only and Both-On clusters was inferior to that of the All-Off and RP-Only clusters (OS median 41 & 45 vs. 52 &63,p=0.06, RemDur 39 & 27 weeks vs. 63 & 53, p=0.008) but this was restricted to Intermediate cytogenetics cases (Fig C “IntCyto” OS 49 & 55 weeks vs. 107& 79 p=0.01, RemDur 37 & 35 weeks vs. 89 & 53 , p = 0.005) that were FLT3 mutation ((Fig C “FLT3-WT” OS p=0.006, RemDur p0.007) and NPM1 mutation negative (Fig C “NPM1-WT”, OS p=0.006, RemDur p=0.001).Conclusions. Activation of EIF4EBP1, with or without RPS6 activation is prognostically adverse in AML, particularly in intermediate cytogenetic cases with wildtype FLT3 and NPM1. This is associated with increased proliferation. Therapy directed against EIF4EBP1 activity, e.g. that block it’s phosphorylation, may have utility in the ~46% of cases of AML that demonstrate high levels of EIF4EBP1 phosphorylation, especially in FLT3/NPM1 wildtype cases. Many agents that inhibit signal transduction pathways are in clinical development, analyzing them for the ability to inhibition the activation of EIF4EBP1 might identify clinically useful molecules. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ribosomal protein S6 mRNA is a biomarker upregulated in multiple sclerosis, downregulated by interferon treatment, and affected by season

Background: Multiple Sclerosis (MS) is an immune-mediated disease of the central nervous system which responds to therapies targeting circulating immune cells. Objective: Our aim was to test if the T-cell activation gene expression pattern (TCAGE) we had previously described from whole blood was replicated in an independent cohort. Methods: We used RNA-seq to interrogate the whole blood transcriptomes of 72 individuals (40 healthy controls, 32 untreated MS). A cohort of 862 control individuals from the Brisbane Systems Genetics Study (BSGS) was used to assess heritability and seasonal expression. The effect of interferon beta (IFNB) therapy on expression was evaluated. Results: The MS/TCAGE association was replicated and rationalized to a single marker, ribosomal protein S6 (RPS6). Expression of RPS6 was higher in MS than controls (p<0.0004), and lower in winter than summer (p<4.6E-06). The seasonal pattern correlated with monthly UV light index (R=0.82, p<0.002), and was also identified in the BSGS cohort (p<0.0016). Variation in expression of RPS6 was not strongly heritable. RPS6 expression was reduced by IFNB therapy. Conclusions: These data support investigation of RPS6 as a potential therapeutic target and candidate biomarker for measuring clinical response to IFNB and other MS therapies, and of MS disease heterogeneity.

Enhanced Translation of mRNAs Encoding Proteins Involved in mRNA Translation during Recovery from Heat Shock

The mRNAs encoding poly (A) binding protein (PABP1), eukaryotic elongation factor 1A (eEF1A) and ribosomal protein S6 (RPS6) belong to the family of terminal oligo pyrimidine tract (TOP) containing mRNAs. Translation of the TOP mRNAs is regulated by growth signals and usually codes for proteins involved in mRNA translation. Previous studies from our laboratory showed that translation of PABP1 mRNA was preferentially enhanced during recovery of HeLa cells from heat shock. Presence of the 5′ TOP cis element was required for the observed increase of PABP1 mRNA translation. In the studies reported here we showed that translation of two additional TOP mRNAs such as, eEF1A and RPS6 was similarly enhanced during recovery. In addition, we showed by in vivo cross-linking experiments that the cellular nucleic acid binding protein ZNF9 binds to all three TOP mRNAs examined in these studies as well as to the β-actin mRNA that lacks a TOP cis element. Binding of ZNF9 to mRNAs was observed in both heat-shocked and non heat- shocked cells. However, depletion of ZNF9 by siRNA prevented the preferred stimulation of PABP1, eEF1A and RPS6 expression during recovery from heat shock. There was no detectable effect of ZNF9 depletion on the basal level of expression of either β-actin or PABP1, eEF1A and RPS6 in HeLa cells following recovery from heat shock.ConclusionAlthough the presence of ZNF9 was required for the translational stimulation of PABP1, eEF1A and RPS6 mRNAs, the mechanistic details of this process are still unclear. Since ZNF9 was shown to bind both TOP and non-TOP mRNAs, it is uncertain whether ZNF9 exerts its stimulatory effect on TOP mRNA translation following recovery from heat shock through the TOP cis-element. Perhaps additional factors or post-translational modification(s) of ZNF9 following heat shock are necessary for the preferred increase of TOP mRNA translation.

Ribosomal Protein S6 Phosphorylation is Associated with Epithelial Dysplasia and Squamous Cell Carcinoma of the Oral Cavity

Ribosomal protein S6 (RPS6), a downstream effector of the mammalian target of rapamycin pathway (mTOR), is activated in many cancers including oral squamous cell carcinoma (OSCC). However, the role of RPS6 in the progression of potentially malignant disorders (or premalignant lesions) to OSCC is unknown. The purpose of this study was to examine the expression of RPS6 in epithelial dysplasia and OSCC to determine the association of RPS6 in tumor progression. In our study, an immunohistochemical analysis of RPS6 was performed on tissue microarrays containing 30 control samples, 15 epithelial dysplasia cases, and 53 OSCC cases. Correlations between the clinicopathologic features of OSCC and RPS6 expression were analyzed using the Chi-square test. We found RPS6 phosphorylation (p-RPS6) in 15/30 (50%) control normal oral mucosa samples, 15/15 (100%) epithelial dysplasia cases, and 47/53 (88.68%) OSCC cases. The frequency of p-RPS6 in epithelial dysplasia or OSCC showed a statistically significant difference compared to control (P < 0.001). However, there were no significant correlations between p-RPS6 and the clinicopathologic features of OSCC. Our findings suggest that RPS6 activation is associated with the early events of tumor progression, suggesting p-RPS6 as a potential marker for early detection of oral cancer.

Abstract P2-09-30: Impact of PIK3CA, PTEN and RPS6 Status on Lapatinib Treatment Outcome in Advanced-Stage Breast Cancer Patients

Abstract Background: Lapatinib is a tyrosine kinase inhibitor targeting growth factor receptors 1 and 2 (HER1, HER2) used in the case of HER2 positive advanced breast cancer. Lapatinib effects may be impaired by disregulation of the PI3K/Akt pathway. However, there are conflicting reports on the impact of the pathway disregulation on treatment efficacy. This study focused on a catalytic subunit of phosphoinositide-3-kinase, alpha polypeptide (PIK3CA) mutation status as well as phosphatase and tensin homolog (PTEN), ribosomal protein S6 (RPS6), the pathway downstream target and its phosphorylated form (P-RPS6) expression in relation to lapatinib plus capecitabine (LC) treatment. Materials and Methods: Retrospective formalin-fixed, paraffin-embedded tumor samples from 28 LC treated patients (Pts) were used. PIK3CA mutations in exon 9 and 20 were investigated in DNA by polymerase chain reaction followed by high-resolution melting analysis. Exact mutation occurring in a sample was estimated by pyrosequencing. PTEN, RPS6 and P-RPS6 expressions were assessed by immunohistochemistry. The data were evaluated by standard statistical methods in correlation with Pts clinical and histopathological data. Results: The majority of tumors were invasive ductal carcinomas 82% (23/28); 43% (12/28) with hormonal receptor positivity. Pts were treated with LC for advanced disease for 1.2 - 15.6 months. LC treatment was ended mostly due to progression (20/28), only in 6 cases owing to toxicity. PIK3CA mutations were found in 21% (4/19), only in exon 20. The mutations were in all cases: c.3140A&amp;gt;G. PTEN, RPS6 and P-RPS6 cytoplasmic expressions were observed in 45 % (10/22), 81% (17/21) and 82% (18/22), respectively. P-RPS6 high expression (3+) was found in 73% (16/22). PTEN loss or PIK3CA mutation was found in 66.7% (16/24) but never in the same tumor. PIK3CA mutation tended to shorter disease free survival (DFS) (p = 0.062). PTEN and RPS6 expression showed a trend to longer LC treatment (p = 0.075 and p = 0.089, respectively). RPS6 expression correlated with longer trastuzumab treatment (p = 0.04). However, P-RPS6 3+ expression was associated with shorter DFS (p = 0.043), overall survival (p = 0.04) and tended to shorter survival after beginning LC treatment (p = 0.077). This parameter also correlated with progression on LC treatment whereas its negativity correlated with withdrawal of LC treatment because of toxicity (p = 0.009). Discussion: PTEN loss or PIK3CA mutation, suggesting PI3K/Akt pathway deregulation, was observed in 66.7 % of Pts. PTEN as well as non-phosphorylated RPS6 positivity tended to longer LC treatment duration which might be due to maintained PI3K/Akt regulation. On the other hand, high level RPS6 phosphorylation associated with poorer patient outcome, and its positivity correlated with progression on LC treatment. Together, these results suggest a negative influence of PI3K/Akt pathway disregulation on disease prognosis and LC treatment response. Supported by a grant of the Faculty of Medicine and Dentistry, Palacky University 91110301, MSM 6198959216, project Biomedreg CZ.1.05/2.1.00/01.0030 and partially IGA NS10286-3. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-30.