RPS6KB1 Research Articles

Prognostic and survival impact of BCL9 and RPS6KB1 copy number variation detected from circulating free DNA in hepatocellular carcinoma

ABSTRACT Background Circulating cell-free DNA (cfDNA) is a noninvasive substitute to liver biopsy for hepatocellular carcinoma (HCC) molecular profiling. This study aimed to use cfDNA to investigate copy number variation (CNV) in the BCL9 and RPS6KB1 genes and its impact on prognosis in HCC. Methods Real-Time Polymerase Chain Reaction was used to determine the CNV and cfDNA integrity index in 100 HCC patients. Results CNV gain in BCL9 and RPS6KB1 genes was detected in 14% and 24% of patients, respectively. Gain in CNV of BCL9 associated with risk of HCC in alcohol drinkers and hepatitis C seropositivity. In patients with RPS6KB1 gain, HCC risk increased with a high body mass index, smoking, schistosomiasis, and Barcelona clinical liver cancer stage (BCLC) A. Gain in both genes showed a high risk of HCC with elevated liver enzymes, Schistosomiasis, BCLC C, and PS > 1. The integrity of cfDNA was higher in patients with CNV gain in RPS6KB1 than those harboring CNV gain in BCL9. Lastly, BCL9 gain and BCL9 + RPS6KB1 gain led to higher mortality rates and reduced survival times. Conclusion cfDNA was used to detect BCL9 and RPS6KB1 CNVs, which influence prognosis and can be used as independent predictors of HCC patient survival.

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis, and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here, we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

Prognostic and Therapeutic Potentials of OncomiRs Modulating mTOR Pathways in Virus-Associated Hepatocellular Carcinoma.

BackgroundDysregulated oncomiRs are attributed to hepatocellular carcinoma (HCC) through targeting mTOR signaling pathway responsible for cell growth and proliferation. The potential of these oncomiRs as biomarker for tumor response or as target for therapy needs to be evaluated.AIMTumor response assessment by OncomiR changes following locoregional therapy (LRT) and targeting of these oncomiRs modulating pathwayMethodsAll consecutive viral-HCC patients of BCLC stage-A/B undergoing LRT were included. OncomiRs (miR-21, -221, and -16) change in circulation and AFP-ratio at 1-month post-LRT to baseline was estimated to differentiate various categories of response as per mRECIST criteria. OncomiR modulating mTOR pathway was studied by generating miR-21 and miR-221 overexpressing Huh7 stable cell lines.ResultsPost-LRT tumor response was assessed in 90 viral-HCC patients (CR, 40%; PR, 31%, and PD, 29%). Significant increase of miRNA-21 and -221 expression was observed in PD (p = 0.040, 0.047) and PR patients (miR-21, p = 0.045). Fold changes of miR-21 can differentiate response in group (CR from PR+PD) at AUROC 0.718 (95% CI, 0.572–0.799) and CR from PD at AUROC 0.734 (95% CI, 0.595–0.873). Overexpression of miR-21 in hepatoma cell line had shown increased phosphorylation p70S6K, the downstream regulator of cell proliferation in mTOR pathway. Upregulation of AKT, mTOR, and RPS6KB1 genes were found significant (P < 0.005) and anti-miR-21 specifically reduced mTOR gene (P = 0.02) expression.ConclusionsThe miR-21 fold change correlates well with imaging in predicting tumor response. Overexpression of miR-21 has a role in HCC through mTOR pathway activation and can be targeted.

Deficiency of the oxidative stress–responsive kinase p70S6K1 restores autophagy and ameliorates neural tube defects in diabetic embryopathy

Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For invitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes invivo and high glucose invitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.

Effects of jugular infusions of isoleucine, leucine, methionine, threonine, and other amino acids on insulin and glucagon concentrations, mammalian target of rapamycin (mTOR) signaling, and lactational performance in goats

The supply and profile of absorbed AA may affect milk protein synthesis through hormonal changes and mammalian target of rapamycin (mTOR) signaling pathways; and Ile, Leu, Met, and Thr (ILMT) are the 4 AA that have been reported to have the greatest effect on mammary mTOR signaling. The extent to which ILMT and the other remaining AA (RAA) differ in their effects on milk protein synthesis needs to be systematically investigated. In this study, 5 lactating goats, averaging 120 ± 10 d in milk, fitted with jugular vein and carotid artery catheters, were fasted for 24 h, followed by intravenous infusions of a mixture containing AA and glucose for 8 h in a 5 × 5 Latin square design. The AA mixtures were formulated according to the profile of casein. The amounts of AA infused were calculated based on supplies of AA when metabolizable protein (MP) was at requirement (MR). Treatments were an infusate containing glucose without AA (NTAA); an infusate containing 3 × the MR of Ile, Leu, Met and Thr (3F0R); and infusates containing 3F0R plus 1, 2, or 3 × MR of RAA (3F1R, 3F2R, and 3F3R, respectively) according to amounts provided when fed to meet MP requirements for maintenance and lactation for each goat. Milk, arterial blood, and mammary tissue samples were collected immediately after halting the infusion. Relative to NTAA, supplementation of ILMT tended to increase milk protein production and plasma glucose concentrations, and increased milk and lactose production, but had no effects on production or content of milk fat. Graded supplementation of RAA tended to quadratically affect production of milk and lactose. Arterial glucose and glucagon concentrations decreased linearly, and plasma insulin concentrations decreased quadratically with increased RAA. Mammary p70-S6K1 phosphorylation was decreased by addition of ILMT compared with NTAA but increased linearly with increased RAA infusion. Furthermore, EIF4EBP1 gene expression was much lower for 3F-treated goats than for the NTAA treatment. Both MTOR and RPS6KB1 gene expressions were decreased quadratically with increased RAA supply. These results suggested that short-term milk protein yield tended to be increased by elevated ILMT availability, and this trend was not explained by variations in mammary mTOR signaling or pancreatic hormone secretions, whereas graded increase of RAA in combination with ILMT appeared to regulate the efficiency of conversion of glucose to lactose in a manner not involving milk protein production.

QTL fine mapping for intramuscular fat and fatty acid composition using high-density SNP chip array on SSC12 in Korean native pig × Yorkshire F2 population

Intramuscular fat (IMF) and fatty acid composition are characteristics that are used as important indicators of evaluating high quality pork and contribute to the economic benefits of the pig farming industry. In this study, quantitative trait loci (QTL) fine mapping of chromosome 12 was performed in a population of F2 intercross between Yorkshire (YS) and Korean native pigs (KNPs) by adopting combined linkage and linkage disequilibrium method using high-density SNP chips. QTLs for IMF (H3GA0034813 to H3GA0034965) and oleic acid (C18:1) (ASGA0054380 to ALGA0066299) were located at 120 cM (54.112–57.610 kb) and 85 cM (36.097–38.601 kb), respectively, within chromosome 12 (Sscrofa11.1 genomic reference). In addition, 31 candidate genes present within the IMF QTL region and 28 candidate genes existing within C18:1 QTL region were chosen. In order to understand the function of these candidate genes at the molecular level, these candidate genes were functionally categorized by studying gene ontology and analyzing network and pathway. Among the 59 candidate genes within the region of IMF QTL and C18:1 QTL, five (MYH1, MYH2, MYH4, ACACA, and RPS6KB1) directly interacting candidate genes were found. Furthermore, the RPS6KB1 gene was assumed to be an important candidate gene that is involved in leptin and insulin signaling pathway and participates in controlling adipogenic differentiation, fat deposition, and fatty acid composition, which is related to obesity of pigs.

Liraglutide alters hepatic metabolism in high-fat fed obese mice: A bioinformatic prediction and functional analysis

Liraglutide, an important pharmacological agent used to improve glycemic status and weight control, has emerged as a therapeutic strategy for hepatic steatosis treatment. ObjectiveThe aim of the present study was to analyze the influence of liraglutide in hepatic steatosis and evaluate the potential target genes involved in the liraglutide action in the hepatic steatosis through a bioinformatics study. Methods & proceduresWe performed an animal study with male mice divided into three groups: standard, high-fat diet and the third group were fed a high-fat diet and treated with liraglutide at a dose of 0.6 mg/kg body weight. Blood parameters (glucose tolerance and insulin sensitivity tests, total cholesterol, high-density lipoprotein-C, triglycerides and glucose levels) were evaluated. mRNA analysis for ACC and FAS were performed. Gene databases, and bioinformatics algorithms were used to generate molecular targets for liraglutide and hepatic steatosis based on in silico investigation. Interactions networks between protein coding were accessed. ResultsThe present study showed that liraglutide decreased glucose levels, total cholesterol and triglycerides in obese animals as compared to the high-fat-fed obese mice. AKT and RPS6KB1 genes presented higher disease-related connectivity. Interaction Network exhibited a power law behavior in correlation: 0.896; R2: 0.796. Ontological analysis demonstrated different mechanisms associated such as regulation of signaling process. ConclusionThe present study reveals relevant information regarding the liraglutide effects in hepatic steatosis. The liraglutide improved hyperglycemia and attenuated hepatic steatosis in mice fed a high-fat diet.

The Gene Expression Status of the PI3K/AKT/mTOR Pathway in Gastric Cancer Tissues and Cell Lines.

The PI3K/AKT/mTOR pathway plays a crucial role in the regulation of multiple cellular functions including cell growth, proliferation, metabolism and angiogenesis. Emerging evidence has shown that deregulation of this pathway has a role promoting gastric cancer (GC). The aim was to assess the expression of genes involved in this pathway by qPCR in 23 tumor and 23 non-tumor gastric mucosa samples from advanced GC patients, and in AGS, MKN28 and MKN45 gastric cancer cell lines. Results showed a slight overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1, EIF4EBP1 and EIF4E genes, and a slightly decreased PTEN and TSC1 expression. In AGS, MKN28 and MKN45 cells a significant gene overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1 and EIF4E, and a significant repression of PTEN gene expression were observed. Immunoblotting showed that PI3K-β, AKT, p-AKT, PTEN, mTOR, p-mTOR, P70S6K1, p-P70S6K1, 4E-BP1, p-4E-BP1, eIF4E and p-eIF4E proteins were present in cell lines at different levels, confirming activation of this pathway in vitro. This is the first time this extensive panel of 9 genes within PI3K/AKT/mTOR pathway has been studied in GC to clarify the biological role of this pathway in GC and develop new strategies for this malignancy.

Abstract B07: Estrogen-related receptor alpha regulates S6K1 expression and tamoxifen sensitivity in breast cancer

Abstract Estrogen related receptor alpha (ERRα) is an orphan nuclear receptor that plays important role in the metabolic activities and proliferation of cancer cells. We investigated the role of ERRα in regulating the expression of S6K1 in breast cancer cells. First, using RT-qPCR and immunoblotting we found that ERRα overexpression in estrogen receptor (ER) positive MCF7 cells results in decreased S6K1 expression and activity. Conversely, downregulation of ERRα expression resulted in increased S6K1 expression and activity in ER-negative MDA-MB-231 cells. Second, overexpression of ERRα in MCF7 cells also resulted in decreased transcriptional activity of the RPS6KB1 gene promoter as revealed by luciferase reporter assay. On the other hand, ERRα knockdown in MDA-MB-231 cells resulted in increased promoter activity. Third, because the proximal 1 kilobase (Kb) promoter region of S6K1 contains several putative estrogen receptor DNA binding elements (EREs), we investigated whether ERRα modulates S6K1 expression by direct promoter binding using chromatin immunoprecipitation (ChIP) technique. We detected two binding peaks approximately 300 and 600 kb away from the transcription start site (TSS). In addition, we showed that downregulation of ERRα caused increased cell proliferation and sensitivity to the mTOR inhibitor rapamycin. Suprisingly, tamoxifen treatment increased the proliferative capacity of these cells. Interestingly, cytoplasmic and nuclear staining of ERRα in tumor specimens derived from patients in the Stockholm breast cancer trial of adjuvant tamoxifen revealed that (1) in triple-negative tumors, the low-expressing cytoplasmic ERRα group had a worse prognosis, whereas a clear improvement in recurrence-free survival was found in the group with high ERRα-expressing tumors; and (2) in the ERα-negative subgroup, high ERRα rendered a clear tamoxifen benefit, which is very interesting, as this group is not receiving tamoxifen today and may constitute a subgroup which could gain from tamoxifen. Overall, our data indicate that ERRα regulates S6K1 expression and affects the sensitivity to rapamycin tamoxifen. Our long-term goal is to establish ERRα as a marker for endocrine and rapamycin therapy response, as well as a novel target in combination therapy approaches. Citation Format: Subrata Manna, Josefine Bostner, Anya Alayev, Olle Stal, Marina K. Holz. Estrogen-related receptor alpha regulates S6K1 expression and tamoxifen sensitivity in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B07.

Chromosome 17 copy number changes in male breast cancer.

BackgroundOverall, HER2-amplified female breast cancer (FBC) is associated with a high grade, an aggressive phenotype and a poor prognosis. In male breast cancer (MBC) amplification of HER2, located on chromosome 17, occurs at a lower frequency than in FBC, where it is part of complex rearrangements. So far, only few studies have addressed the occurrence of chromosome 17 alterations in small MBC cohorts.MethodsMultiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) were used to detect and characterize copy number changes on chromosome 17 in a cohort of 139 MBC. The results obtained were compared to those in FBC, and were correlated with clinicopathological features and patient outcome data.ResultsWe observed a lower frequency of chromosome 17 copy number changes with less complex rearrangement patterns in MBC compared to FBC. Chromosome 17 changes in MBC included gains of 17q and losses of 17p. Whole chromosome 17 polyploidies were not encountered. Two recurrent chromosome 17 amplicons were detected: on 17q12 (encompassing the NEUROD2, HER2, GRB7 and IKZF3 gens) and on 17q23.1 (encompassing the MIR21 and RPS6KB1 genes). Whole arm copy number gains of 17q were associated with decreased 5 year survival rates (p = 0.010). Amplification of HER2 was associated with a high tumor grade, but did not predict patient survival. Although copy number gains of HER2 and NEUROD2 were associated with a high tumor grade, a high mitotic count and a decreased 5 year survival rate (p = 0.015), only tumor size and NEUROD2 copy number gains emerged as independent prognostic factors.ConclusionsIn MBC chromosome 17 shows less complex rearrangements and fewer copy number changes compared to FBC. Frequent gains of 17q, encompassing two distinct amplicons, and losses of 17p were observed, but no whole chromosome 17 polyploidies. Only NEUROD2 gains seem to have an independent prognostic impact. These results suggest different roles of chromosome 17 aberrations in male versus female breast carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-015-0227-7) contains supplementary material, which is available to authorized users.

Estrogen‐Related Receptor (ERR)α Negatively Regulates Expression of the 40S Ribosomal S6 Kinase 1 (p70 S6K1) in Breast Cancer Cells

rEstrogen related receptor alpha (ERRa) is an orphan nuclear receptor that plays important role in the metabolic activities and proliferation of cancer cells. We investigated the role of ERRa in regulating the expression of 40S Ribosomal S6 Kinase 1 (S6K1) in breast cancer cells. First, using RT‐qPCR and immunoblotting we found that ERRa overexpression in estrogen receptor (ER) positive MCF7 cells results in decreased S6K1 expression and activity. Conversely, downregulation of ERRa expression resulted in increased S6K1 expression and activity in ER‐negative MDA‐MB‐231 cells. Second, overexpression of ERRa in MCF7 cells also resulted in decreased transcriptional activity of the RPS6KB1 gene promoter as revealed by luciferase reporter assay. On the other hand, ERRa knockdown in MDA‐MB‐231 cells resulted in increased promoter activity. Third, because the proximal 1 kilobase (Kb) promoter region of S6K1 contains several putative estrogen receptor DNA binding elements (EREs), we investigated whether ERRa modulates S6K1 expression by direct promoter binding using chromatin immunoprecipitation (ChIP) technique. We detected two binding peaks approximately 300 and 600 kb away from the transcription start site (TSS). In addition, we showed that downregulation of ERRa caused increased cell proliferation and sensitivity to the mTOR inhibitor rapamycin. Surprisingly, tamoxifen treatment increased the proliferative capacity of these cells. Overall, our data indicate that ERRα regulates S6K1 expression and affects the sensitivity to rapamycin and tamoxifen. Our long‐term goal is to establish ERRa as a marker for endocrine and rapamycin therapy response, as well as a novel target in combination therapy approaches.

Expression of S6K1 in human visceral adipose tissue is upregulated in obesity and related to insulin resistance and inflammation.

The ribosomal protein S6 kinase 1 (S6K1) is a component of the insulin signalling pathway that has been proposed as a key molecular factor in insulin resistance development under conditions of nutrient overload. The aim was to evaluate the involvement of S6K1 in obesity as well as to explore their association with visceral adipose tissue (VAT) inflammation. Samples obtained from 40 subjects were used. Gene expression levels of RPS6KB1 and key inflammatory markers were analysed in VAT. The effect of insulin on transcript levels of RPS6KB1 in human differentiated adipocytes was also explored. RPS6KB1 mRNA levels in VAT were increased (P < 0.05) in obese patients. Insulin treatment significantly enhanced (P < 0.01) gene expression levels of RPS6KB1 and a positive association (P < 0.05) of RPS6KB1 expression with different markers of insulin resistance was observed. Moreover, RPS6KB1 gene expression levels were positively correlated with VAT gene expression levels of the inflammatory markers CCL2, CD68, MMP2, MMP9, VEGFA and CHI3L1 as well as with mRNA levels of MTOR and MAPK8, representative players involved in signalling pathways related to S6K1. The increased levels of S6K1 in obesity and its positive association with insulin resistance and inflammation suggest a role for this protein in the changes that take place in VAT in obesity establishing a link between inflammation and a higher risk for the development of metabolic diseases.

P70 Ribosomal protein S6 kinase (Rps6kb1): an update

The Rps6kb1 gene encodes the 70 kDa ribosomal protein S6 kinase (p70S6K), which is a serine/threonine kinase regulated by phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. p70S6K plays a...

Abstract A068: Targeting Akt reactivation following mTOR inhibition in breast cancer treatment

Abstract Breast cancer is the second leading cause of cancer related deaths in women and up to 60% of diagnosed cases are estrogen receptor (ER)-positive. ER-positive status is associated with a poor prognosis. While ER-positive cancers could be targeted with endocrine therapy, such as aromatase inhibitors, resistance often develops. Therefore, there is a need to better understand molecular contributions of other signaling pathways with the goal of treating patients with personalized combination therapies. The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell cycle progression and proliferation and is a target of a naturally occurring inhibitor rapamycin. The 40S ribosomal S6 Kinase 1 (S6K1) is one of the best characterized downstream targets of mTORC1. The mTORC1/S6K1 pathway is often hyperactivated in breast cancer, promoting cell growth and proliferation. S6K1 is encoded by the RPS6KB1 gene, located on the 17q23 chromosomal region. While this region is amplified in several types of cancer, multi-copy amplicons are found specifically in breast cancer, underscoring the importance of S6K1 in this disease. Importantly, there is a direct link between the mTORC1/S6K1 and ER signaling pathways. S6K1 directly phosphorylates and activates ER, promoting ligand-independent ER activation and endocrine resistance. Based on this data, current FDA approved treatment of advaced ER-positive breast cancer combines mTORC1 inhibitors with endocrine therapy. Some of the concerns that arise with the use of mTORC1 inhibitors (rapamycin analogs or rapalogs) is that they promote induction of autophagy and relieve the negative feedback signaling to Akt effected by S6K1, which promotes oncogenic Akt signaling, survival, and further stimulates mTORC1/S6K1 signaling, which may contribute to acquired drug resistance and patient relapse. Our research focuses on identifying agents that could be combined with mTORC1 inhibitors to block S6K1 while maintaining negative feedback signaling to Akt. Our results show that such combination treatment does in fact prevent rapamycin-induced upregulation of Akt while maintaining inhibition of the S6K1 signaling pathway, as assayed by analysis of S6K1 targets PDCD4, eIF4B and S6. Moreover, the combination treatment inhibits autophagy and promotes apoptosis. Thus, the combined inhibition of S6K1 and Akt may prevent or reverse drug resistance in breast cancer. Citation Format: Anya Alayev, Rose B. Snyder, Marina K. Holz. Targeting Akt reactivation following mTOR inhibition in breast cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A068.

Abstract 2894: Co-regulatory relationship between estrogen receptor alpha and the mTOR/S6K1 signaling pathways

Abstract ERα mediates the proliferative effects of estrogen and represents an important clinical target in treatment of breast cancer. Tamoxifen is an anti-estrogen that has become the standard agent for the treatment of ER-positive breast cancer, where it acts as an antagonist. However, resistance to tamoxifen, and other endocrine or anti-estrogen therapies develops in many cases. One mechanism by which resistance develops is through phosphorylation of ERα, allowing it to act in estrogen-independent manner. Phosphorylation of Ser118 and Ser167 is important for transcriptional activity of ERα. The regulation of these sites is complex, involving inputs from the mTOR and MAPK signaling pathways. Importantly, ERα phosphorylation correlates with resistance to tamoxifen, and is a prognostic marker for disease progression and survival. Thus, the mechanism of these phosphorylation events, as well as the identity of the kinases responsible may have important clinical consequences. In this study we demonstrate that the mTOR/S6K1 pathway controls activation of ERα by phopshorylating it on these two critical residues. Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types is not well understood. In this study, we demonstrate that in response to estrogen activation, ERα activates expression of S6K1 by upregulating transcription of the RPS6KB1 gene. Increased levels of S6K1 promote further phosphorylation and activation of ERα, creating a positive feed-forward loop. We hypothesize that this relationship promotes breast cancer cell growth, thus providing a selective advantage to S6K1- and ERα-overexpressing cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2894. doi:10.1158/1538-7445.AM2011-2894

Abstract 3167: Estrogenic regulation of S6 kinase 1 expression creates a positive feed-forward loop in control of breast cancer cell proliferation

Abstract The 40S ribosomal S6 kinase 1 (S6K1) acts downstream of the mammalian Target of Rapamycin (mTOR), and is sensitive to inhibition by rapamycin. Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types is not well understood. We have previously reported that S6K1 regulates growth of breast cancer cells by phosphorylating Estrogen Receptor α (ERα), and co-expression of S6K1 and ERα in breast cancer cells renders them sensitive to combination therapy with rapamycin and tamoxifen. In this study, we demonstrate that in response to estrogen activation, ERα activates expression of S6K1 by upregulating transcription of the RPS6KB1 gene. Increased levels of S6K1 promote further phosphorylation and activation of ERα, creating a positive feed-forward loop. We hypothesize that this relationship promotes breast cancer cell growth, thus providing a selective advantage to S6K1- and ERα-overexpressing cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3167.

S6 Kinase 1 Regulates Estrogen Receptor α in Control of Breast Cancer Cell Proliferation

The 40 S ribosomal S6 kinase 1 (S6K1) acts downstream of mTOR (mammalian target of rapamycin) and is sensitive to inhibition by rapamycin. The chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, leading to S6K1 overexpression. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the identity of mammary cell-specific S6K1 targets is not well understood. In this study, we report that overexpression of S6K1 endows breast cancer cells with a proliferative advantage in low serum conditions and enhanced sensitivity to rapamycin. We investigate the molecular mechanism behind this observation to show that S6K1 regulates estrogen receptor alpha (ERalpha) by phosphorylating it on serine 167, leading to transcriptional activation of ERalpha. By contributing to the activation of ERalpha, S6K1 promotes ERalpha-mediated cell proliferation and may be a target of therapeutic intervention in breast cancer.

Phospho-p70S6K/p85S6K and cdc2/cdk1 Are Novel Targets for Diffuse Large B-Cell Lymphoma Combination Therapy

This study aimed to identify and evaluate molecular targets for the development of a novel combination chemotherapy to treat refractory and recurrent diffuse large B-cell lymphoma (DLBCL). Lymphoma samples from 38 cases of primary and recurrent DLBCL were analyzed using real-time quantitative PCR of the RPS6KB1 and CDC2 genes, and immunohistochemistry for their gene products p70S6K/p85S6K and cdc2/cdk1. The Farage, Karpas422, Pfeiffer, and Toledo DLBCL cell lines were subsequently treated with rapamycin and UCN-01 alone or in combination. Cell proliferation, apoptosis, and cell cycle progression were analyzed after the drug treatment. In addition, the levels of several key protein kinases involved in the phosphoinositide 3'-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, apoptosis, and cell cycle progression were analyzed in the presence and absence of the drugs. Amplification of the RPS6KB1 and CDC2 genes was found in both primary and recurrent DLBCL. Moreover, the vast majority of these lymphomas (approximately 94%) were strongly positive for phospho-p70S6K and cdc2/cdk1 proteins. The combination of rapamycin and UCN-01 synergistically inhibited the DLBCL cell proliferation by inducing G1 arrest as well as apoptosis by suppressing the phosphorylation of p70S6K/p85S6K and CDC2 expression. RPS6KB1 and CDC2 overexpression is common in DLBCL. Simultaneously targeting the RPS6KB1 and CDC2 products phospho-p70S6K/p85S6K and cdc2/cdk1 is very effective in inhibiting DLBCL proliferation and overcoming drug resistance. This work suggests that multilevel inhibition of the PI3K/Akt/mTOR pathway and double-block of cell cycle progression are effective strategies for DLBCL therapy.

Estrogen receptor α is a target of mTOR/S6K1 signaling in control of breast cancer cell proliferation.

Abstract Abstract #4062 Background: The 40S ribosomal S6 kinase 1 (S6K1) acts downstream of the mammalian Target of Rapamycin (mTOR), and is sensitive to inhibition by rapamycin. Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types, and the identity of breast cell-specific S6K1 targets is not well understood. In this study, we report that S6K1 regulates growth of breast cancer cells by phosphorylating Estrogen Receptor α (ERα), and co-expression of S6K1 and ERα in breast cancer cells renders them sensitive to combination therapy with rapamycin and tamoxifen.&amp;#x2028; Materials and Methods: Using immunoblot analysis we determined expression levels of S6K1 in breast cancer cell lines, and the phosphorylation status of ERα on Ser167 after serum or mitogen stimulation. We compared the effect of low-serum media, and rapamycin and/or 4-hydroxytamoxifen (4-HT) addition on breast cancer cell proliferation using neutral red uptake assay.&amp;#x2028; Results: We determined that overexpression of S6K1 in breast cancer cells confers proliferative advantage in low serum, contributing to neoplastic transformation. Next, we wished to identify targets of S6K1 in control of proliferation of breast cancer cells. One of the proteins we identified as a target of S6K1 is ERα. ERα is frequently co-overexpressed with S6K1 in breast cancer cells, which could be an indication of co-selection, and ERα contains a putative S6K1 phosphorylation motif RERLAS167. We confirmed that ERα phosphorylation may be mediated by S6K1. We shown that in S6K1-overexpressing MCF7 cells, serum-stimulated ERα phosphorylation is rapamycin-sensitive. Moreover, ERα phosphorylation stimulated by insulin and tumor-promoting phorbol esters is also rapamycin-sensitive. S6K1 can phosphorylate ERα in an in vitro kinase assay. Lastly, shRNA-mediated suppression of S6K1 expression in MCF7 cells reduces ERα phosphorylation. Subsequently, we examined whether co-overexpression of S6K1 and ERα in breast cancer cells sensitizes them to combination therapy with rapamycin and tamoxifen. We analyzed proliferation of cells treated with rapamycin and/or 4-HT. We observed that in S6K1- and ERα-overexpressing cells, treatment with rapamycin and 4-HT resulted in greater inhibition of cell growth.&amp;#x2028; Discussion: Our data suggest that one of the targets of S6K1 signaling in breast cancer cells is ERα, and together, these proteins promote breast cancer cell proliferation. The ultimate objective would be to determine whether combined inhibition of S6K1 and the estrogen receptor could be used as targeted therapy for patients with double positive expression of S6K1 and ER. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4062.

Identification of genes associated with tumorigenesis of retinoblastoma by microarray analysis

There is no report on the gene expression profile of retinoblastoma (Rb). We analyzed the gene expression profile of Rb by the microarray technique. One thousand four genes were upregulated and 481 genes were downregulated. Microarray data were confirmed by semiquantitative RT-PCR for 5 genes in Rb samples: CDC25A, C17orf75, ERBB3, LATS2, and CHFR. Clusters of differentially expressed genes were identified on chromosomes 1, 16, and 17. Based on the expression profile, we hypothesized that the PI3K/AKT/mTOR (insulin signaling) pathway might be dysregulated in Rb. Our semiquantitative RT-PCR analysis of the PIK3CA, AKT1, FRAP1, and RPS6KB1 genes in Rb samples supported this hypothesis. We suggest that known inhibitors of this pathway could be evaluated for the treatment of Rb.