PSAT1 is regulated by ATF4 and enhances cell proliferation via the GSK3?/?-catenin/cyclin D1 signaling pathway in ER-negative breast cancer.
ABSTRACT: A growing amount of evidence has indicated that PSAT1 is an oncogene that plays an important role in cancer progression and metastasis. In this study, we explored the expression and function of PSAT1 in estrogen receptor (ER)-negative breast cancer.The expression level of PSAT1 in breast cancer tissues and cells was analyzed using real-time-PCR (RT-PCR), TCGA datasets or immunohistochemistry (IHC). The overall survival of patients with ER-negative breast cancer stratified by the PSAT1 expression levels was evaluated using Kaplan-Meier analysis. The function of PSAT1 was analyzed using a series of in vitro assays. Moreover, a nude mouse model was used to evaluate the function of PSAT1 in vivo. qRT-PCR and western blot assays were used to evaluate gene and protein expression, respectively, in the indicated cells. In addition, we demonstrated that PSAT1 was activated by ATF4 by chromatin immunoprecipitation (ChIP) assays.mRNA expression of PSAT1 was up-regulated in ER-negative breast cancer. A tissue microarray that included 297 specimens of ER-negative breast cancer was subjected to an immunohistochemistry assay, which demonstrated that PSAT1 was overexpressed and predicted a poor clinical outcome of patients with this disease. Our data showed that PSAT1 promoted cell proliferation and tumorigenesis in vitro and in vivo. We further found that PSAT1 induced up-regulation of cyclin D1 via the GSK3?/?-catenin pathway, which eventually led to the acceleration of cell cycle progression. Furthermore, ATF4 was also overexpressed in ER-negative breast cancers, and a positive correlation between the ATF4 and PSAT1 mRNA levels was observed in ER-negative breast cancers. We further demonstrated that knockdown of ATF4 by siRNA reduced PSAT1 expression. Finally, chromatin immunoprecipitation (ChIP) assays showed that PSAT1 was a target of ATF4.PSAT1, which is overexpressed in ER-negative breast cancers, is activated by ATF4 and promotes cell cycle progression via regulation of the GSK3?/?-catenin/cyclin D1 pathway.
Project description:I?B kinase ? (IKK?) is a key molecule at the crossroads of inflammation and cancer. Known to regulate cytokine secretion via NF?B and IRF3, the kinase is also a breast cancer oncogene, overexpressed in a variety of tumours. However, to what extent IKK? remodels cellular metabolism is currently unknown. Here we used metabolic tracer analysis to show that IKK? orchestrates a complex metabolic reprogramming that affects mitochondrial metabolism and consequently serine biosynthesis independently of its canonical signalling role. We found that IKK? upregulates the serine biosynthesis pathway (SBP) indirectly, by limiting glucose derived pyruvate utilisation in the TCA cycle, inhibiting oxidative phosphorylation. Inhibition of mitochondrial function induces Activating Transcription Factor 4 (ATF4), which in turn drives upregulation of the expression of SBP genes. Importantly, pharmacological reversal of the IKK?-induced metabolic phenotype reduces proliferation of breast cancer cells. Finally, we show that in a highly proliferative set of ER negative, basal breast tumours, IKK? and PSAT1 are both overexpressed, corroborating the link between IKK? and the SBP in the clinical context.
Project description:I?B kinase ? (IKK?) is a key molecule at the crossroads of inflammation and cancer. Known to regulate cytokine secretion via NF?B and IRF3, the kinase is also a breast cancer oncogene, overexpressed in a variety of tumours. However, to what extent IKK? remodels cellular metabolism is currently unknown. Here, we used metabolic tracer analysis to show that IKK? orchestrates a complex metabolic reprogramming that affects mitochondrial metabolism and consequently serine biosynthesis independently of its canonical signalling role. We found that IKK? upregulates the serine biosynthesis pathway (SBP) indirectly, by limiting glucose-derived pyruvate utilisation in the TCA cycle, inhibiting oxidative phosphorylation. Inhibition of mitochondrial function induces activating transcription factor 4 (ATF4), which in turn drives upregulation of the expression of SBP genes. Importantly, pharmacological reversal of the IKK?-induced metabolic phenotype reduces proliferation of breast cancer cells. Finally, we show that in a highly proliferative set of ER negative, basal breast tumours, IKK? and PSAT1 are both overexpressed, corroborating the link between IKK? and the SBP in the clinical context.
Project description:Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor ?-negative (ER?(-)) invasive breast cancers, its role in ER?-positive (ER?(+)) breast cancers is relatively unknown. We investigated the significance of S100A7 in ER?(+) breast cancer cells and observed that S100A7 overexpression in ER?(+) breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the ?-catenin/TCF4 pathway and an enhanced interaction of ?-catenin and E-cadherin in S100A7-overexpressing ER?(+) breast cancer cells. We observed down-regulation of ?-catenin, p-GSK3?, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ER?(+) breast cancer cells. In addition, we observed increased expression of GSK3?. Treatment with GSK3? inhibitor CHIR 99021 increased the expression of ?-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the ?-catenin/TCF4 pathway. Therefore, our studies reveal for the first time that S100A7-overexpressing ER?(+) breast cancer cells exhibit tumor suppressor capabilities through down-modulation of the ?-catenin/TCF4 pathway both in vitro and in vivo. Because S100A7 has been shown to enhance tumorigenicity in ER?(-) cells, our studies suggest that S100A7 may possess differential activities in ER?(+) compared with ER?(-) cells.
Project description:BACKGROUND:BRK is, a non-receptor tyrosine kinase, overexpressed in approximately 85% of human invasive ductal breast tumors. It is not clear whether BRK expression correlates with breast cancer subtypes, or the expression has prognostic or diagnostic significance. Herein, we investigated the correlation of BRK with any breast cancer subtypes and clinicopathological significance of BRK expression in breast cancer. METHODS:In this study, we examined BRK expression in 120 breast tumor samples and 29 breast cancer cell lines to explore the positive correlation between BRK and the expression of ER?. We used immunohistochemistry, RT-PCR, and immunoblotting to analyse our experimental samples. RESULT:We demonstrate that estrogen induces BRK gene and protein expression in ER+ breast cancer cells. Over-expression of ER? in the ER-negative breast cancer cell line increased BRK expression, and knock-down of ESR1 in MCF7 cells reduced BRK levels. Further, we provide evidence that BRK is regulated by ER? signaling and the presence of ER antagonists (tamoxifen and fulvestrant) reduce the expression of BRK in ER-positive breast cancer cells. Finally, we demonstrate that the overall survival of ER-positive breast cancer patients is poor when their cancers express high levels of BRK. CONCLUSION:Our data indicate that BRK is a prognostic marker for ER+ breast cancers and provide a strong rationale for targeting BRK to improve patients' survival.
Project description:Burkitt lymphoma (BL) is a rapidly growing tumor, characterized by high anabolic requirements. The MYC oncogene plays a central role in the pathogenesis of this malignancy, controlling genes involved in apoptosis, proliferation, and cellular metabolism. Serine biosynthesis pathway (SBP) couples glycolysis to folate and methionine cycles, supporting biosynthesis of certain amino acids, nucleotides, glutathione, and a methyl group donor, S-adenosylmethionine (SAM). We report that BLs overexpress SBP enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). Both genes are controlled by the MYC-dependent ATF4 transcription factor. Genetic ablation of PHGDH/PSAT1 or chemical PHGDH inhibition with NCT-503 decreased BL cell lines proliferation and clonogenicity. NCT-503 reduced glutathione level, increased reactive oxygen species abundance, and induced apoptosis. Consistent with the role of SAM as a methyl donor, NCT-503 decreased DNA and histone methylation, and led to the re-expression of ID4, KLF4, CDKN2B and TXNIP tumor suppressors. High H3K27me3 level is known to repress the MYC negative regulator miR-494. NCT-503 decreased H3K27me3 abundance, increased the miR-494 level, and reduced the expression of MYC and MYC-dependent histone methyltransferase, EZH2. Surprisingly, chemical/genetic disruption of SBP did not delay BL and breast cancer xenografts growth, suggesting the existence of mechanisms compensating the PHGDH/PSAT1 absence in vivo.
Project description:Altered choline phospholipid metabolism is a hallmark of cancer, leading to malignant choline metabolite profiles consisting of low glycerophosphocholine (GPC) and high phosphocholine (PC) in human breast cancers. Glycerophosphocholine phosphodiesterase (GPC-PDE) catalyzes the degradation of GPC to free choline and glycerol-3-phosphate. The gene(s) encoding for the GPC-PDE(s) responsible for GPC degradation in breast cancers have not yet been identified. Here, we demonstrate for the first time that the GPC-PDE encoded by glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) is associated with breast cancer malignancy. Two human breast cancer cell lines (n = 8 and n = 10) and primary human breast tumor samples (n = 19) were studied with combined MRS and quantitative reverse transcription-polymerase chain reaction to investigate several isoforms of GDPD expression with respect to choline phospholipid metabolite levels. Of the five GDPDs tested, GDPD5 was found to be significantly overexpressed in highly malignant estrogen receptor negative (ER(-)) compared with weakly malignant estrogen receptor positive (ER(+)) human breast cancer cells (p = 0.027) and breast tumors from patients (p = 0.015). GDPD5 showed significantly positive correlations with PC (p < 0.001), total choline (tCho) (p = 0.007) and PC/GPC (p < 0.001) levels in human breast tumors. GDPD5 showed a trend towards a negative correlation with GPC levels (p = 0.130). Human breast cancers with malignant choline metabolite profiles consisting of low GPC and high PC levels highly co-expressed GDPD5, choline kinase alpha (CHKA) and phosphatidylcholine-specific phospholipase D1 (PLD1), whereas cancers containing high GPC and relatively low PC levels displayed low co-expression of GDPD5, CHKA and PLD1. GDPD5, CHKA and PLD1 were significantly overexpressed in highly malignant ER(-) tumors in our patient cohort. Our study identified GDPD5 as a GPC-PDE that probably participates in the regulation of choline phospholipid metabolism in breast cancer, which possibly occurs in cooperation with CHKA and PLD1.
Project description:MDM2 and MDM4 are heterodimeric, non-redundant oncoproteins that potently inhibit the p53 tumor suppressor protein. MDM2 and MDM4 also enhance the tumorigenicity of breast cancer cells in in vitro and in vivo models and are overexpressed in primary human breast cancers. Prior studies have characterized Estrogen Receptor Alpha (ER?/ESR1) as a regulator of MDM2 expression and an MDM2- and p53-interacting protein. However, similar crosstalk between ER? and MDM4 has not been investigated. Moreover, signaling pathways that mediate the overexpression of MDM4 in human breast cancer remain to be elucidated. Using the Cancer Genome Atlas (TCGA) breast invasive carcinoma patient cohort, we have analyzed correlations between ER? status and MDM4 and MDM2 expression in primary, treatment-naïve, invasive breast carcinoma samples. We report that the expression of MDM4 and MDM2 is elevated in primary human breast cancers of luminal A/B subtypes and associates with ER?-positive disease, independently of p53 mutation status. Furthermore, in cell culture models, ER? positively regulates MDM4 and MDM2 expression via p53-independent mechanisms, and these effects can be blocked by the clinically-relevant endocrine therapies fulvestrant and tamoxifen. Additionally, ER? also positively regulates p53 expression. Lastly, we report that endogenous MDM4 negatively regulates ER? expression and forms a protein complex with ER? in breast cancer cell lines and primary human breast tumor tissue. This suggests direct signaling crosstalk and negative feedback loops between ER? and MDM4 expression in breast cancer cells. Collectively, these novel findings implicate ER? as a central component of the p53-MDM2-MDM4 signaling axis in human breast cancer.
Project description:In human breast cancer, estrogen receptor-? (ER?) suppresses epithelial-mesenchymal transition (EMT) and stemness, two crucial parameters for tumor metastasis; however, the underlying mechanism by which ER? regulates these two processes remains largely unknown. Bmi1, the polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog, regulates EMT transition, maintains the self-renewal capacity of stem cells, and is frequently overexpressed in human cancers. In the present study, ER? upregulated the expression of the epithelial marker, E-cadherin, in breast cancer cells through the transcriptional down-regulation of Bmi1. Furthermore, ER? overexpression suppressed the migration, invasion, and EMT of breast cancer cells. Notably, overexpression of ER? significantly decreased the CD44high/CD24low cell population and inhibited the capacity for mammosphere formation in ER?-negative breast cancer cells. In addition, overexpression of Bmi1 attenuated the ER?-mediated suppression of EMT and cell stemness. Immunohistochemistry revealed an inverse association of ER? and Bmi1 expression in human breast cancer tissue. Taken together, our findings suggest that ER? inhibits EMT and stemness through the downregulation of Bmi1.
Project description:Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor ? (ER?)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ER?-positive disease than in ER?-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ER?-positive disease by showing that Lrig1 is a target of ER?. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ER?-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ER?-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.