Level Of Protein Stability Research Articles

Abstract PO4-27-12: Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence

Abstract Background: Resistance to endocrine therapy (ET; tamoxifen, aromatase inhibitors, AI, or fulvestrant) in ER+ breast cancer (BC) could be due to survival of breast cancer stem cells (BCSCs). BCSCs contribute to relapse of ER+ BC. We discovered a novel and potent anti-BCSC gene, Death Associated Protein 6 (DAXX) through a pre-surgical biomarker window study combining ET plus a Notch inhibitor [MK-0752, a g-secretase inhibitor (GSI)]. We also found that ET enhances the BCSC population by decreasing DAXX protein levels. In this current study, we measured DAXX protein levels in ER+ PDX tumors and human tumor tissues treated with ET. We determined the mechanism by which ET regulates DAXX expression at the level of protein stability. The goal of this study was to identify novel therapeutic strategies to prevent ET-mediated degradation of the DAXX protein, eliminate BCSCs, and prevent ER+ recurrence. Methods: ER+ BC cell lines (MCF-7 and T47D), an ER+ PDX BCM 5097 tumor line, and human breast cancer tumor samples were used. The ER+ PDX tumor (BCM 5097) was implanted into NSG mice and passaged into female athymic, nude mice-supplemented with an estradiol capsule. After one week, the estradiol capsule was removed from 10 mice, to mimic the use of AI. The remaining 10 mice retained the estradiol capsule. Tumor area was measured weekly up to 50 weeks. Western blotting detected ERa, the stem cell factor Notch4, and DAXX levels. Immunohistochemistry (IHC) detected DAXX protein levels. ER+ human tumor samples were collected prior to and after neoadjuvant treatment with ET (N=11) or chemotherapy (N=14) and stained for the DAXX protein. Correlations were made between DAXX and Ki67. Full length and deletion mutants of DAXX were expressed in ER+ BC cells. BCSCs were measured using the mammosphere forming assay. Liquid chromatography followed by mass spectrometry were used to detect phosphorylated residues of DAXX in response to ET. Kinases that phosphorylate these residues on DAXX were identified using PhosphoSite.org. ER+ cells were treated with kinase inhibitors for AURKA (alisertib), AURKB (barasertib), CK1 (CK-IN-1), or CK2 (CX-4945) and DAXX protein was detected by western blotting. BCSC survival was measured using mammosphere forming assay. Based on results from the kinase inhibitor screen, barasertib was selected for pre-clinical testing in mice. ER+ MCF-7 xenograft tumors were treated with vehicle or barasertib in vivo up to 120 days. Results: DAXX protein levels decreased in ER+ PDX tumors in response to ET. Similar trends were detected in human tumor tissue after ET or chemotherapy alone. DAXX protein levels inversely correlated with Ki67 in ER+ human tumor samples. Analysis of DAXX deletion mutants demonstrated that amino acids 400-740 of the DAXX protein were required to inhibit BCSC survival. This region of the DAXX protein had high levels of phosphorylated residues in response to ET. Only the AURKB (barasertib) inhibitor increased DAXX protein expression and inhibited BCSCs in a DAXX-dependent manner. Barasertib inhibited growth of ER+ MCF-7 tumor xenografts compared to the vehicle control. After treatment ceased, tumor recurrence was measured up to 120 days. Regrowth of tumors was partially delayed in response to barasertib. Conclusions: ET decreased DAXX protein levels in ER+ PDX and human tumors. Downregulation of the DAXX protein by ET was through activation of AURKB and hyper-phosphorylation of DAXX which resulted in protein degradation and enhanced survival of BCSCs. Therefore, Inhibition of AURKB using barasertib partially restored DAXX expression, inhibited BCSCs, and delayed tumor recurrence. A combination approach of ET plus an AURKB inhibitor might be a novel therapeutic strategy to prevent ER+ breast cancer relapse by increasing DAXX in order to eradicate BCSCs. Support: Breast Cancer Research Foundation Citation Format: Clodia Osipo, Debra Wyatt, Kathy Albain. Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-27-12.

KEAP1/NRF2 kao potencijalno ciljno mesto dejstva lekova

Nuclear factor erythroid 2-related factor 2 (NRF2; encoded by NFE2L2) is an inducible transcription factor that regulates the expression of a large network of genes encoding proteins with cytoprotective functions. NRF2 also has a role in the maintenance of mitochondrial and protein homeostasis, and its activation allows adaptation to numerous types of cellular stress. NRF2 is principally regulated at the protein stability level by three main ubiquitin ligase systems, of which the regulation by Kelch-like ECH-associated protein 1 (KEAP1), a substrate adaptor protein for Cul3/Rbx1-based ubiquitin ligase, is best understood. KEAP1 is a multi-functional protein and, in addition to being a substrate adaptor, it is a sensor for electrophiles and oxidants. Pharmacological inactivation of KEAP1 has protective effects in animal models of human disease, and KEAP1 is now widely recognized as a drug target, particularly for chronic diseases, where oxidative stress and inflammation underlie pathogenesis. Many compounds that target KEAP1 have been developed, including electrophiles that bind covalently to cysteine sensors in KEAP1, non-electrophilic protein-protein interaction inhibitors that bind to the Kelch domain of KEAP1, disrupting its interaction with NRF2, and most recently, heterobifunctional proteolysistargeting chimeras (PROTACs) that promote the proteasomal degradation of KEAP1. The drug development of KEAP1-targeting compounds has led to the entry of two compounds, dimethyl fumarate (BG-12, Tecfidera®) and RTA-408 (omaveloxolone, SKYCLARYS®), in clinical practice. In 2013, dimethyl fumarate was licenced as the first oral first-line therapy for relapsingremitting multiple sclerosis and is also used for the treatment of moderate-to-severe plaque psoriasis. In February 2023, omaveloxolone was approved by the United States Food and Drug Administration as the first and only drug for patients with Friedreich's ataxia.

Efficient gene transfection to lung cancer cells via Folate-PEI-Sorbitol gene transporter.

Lung cancer is known to be one of the fatal diseases in the world and is experiencing treatment difficulties. Many treatments have been discovered and implemented, but death rate of patients with lung cancer continues to remain high. Current treatments for cancer such as chemotherapy, immunotherapy, and radiotherapy have shown considerable results, yet they are accompanied by side effects. One effective method for reducing the cytotoxicity of these treatments is via the use of a nanoparticle-mediated siRNA delivery strategy with selective silencing effects and non-viral vectors. In this study, a folate (FA) moiety ligand-conjugated poly(sorbitol-co-PEI)-based gene transporter was designed by combining low-molecular weight polyethyleneimine (LMW PEI) and D-sorbitol with FA to form FPS. Since folate receptors are commonly overexpressed in various cancer cells, folate-conjugated nanoparticles may be more effectively delivered to selective cancer cells. Additionally, siOPA1 was used to induce apoptosis through mitochondrial fusion. The OPA1 protein stability level is important for maintaining normal mitochondrial cristae structure and function, conserving the inner membrane structure, and protecting cells from apoptosis. Consequently, when FPS/siOPA1 was used for lung cancer in-vitro and in-vivo, it improved cell viability and cellular uptake.

Glucose regulation of the paralogous glucose sensing receptors Rgt2 and Snf3 of the yeast Saccharomyces cerevisiae

BackgroundThe yeast Saccharomyces cerevisiae senses extracellular glucose levels through the two paralogous glucose sensing receptors Rgt2 and Snf3, which appear to sense high and low levels of glucose, respectively. MethodsWestern blotting and qRT-PCR were used to determine expression levels of the glucose sensing receptors. ResultsRgt2 and Snf3 are expressed at different levels in response to different glucose concentrations. SNF3 expression is repressed by high glucose, whereas Rgt2 is turned over in response to glucose starvation. As a result, Rgt2 is predominant in cells grown on high glucose, whereas Snf3 is more abundant of the two paralogs in cells grown on low glucose. When expressed from a constitutive promoter, however, Snf3 behaves like Rgt2, being able to transduce the high glucose signal that induces HXT1 expression. Of note, constitutively active Rgt2 does not undergo glucose starvation-induced endocytic downregulation, whereas signaling defective Rgt2 is constitutively targeted for vacuolar degradation. These results suggest that glucose protects Rgt2 from endocytic degradation and reveal a previously unknown function of glucose as a signaling molecule that regulates the stability of its receptor. ConclusionExpression of Rgt2 and Snf3 is regulated by different mechanisms: Rgt2 expression is highly regulated at the level of protein stability; Snf3 expression is mainly regulated at the level of transcription. General significanceThe difference in the roles of Rgt2 and Snf3 in glucose sensing is a consequence of their cell surface abundance rather than a result of the two paralogous proteins having different functions.

Blue Light-Operated CRISPR/Cas13b-Mediated mRNA Knockdown (Lockdown).

The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light-regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas-related methods. However, these approaches lack the key characteristics and advantages provided by optical control. "Lockdown" introduces optical control of RNA levels utilizing a blue light-dependent switch to induce expression of CRISPR/Cas13b, which mediates sequence-specific mRNA knockdown. Combining Lockdown with optogenetic tools to repress gene-expression and induce protein destabilization with blue light yields efficient triple-controlled downregulation of target proteins. Implementing Lockdown to degrade endogenous mRNA levels of the cyclin-dependent kinase 1 (hCdk1) leads to blue light-induced G2/M cell cycle arrest and inhibition of cell growth in mammalian cells.

Abstract 5192: EGFR inhibition enhances the cellular uptake and antitumor activity of the novel HER3 antibody drug conjugate U3-1402

Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have changed the treatment landscape for EGFR-mutant non-small cell lung cancers (NSCLC); however, most patients develop resistance over time. HER3 is a unique pseudokinase member of the ERBB family which functions through dimerization with other ERBB family members (EGFR and HER2) and has been difficult to target with conventional kinase inhibitor strategies. HER3 is frequently over expressed in EGFR-mutant NSCLC. U3-1402 is an antibody-drug conjugate (ADC) comprised of HER3-targeting antibody (patritumab) linked to a topoisomerase I inhibitor (DX-8951 derivative, or DXd). U3-1402 is currently in clinical development as a single agent in metastatic EGFR-mutant NSCLC (NCT03260491). We aimed to develop a preclinical strategy to enhance the efficacy of U3-1402. Pre-treatment with EGFR TKIs (gefitinib or osimertinib) increased HER3 membrane levels in six different EGFR-mutant cell lines by increasing both the amount of HER3 positive cells and the intensity of HER3 expression. Furthermore, using a proximity-ligation assay (PLA), we noted an increase in membrane EGFR:HER3 interaction following EGFR inhibitor treatment. HER3 protein levels and EGFR:HER3 interaction were similarly increased in four EGFR-mutant patient-derived xenograft (PDX) models following osimertinib treatment. HER3 expression peaked between 24 and 48 hours, and the increase in HER3 was regulated by both increased gene expression and in the level of protein stability. We further evaluated the biological consequences of increased HER3 expression on U3-1402 intake and activity. We used pH-sensitive pHrodo Red conjugate to label U3-1402 to monitor the intake of the ADC using Incucyte live cell imaging. In three EGFR-mutant cell lines, there was a 3- to 5-fold higher intake of U3-1402 over time in cell lines pre-treated with osimertinib, compared to cell lines without pre-treatment. Osimertinib pre-treatment also increased the uptake of U3-1402 in HER3-low expressing EGFR-mutant models. In contrast, osimertinib pre-treatment did not increase the uptake of a control IgG-ADC. In long-term in vitro cell growth assays, the combination of osimertinib and U3-1402 was superior to single agent U3-1402 in four EGFR-mutant cell lines. Preliminary in vivo studies demonstrated no significant toxicities as measured by mouse body weights and complete blood counts. In vivo experiments evaluating the antitumor efficacy of the combination of osimertinib and U3-1402 are currently underway. Our studies reveal that EGFR inhibitor treatment increased membrane expression of HER3 which was associated with enhanced internalization of U3-1402 in EGFR-mutant NSCLC. The combination of osimertinib and U3-1402 may be an effective treatment approach and should be evaluated in future clinical trials in patients with EGFR-mutant NSCLC. Citation Format: Heidi M. Haikala, Jens Köhler, Timothy Lopez, Pinar Eser, Man Xu, Channing Yu, Yoshinobu Shiose, Yang Qiu, Prafulla Gokhale, Pasi A. Jänne. EGFR inhibition enhances the cellular uptake and antitumor activity of the novel HER3 antibody drug conjugate U3-1402 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5192.

Regulation of the p53 Family Proteins by the Ubiquitin Proteasomal Pathway.

The tumor suppressor p53 and its homologues, p63 and p73, play a pivotal role in the regulation of the DNA damage response, cellular homeostasis, development, aging, and metabolism. A number of mouse studies have shown that a genetic defect in the p53 family could lead to spontaneous tumor development, embryonic lethality, or severe tissue abnormality, indicating that the activity of the p53 family must be tightly regulated to maintain normal cellular functions. While the p53 family members are regulated at the level of gene expression as well as post-translational modification, they are also controlled at the level of protein stability through the ubiquitin proteasomal pathway. Over the last 20 years, many ubiquitin E3 ligases have been discovered that directly promote protein degradation of p53, p63, and p73 in vitro and in vivo. Here, we provide an overview of such E3 ligases and discuss their roles and functions.

Inflammation-induced reactive nitrogen species cause proteasomal degradation of dimeric peroxiredoxin-1 in a mouse macrophage cell line

Peroxiredoxin 1 (PRDX1) is an antioxidant enzyme that, when secreted, can act as a proinflammatory signal. Here we studied the regulation of intracellular PRDX1 by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in the RAW 264.7 mouse macrophage cell line. While LPS or IFN-γ alone did not affect PRDX1 protein levels, their combination led to an almost complete loss of the PRDX1 dimer. This was likely mediated by the increased production of nitric oxide (NO) as it was reversed by the NO synthase inhibitor L-N-methylarginine (L-NMMA), while a NO-releasing agent decreased PRDX1 levels. Inhibition of the proteasome with MG132 also prevented the loss of the PRDX1 dimer, suggesting that the decrease is due to a NO-activated proteasomal degradation pathway. By contrast with the decrease in protein levels, LPS increased PRDX1 mRNA and this effect was amplified by IFN-γ. Two other Nrf2 target genes, thioredoxin reductase (TXNRD1) and haem oxygenase (HMOX1), were also induced by LPS but IFN-γ did not increase their expression further. This study shows that inflammation differentially regulates PRDX1 at the levels of protein stability and gene expression, and that NO plays a key role in this mechanism.

Extensive Posttranscriptional Regulation of Nuclear Gene Expression by Plastid Retrograde Signals.

Retrograde signals emanate from the DNA-containing cell organelles (plastids and mitochondria) and control the expression of a large number of nuclear genes in response to environmental and developmental cues. Previous studies on retrograde signaling have mainly analyzed the regulation of nuclear gene expression at the transcript level. To determine the contribution of translational and posttranslational regulation to plastid retrograde signaling, we combined label-free proteomics with transcriptomic analysis of Arabidopsis (Arabidopsis thaliana) seedlings and studied their response to interference with the plastid gene expression pathway of retrograde signaling. By comparing the proteomes of the genomes uncoupled1 (gun1) and gun5 mutants with the wild type, we show that GUN1 is critical in the maintenance of plastid protein homeostasis (proteostasis) when plastid translation is blocked. Combining transcriptomic and proteomic analyses of the wild type and gun1, we identified 181 highly translationally or posttranslationally regulated (HiToP) genes. We demonstrate that HiToP photosynthesis-associated nuclear genes (PhANGs) are largely regulated by translational repression, while HiToP ribosomal protein genes are regulated posttranslationally, likely at the level of protein stability without the involvement of GUN1. Our findings suggest distinct posttranscriptional control mechanisms of nuclear gene expression in response to plastid-derived retrograde signals. They also reveal a role for GUN1 in the translational regulation of several PhANGs and highlight extensive posttranslational regulation that does not necessitate GUN1. This study advances our understanding of the molecular mechanisms underlying intracellular communication and provides new insight into cellular responses to impaired plastid protein biosynthesis.

Cell cycle regulation of condensin Smc4.

The condensin complex is a conserved ATPase which promotes the compaction of chromatin during mitosis in eukaryotic cells. Condensin complexes have in addition been reported to contribute to interphase processes including sister chromatid cohesion. It is not understood how condensins specifically become competent to facilitate chromosome condensation in preparation for chromosome segregation in anaphase. Here we describe evidence that core condensin subunits are regulated at the level of protein stability in budding yeast. In particular, Smc2 and Smc4 abundance is cell cycle regulated, peaking at mitosis and falling to low levels in interphase. Smc4 degradation at the end of mitosis is dependent on the Anaphase Promoting Complex/Cyclosome and is mediated by the proteasome. Overproduction of Smc4 results in delayed decondensation, but has a limited ability to promote premature condensation in interphase. Unexpectedly, the Mad2 spindle checkpoint protein is required for mitotic Smc4 degradation. These studies have revealed the novel finding that condensin protein levels are cell cycle regulated and have identified the factors necessary for Smc4 proteolysis.

Towards continuous wine fining: Materials characterisation and crossflow performance testing of polymer–bentonite mixed matrix membranes

AbstractThis study investigates the feasibility of using bentonite‐embedded mixed matrix membranes to perform wine fining on white wines to eliminate protein instability. Membranes made using polyethersulphone or polyvinylidene fluoride were fabricated using the wet phase inversion technique and used to filter unfined New Zealand Sauvignon Blanc wine in a crossflow filtration system.The morphologies of all the fabricated membranes were characteristic of asymmetric phase inversion membranes. Confocal laser scanning microscopy analysis on the membranes suggests that the two major mechanisms of protein removal are adsorption by the embedded bentonite particles and size exclusion via the membrane morphology. Polyethersulphone membranes were found to produce wine of similar levels of protein stability (around 90% protein removal) and polyphenol content as polyvinylidene fluoride membranes of the same membrane composition and operating conditions but with as much as 80% higher flux. Stabilising wine in one filtration pass was possible with polyethersulphone membrane with 25 wt% bentonite while maintaining high flux. These promising results show that the tested process has the potential to replace the current batch‐wise fining process with continuous crossflow membrane filtration with benefits such as increased processing speed and reduced wine loss.

Endoplasmic reticulum stress–induced degradation of DNAJB12 stimulates BOK accumulation and primes cancer cells for apoptosis

DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.

Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells.

Paraoxonase-1 (PON1) gene polymorphisms have been closely associated with the development of advanced cancers while PON1 secretion to the serum is linked with inhibition of oxidized high-density lipoprotein by its antioxidative function. Our group previously demonstrated that post-translational modification of serum PON1 in form of fucosylated PON1 is a potential biomarker of small cell lung cancer. Here, we interrogated the role of PON1 in the pathobiology of lung cancer (LC) by addressing cell-autonomous mechanisms using gain-of-function and loss-of-function approaches and protein expression profiling of tissue samples in our clinical biobank. PON1 expression in LC patient tissues varied between overexpression in squamous cell carcinoma and minimal loss in adenocarcinoma sub-types. Simultaneous overexpression of PON1 both at the gene and protein stability levels induced pro-oncogenic characteristics in LC cells and xenografts. PON1 overexpression supported metastatic progression of LC by decreasing G1/S ratio and LC cell senescence involving p21Waf1/Cip1. PON1 suppressed drug- and ligand-induced cell death and protected LC cells from genotoxic damages with maintained ATP levels, requiring p53-directed signals. PON1 promoted ROS deregulation protecting the mitochondria from dysregulation. PON1 knockdown resulted in the blockage of its antioxidant function in LC cells through Akt signaling with reduced invasive signature as a consequence of scant expression. Targeted glycolysis stimulated PON1 antioxidant activity regulating phosphorylation of AMPK-α. The functional data imply that exploitation of the antioxidative function of PON1 is consequential in driving LC pathogenesis at the cell-autonomous mechanistic level with consequences on tumor growth.

Abstract 3673: BVES dependent regulation of YAP1 in colorectal cancer cells

Abstract Colorectal cancer (CRC) has a US incidence rate of almost 150,000 cases and accounts for over 10% of new cancer diagnoses. Dysfunction of epithelial adherens junctions and tight junctions (TJs) have long been implicated in CRC growth and metastasis The Popeye domain containing family of proteins are TJ-associated and appear to nucleate TJ formation. BVES or POPDC1 is one member of this family and is under-expressed in all stages of human colorectal carcinoma (CRC). On the other hand, YAP1 a novel oncogene in the Hippo pathway is up-regulated in many cancers including colon cancer. In the present study, we report an inverse correlation between tumor suppressor protein BVES and oncogene YAP1 in human colorectal carcinoma tumor samples and human colon cancer cell lines. We demonstrate that ectopic expression of BVES in BVES deficient cells decreases YAP1 protein while knocking down BVES in cells expressing BVES increases YAP1 levels. Cycloheximide (CHX) experiments indicate that this is likely occurring at the level of protein stability. CDK6 is a cell cycle regulator and YAP1 transcriptional target, and its levels are increased in the setting of BVES-knockdown in CACO2 cells. Concurrent with this there is increased cell growth. Conversely, when BVES is overexpressed, CDK6 transcription is reduced. Further knocking down of YAP1 in BVES knocked down cells reverses the cell growth advantage suggesting that BVES regulation of cell growth in part is dependent upon YAP1 protein levels. Co-immunoprecipitation (Co-IP) and yeast-2-hybrid (Y2H) assay also shows a direct interaction of BVES and YAP1. In sum, these findings suggest a novel mechanism underlying BVES tumor-suppressive function through regulation of a potential growth modulator, YAP1 in colorectal cancer. Citation Format: Mukul K. Mittal, Anthony Bilotta, Cody E. Keating, Sarah P. Short, Christopher S. Williams. BVES dependent regulation of YAP1 in colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3673.

The C/EBPδ protein is stabilized by estrogen receptor α activity, inhibits SNAI2 expression and associates with good prognosis in breast cancer.

Hypoxia and inflammatory cytokines like interleukin-6 (IL6) are strongly linked to cancer progression, and signal in part through the transcription factor Ccaat/enhancer binding protein δ (C/EBPδ, CEBPD), which has been shown to promote mesenchymal features and malignant progression of glioblastoma. Here we report a different role for C/EBPδ in breast cancer. We found that the C/EBPδ protein is expressed in normal breast epithelial cells and in low-grade cancers. C/EBPδ protein (but not mRNA) expression correlates with estrogen receptor (ER+) and progesterone receptor (PGR) expression and longer progression-free survival of breast cancer patients. Specifically in ER+ breast cancers, CEBPD–but not the related CEBPB–mRNA in combination with IL6 correlated with lower risk of progression. Functional studies in cell lines showed that ERα promotes C/EBPδ expression at the level of protein stability by inhibition of the FBXW7 pathway. Furthermore, we found that C/EBPδ attenuates cell growth, motility and invasiveness by inhibiting expression of the SNAI2 (Slug) transcriptional repressor, which leads to expression of the cyclin dependent kinase inhibitor CDKN1A (p21CIP1/WAF1). These findings identify a molecular mechanism by which ERα signaling reduces the aggressiveness of cancer cells, and demonstrate that C/EBPδ can have different functions in different types of cancer. Furthermore, our results support a potentially beneficial role for the IL-6 pathway specifically in ER+ breast cancer and call for further evaluation of the role of intra-tumoral IL-6 expression and of which cancers might benefit from current attempts to target the IL-6 pathway as a therapeutic strategy.

BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation

The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolutely required for this process. Here, we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum (ER)-associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

Hypoxia-Inducible Factors (HIFs) and Phosphorylation: Impact on Stability, Localization, and Transactivity.

The hypoxia-inducible factor α-subunits (HIFα) are key transcription factors in the mammalian response to oxygen deficiency. The HIFα regulation in response to hypoxia occurs primarily on the level of protein stability due to posttranslational hydroxylation and proteasomal degradation. However, HIF α-subunits also respond to various growth factors, hormones, or cytokines under normoxia indicating involvement of different kinase pathways in their regulation. Because these proteins participate in angiogenesis, glycolysis, programmed cell death, cancer, and ischemia, HIFα regulating kinases are attractive therapeutic targets. Although numerous kinases were reported to regulate HIFα indirectly, direct phosphorylation of HIFα affects HIFα stability, nuclear localization, and transactivity. Herein, we review the role of phosphorylation-dependent HIFα regulation with emphasis on protein stability, subcellular localization, and transactivation.

Functional dissection of proliferating-cell nuclear antigens (1 and 2) in human malarial parasite Plasmodium falciparum: possible involvement in DNA replication and DNA damage response.

Eukaryotic PCNAs (proliferating-cell nuclear antigens) play diverse roles in nucleic acid metabolism in addition to DNA replication. Plasmodium falciparum, which causes human malaria, harbours two PCNA homologues: PfPCNA1 and PfPCNA2. The functional role of two distinct PCNAs in the parasite still eludes us. In the present study, we show that, whereas both PfPCNAs share structural and biochemical properties, only PfPCNA1 functionally complements the ScPCNA mutant and forms distinct replication foci in the parasite, which PfPCNA2 fails to do. Although PfPCNA1 appears to be the primary replicative PCNA, both PfPCNA1 and PfPCNA2 participate in an active DDR (DNA-damage-response) pathway with significant accumulation in the parasite upon DNA damage induction. Interestingly, PfPCNA genes were found to be regulated not at the transcription level, but presumably at the protein stability level upon DNA damage. Such regulation of PCNA has not been shown in eukaryotes before. Moreover, overexpression of PfPCNA1 and PfPCNA2in the parasite confers a survival edge on the parasite in a genotoxic environment. This is the first evidence of a PfPCNA-mediated DDR in the parasite and gives new insights and rationale for the presence of two PCNAs as a parasite survival strategy and its probable success.

Regulation of MDM2 Stability After DNA Damage.

Cells in our body are constantly exposed to various stresses and threats to their genomic integrity. The tumor suppressor protein p53 plays a critical role in successful defense against these threats by inducing apoptotic cell death or cell cycle arrest. In unstressed conditions, p53 levels and activity must be kept low to prevent lethal activation of apoptotic and senescence pathways. However, upon DNA damage or other stressors, p53 is released from its inhibitory state to induce an array of apoptosis and cell cycle genes. Conversely, inactivation of p53 could promote unrestrained tumor proliferation and failure to appropriately undergo apoptotic cell death, which could, in turn, lead to carcinogenesis. The ubiquitin E3 ligase MDM2 is the most critical inhibitor of p53 that determines the cellular response to various p53-activating agents, including DNA damage. MDM2 activity is controlled by post-translational modifications, especially phosphorylation. However, accumulating evidence suggests that MDM2 is also regulated at the level of protein stability, which is controlled by the ubiquitin-proteasome pathway. Here, we discuss how MDM2 can be regulated in response to DNA damage with particular focus on the regulation of MDM2 protein stability.

Regulation and biological function of a flagellar glucose transporter in Leishmania mexicana: a potential glucose sensor.

In Leishmania mexicana parasites, a unique glucose transporter, LmxGT1, is selectively targeted to the flagellar membrane, suggesting a possible sensory role that is often associated with ciliary membrane proteins. Expression of LmxGT1 is down-regulated ∼20-fold by increasing cell density but is up-regulated ∼50-fold by depleting glucose from the medium, and the permease is strongly down-regulated when flagellated insect-stage promastigotes invade mammalian macrophages and transform into intracellular amastigotes. Regulation of LmxGT1 expression by glucose and during the lifecycle operates at the level of protein stability. Significantly, a ∆lmxgt1 null mutant, grown in abundant glucose, undergoes catastrophic loss of viability when parasites deplete glucose from the medium, a property not exhibited by wild-type or add-back lines. These results suggest that LmxGT1 may function as a glucose sensor that allows parasites to enter the stationary phase when they deplete glucose and that in the absence of this sensor, parasites do not maintain viability when they run out of glucose. However, alternate roles for LmxGT1 in monitoring glucose availability are considered. The absence of known sensory receptors with defined ligands and biologic functions in Leishmania and related kinetoplastid parasites underscores the potential significance of these observations.