Cyclin D degradation by E3 ligases in cancer progression and treatment.

D cyclins include three isoforms: D1, D2, and D3. D cyclins heterodimerize with cyclin-dependent kinase 4/6 (CDK4/6) to form kinase complexes that can phosphorylate and inactivate Rb. Inactivation of Rb triggers the activation of E2F transcription factors, which in turn regulate the expression of genes whose products drive cell cycle progression. Because D-type cyclins function as mitogenic sensors that link growth factor signaling directly with G1 phase progression, it is not surprising that D cyclin accumulation is dysregulated in a variety of human tumors. Elevated expression of D cyclins results from gene amplification, increased gene transcription and protein translation, decreased microRNA levels, and inefficiency or loss of ubiquitylation-mediated protein degradation. This review focuses on the clinicopathological importance of D cyclins, how dysregulation of Ubiquitin-Proteasome System (UPS) contributes to the overexpression of D cyclins, and the therapeutic potential through targeting D cyclin-related machinery in human tumors.

Control of CCND1 ubiquitylation by the catalytic SAGA subunit USP22 is essential for cell cycle progression through G1 in cancer cells.

Overexpression of the deubiquitylase ubiquitin-specific peptidase 22 (USP22) is a marker of aggressive cancer phenotypes like metastasis, therapy resistance, and poor survival. Functionally, this overexpression of USP22 actively contributes to tumorigenesis, as USP22 depletion blocks cancer cell cycle progression in vitro, and inhibits tumor progression in animal models of lung, breast, bladder, ovarian, and liver cancer, among others. Current models suggest that USP22 mediates these biological effects via its role in epigenetic regulation as a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional cofactor complex. Challenging the dogma, we report here a nontranscriptional role for USP22 via a direct effect on the core cell cycle machinery: that is, the deubiquitylation of the G1 cyclin D1 (CCND1). Deubiquitylation by USP22 protects CCND1 from proteasome-mediated degradation and occurs separately from the canonical phosphorylation/ubiquitylation mechanism previously shown to regulate CCND1 stability. We demonstrate that control of CCND1 is a key mechanism by which USP22 mediates its known role in cell cycle progression. Finally, USP22 and CCND1 levels correlate in patient lung and colorectal cancer samples and our preclinical studies indicate that targeting USP22 in combination with CDK inhibitors may offer an approach for treating cancer patients whose tumors exhibit elevated CCND1.

RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC.

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells.

Correction: RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC.

[This corrects the article DOI: 10.1371/journal.pgen.1006306.].

PERK Is a Haploinsufficient Tumor Suppressor: Gene Dose Determines Tumor-Suppressive Versus Tumor Promoting Properties of PERK in Melanoma.

The unfolded protein response (UPR) regulates cell fate following exposure of cells to endoplasmic reticulum stresses. PERK, a UPR protein kinase, regulates protein synthesis and while linked with cell survival, exhibits activities associated with both tumor progression and tumor suppression. For example, while cells lacking PERK are sensitive to UPR-dependent cell death, acute activation of PERK triggers both apoptosis and cell cycle arrest, which would be expected to contribute tumor suppressive activity. We have evaluated these activities in the BRAF-dependent melanoma and provide evidence revealing a complex role for PERK in melanoma where a 50% reduction is permissive for BrafV600E-dependent transformation, while complete inhibition is tumor suppressive. Consistently, PERK mutants identified in human melanoma are hypomorphic with dominant inhibitory function. Strikingly, we demonstrate that small molecule PERK inhibitors exhibit single agent efficacy against BrafV600E-dependent tumors highlighting the clinical value of targeting PERK.

ErbB2 activation upregulates glutaminase 1 expression which promotes breast cancer cell proliferation.

Active glutamine utilization is critical for tumor cell proliferation. Glutaminolysis represents the first and rate-limiting step of glutamine utilization and is catalyzed by glutaminase (GLS). Activation of ErbB2 is one of the major causes of breast cancers, the second most common cause of death for women in many countries. However, it remains unclear whether ErbB2 signaling affects glutaminase expression in breast cancer cells. In this study, we show that MCF10A-NeuT cell line has higher GLS1 expression at both mRNA and protein levels than its parental line MCF10A, and knockdown of ErbB2 decreases GLS1 expression in MCF10A-NeuT cells. We further show that in these cells, ErbB2-mediated upregulation of GLS1 is not correlated to c-Myc expression. Moreover, activation of neither PI3K-Akt nor MAPK pathway is sufficient to upregulate GLS1 expression. Interestingly, inhibition of NF-κB blocks ErbB2-stimulated GLS1 expression, whereas stimulation of NF-κB is sufficient to enhance GLS1 levels in MCF10A cells, suggesting a PI3K-Akt-independent activation of NF-κB upregulates GLS1 in ErbB2-positive breast cancer cells. Finally, knockdown or inhibition of GLS1 significantly decreased the proliferation of breast cancer cells with high GLS1 levels. Taken together, our data indicate that ErbB2 activation promotes GLS1 expression via a PI3K-Akt-independent NF-κB pathway in breast cancer cells, identifying another oncogenic signaling pathway which stimulates GLS1 expression, and thus promoting glutamine utilization in cancer cells. These findings, if validated by in vivo model, may facilitate the identification of novel biochemical targets for cancer prevention and therapy.

Stanniocalcin 2 governs cancer cell adaptation to nutrient insufficiency through alleviation of oxidative stress.

Solid tumours often endure nutrient insufficiency during progression. How tumour cells adapt to temporal and spatial nutrient insufficiency remains unclear. We previously identified STC2 as one of the most upregulated genes in cells exposed to nutrient insufficiency by transcriptome screening, indicating the potential of STC2 in cellular adaptation to nutrient insufficiency. However, the molecular mechanisms underlying STC2 induction by nutrient insufficiency and subsequent adaptation remain elusive. Here, we report that STC2 protein is dramatically increased and secreted into the culture media by Gln-/Glc-deprivation. STC2 promoter contains cis-elements that are activated by ATF4 and p65/RelA, two transcription factors activated by a variety of cellular stress. Biologically, STC2 induction and secretion promote cell survival but attenuate cell proliferation during nutrient insufficiency, thus switching the priority of cancer cells from proliferation to survival. Loss of STC2 impairs tumour growth by inducing both apoptosis and necrosis in mouse xenografts. Mechanistically, under nutrient insufficient conditions, cells have increased levels of reactive oxygen species (ROS), and lack of STC2 further elevates ROS levels that lead to increased apoptosis. RNA-Seq analyses reveal STC2 induction suppresses the expression of monoamine oxidase B (MAOB), a mitochondrial membrane enzyme that produces ROS. Moreover, a negative correlation between STC2 and MAOB levels is also identified in human tumour samples. Importantly, the administration of recombinant STC2 to the culture media effectively suppresses MAOB expression as well as apoptosis, suggesting STC2 functions in an autocrine/paracrine manner. Taken together, our findings indicate that nutrient insufficiency induces STC2 expression, which in turn governs the adaptation of cancer cells to nutrient insufficiency through the maintenance of redox homeostasis, highlighting the potential of STC2 as a therapeutic target for cancer treatment.

Stanniocalcin 2 governs cancer cell adaptation to nutrient insufficiency through alleviation of oxidative stress.

Solid tumours often endure nutrient insufficiency during progression. How tumour cells adapt to temporal and spatial nutrient insufficiency remains unclear. We previously identified STC2 as one of the most upregulated genes in cells exposed to nutrient insufficiency by transcriptome screening, indicating the potential of STC2 in cellular adaptation to nutrient insufficiency. However, the molecular mechanisms underlying STC2 induction by nutrient insufficiency and subsequent adaptation remain elusive. Here, we report that STC2 protein is dramatically increased and secreted into the culture media by Gln-/Glc- deprivation. STC2 promoter contains cis-elements that are activated by ATF4 and p65/RelA, two transcription factors activated by a variety of cellular stress. Biologically, STC2 induction and secretion promote cell survival but attenuate cell proliferation during nutrient insufficiency, thus switching the priority of cancer cells from proliferation to survival. Loss of STC2 impairs tumour growth by inducing both apoptosis and necrosis in mouse xenografts. Mechanistically, under nutrient insufficient conditions, cells have increased levels of reactive oxygen species (ROS), and lack of STC2 further elevates ROS levels that lead to increased apoptosis. RNA-Seq analyses reveal STC2 induction suppresses the expression of monoamine oxidase B (MAOB), a mitochondrial membrane enzyme that produces ROS. Moreover, a negative correlation between STC2 and MAOB levels is also identified in human tumour samples. Importantly, the administration of recombinant STC2 to the culture media effectively suppresses MAOB expression as well as apoptosis, suggesting STC2 functions in an autocrine/paracrine manner. Taken together, our findings indicate that nutrient insufficiency induces STC2 expression, which in turn governs the adaptation of cancer cells to nutrient insufficiency through the maintenance of redox homoeostasis, highlighting the potential of STC2 as a therapeutic target for cancer treatment.

Bioinformatic Analysis of The Prognostic Value of A Panel of Six Amino Acid Transporters in Human Cancers.

OBJECTIVE: Solid tumor cells utilize amino acid transporters (AATs) to increase amino acid uptake in response to nutrient-insufficiency. The upregulation of AATs is therefore critical for tumor development and progression. This study identifies the upregulated AATs under amino acid deprived conditions, and further determines the clinicopathological importance of these AATs in evaluating the prognosis of patients with cancers. MATERIALS AND METHODS: In this experimental study, the Gene Expression Omnibus (GEO) datasets (GSE62673, GSE26370, GSE125782 and GSE150874) were downloaded from the NCBI website and utilized for integrated differential expression and pathway analysis v0.96, Gene Set Enrichment Analysis (GSEA), and REACTOME analyses to identify the AATs upregulated in response to amino acid deprivation. In addition, The Cancer Genome Atlas (TCGA) datasets with prognostic information were assessed and employed to evaluate the association of identified AATs with patients' prognoses using SurvExpress analysis. RESULTS: Using analysis of NCBI GEO data, this study shows that amino acid deprivation leads to the upregulation of six AAT genes; SLC3A2, SLC7A5, SLC7A1, SLC1A4, SLC7A11 and SLC1A5. GSEA and REACTOME analyses identified altered signaling in cells exposed to amino acid deprivation, such as pathways related to stress responses, the cell cycle and apoptosis. In addition, Principal Component Analysis showed these six AAT genes to be well divided into two distinct clusters in relation to TCGA tumor tissues versus normal counterparts. Finally, Log-Rank analysis confirmed the upregulation of this panel of six AAT genes is correlated with poor prognosis in patients with colorectal, esophageal, kidney and lung cancers. CONCLUSION: The upregulation of a panel of six AATs is common in several human cancers and may provide a valuable diagnostic tool to evaluate the prognosis of patients with colorectal, esophageal, kidney and lung cancers.

SYVN1-mediated ubiquitylation directs localization of MCT4 in the plasma membrane to promote the progression of lung adenocarcinoma.

Tumour cells mainly generate energy from glycolysis, which is commonly coupled with lactate production even under normoxic conditions. As a critical lactate transporter, monocarboxylate transporter 4 (MCT4) is highly expressed in glycolytic tissues, such as muscles and tumours. Overexpression of MCT4 is associated with poor prognosis for patients with various tumours. However, how MCT4 function is post-translationally regulated remains largely unknown. Taking advantage of human lung adenocarcinoma (LUAD) cells, this study revealed that MCT4 can be polyubiquitylated in a nonproteolytic manner by SYVN1 E3 ubiquitin ligase. The polyubiquitylation facilitates the localization of MCT4 into the plasma membrane, which improves lactate export by MCT4; in accordance, metabolism characterized by reduced glycolysis and lactate production is effectively reprogrammed by SYVN1 knockdown, which can be reversed by MCT4 overexpression. Biologically, SYVN1 knockdown successfully compromises cell proliferation and tumour xenograft growth in mouse models that can be partially rescued by overexpression of MCT4. Clinicopathologically, overexpression of SYVN1 is associated with poor prognosis in patients with LUAD, highlighting the importance of the SYVN1-MCT4 axis, which performs metabolic reprogramming during the progression of LUAD.

Carbon source metabolism and its regulation in cancer cells.

Cancer cell proliferation and progression require sufficient supplies of nutrients including carbon sources, nitrogen sources, and molecular oxygen. Particularly, carbon sources and molecular oxygen are critical for the generation of ATP and building blocks, and for the maintenance of intracellular redox status. However, solid tumors frequently outgrow the blood supply, resulting in nutrient insufficiency. Accordingly, cancer cell metabolism shows aberrant biochemical features that are consequences of oncogenic signaling and adaptation. Those adaptive metabolism features, including the Warburg effect and addiction to glutamine, may form the biochemical basis for resistance to chemotherapy and radiation. A better understanding of the regulatory mechanisms that link the signaling pathways to adaptive metabolic reprogramming may identify novel biomarkers for drug development. In this review, we focus on the regulation of carbon source utilization at a cellular level, emphasizing its relevance to proliferative biosynthesis in cancer cells. We summarize the essential needs of proliferating cells and the metabolic features of glucose, lipids, and glutamine, and we review the roles of transcription regulators (i.e., HIF-1, c-Myc, and p53) and two major oncogenic signaling pathways (i.e., PI3K-Akt and MAPK) in regulating the utilization of carbon sources. Finally, the effects of glucose on cell proliferation and perspective from both biochemical and cellular angles are discussed.

The E3 Ubiquitin Ligase Fbxo4 Functions as a Tumor Suppressor: Its Biological Importance and Therapeutic Perspectives.

Fbxo4, also known as Fbx4, belongs to the F-box protein family with a conserved F-box domain. Fbxo4 can form a complex with S-phase kinase-associated protein 1 and Cullin1 to perform its biological functions. Several proteins are identified as Fbxo4 substrates, including cyclin D1, Trf1/Pin2, p53, Fxr1, Mcl-1, ICAM-1, and PPARγ. Those factors can regulate cell cycle progression, cell proliferation, survival/apoptosis, and migration/invasion, highlighting their oncogenic or oncogene-like activities. Therefore, Fbxo4 is defined as a tumor suppressor. The biological functions of Fbxo4 make it a potential candidate for developing new targeted therapies. This review summarizes the gene and protein structure of Fbxo4, the mechanisms of how its expression and activity are regulated, and its substrates, biological functions, and clinicopathological importance in human cancers.

Stanniocalcin 2 (STC2): a universal tumour biomarker and a potential therapeutical target.

Stanniocalcin 2 (STC2) is a glycoprotein which is expressed in a broad spectrum of tumour cells and tumour tissues derived from human breast, colorectum, stomach, esophagus, prostate, kidney, liver, bone, ovary, lung and so forth. The expression of STC2 is regulated at both transcriptional and post-transcriptional levels; particularly, STC2 is significantly stimulated under various stress conditions like ER stress, hypoxia and nutrient deprivation. Biologically, STC2 facilitates cells dealing with stress conditions and prevents apoptosis. Importantly, STC2 also promotes the development of acquired resistance to chemo- and radio- therapies. In addition, multiple groups have reported that STC2 overexpression promotes cell proliferation, migration and immune response. Therefore, the overexpression of STC2 is positively correlated with tumour growth, invasion, metastasis and patients' prognosis, highlighting its potential as a biomarker and a therapeutic target. This review focuses on discussing the regulation, biological functions and clinical importance of STC2 in human cancers. Future perspectives in this field will also be discussed.

Aurora kinase a promotes the progression of papillary thyroid carcinoma by activating the mTORC2-AKT signalling pathway.

BACKGROUND: Treatment failure is the main cause of death from papillary thyroid carcinoma (PTC). It is urgent to look for new intervention targets and to develop new therapies for treating PTC. Aurora-A kinase (AURKA) functionally regulates cell mitosis and is closely related to the occurrence and development of a variety of tumours. However, the expression and potential functions of AURKA in PTC remain largely elusive. RESULTS: Clinicopathologically, AURKA is highly expressed in PTC tissues compared to normal tissues and is correlated with lymph node metastasis, TNM stage and patient prognosis. Biologically, AURKA functions as an oncoprotein to promote the proliferation and migration of PTC cells. Mechanistically, AURKA directly binds to SIN1 and compromises CUL4B-based E3 ligase-mediated ubiquitination and subsequent degradation of SIN1, leading to hyperactivation of the mTORC2-AKT pathway in PTC cells. CONCLUSIONS: We found that AURKA plays critical roles in regulating the progression of PTC by activating the mTORC2-AKT pathway, highlighting the potential of targeting AURKA to treat PTC.

Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Fbxl8 suppresses lymphoma growth and hematopoietic transformation through degradation of cyclin D3.

Overexpression of D-type cyclins in human cancer frequently occurs as a result of protein stabilization, emphasizing the importance of identification of the machinery that regulates their ubiqutin-dependent degradation. Cyclin D3 is overexpressed in ~50% of Burkitt's lymphoma correlating with a mutation of Thr-283. However, the E3 ligase that regulates phosphorylated cyclin D3 and whether a stabilized, phosphorylation deficient mutant of cyclin D3, has oncogenic activity are undefined. We describe the identification of SCF-Fbxl8 as the E3 ligase for Thr-283 phosphorylated cyclin D3. SCF-Fbxl8 poly-ubiquitylates p-Thr-283 cyclin D3 targeting it to the proteasome. Functional investigation demonstrates that Fbxl8 antagonizes cell cycle progression, hematopoietic cell proliferation, and oncogene-induced transformation through degradation of cyclin D3, which is abolished by expression of cyclin D3T283A, a non-phosphorylatable mutant. Clinically, the expression of cyclin D3 is inversely correlated with the expression of Fbxl8 in lymphomas from human patients implicating Fbxl8 functions as a tumor suppressor.

[Correlation between expressions of matrix metalloproteinase-2 & 9 and vasculogenic mimicry in gastrointestinal stromal tumors].

OBJECTIVE: To study the correlation between the expression of matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor (VEGF) and vasculogenic mimicry (VM) in gastrointestinal stromal tumors (GIST). METHODS: The immunohistochemical staining indices (SI) of MMP-2, MMP-9, VEGF were assessed on specimens of 84 human cases with GIST (21 VM-positive cases). Gelatin zymography analysis of the activity of MMP-2 and MMP-9 activities were performed on another 42 human cases of GIST with fresh tissue (22 VM-positive cases). RESULTS: The staining indices (SI) of MMP-2 and MMP-9 were higher in the VM-positive group (4.10 +/- 2.05 and 3.43 +/- 1.89 respectively) than in the VM-negative group (2.98 +/- 1.97 and 2.38 +/- 1.84 respectively, both P < 0.05); there was no statistic difference in the SI of VEGF between VM-positive and VM-negative group. Gelatin zymography analysis showed that the activity of MMP-2 and MMP-9 were significantly higher in the VM-positive group (3.62 +/- 3.95 and 4.77 +/- 5.29 respectively) than in the VM-negative group (1.26 +/- 1.21 and 2.11 +/- 1.54 respectively, both P < 0.05). CONCLUSION: The expression of MMP-2 and MMP-9 correlates with VM formation in GIST.

Glutamine addiction: an Achilles heel in esophageal cancers with dysregulation of CDK4/6.

Understanding and overcoming resistance to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors will be challenging. Recent work reveals that dysregulation of F-Box Protein 4 (FBXO4)-Cyclin D1 axis leads to mitochondrial dysfunction and drives glutamine-addiction in esophageal squamous cell carcinoma. This metabolism feature makes these tumors susceptible to combined treatment with glutaminase (GLS) inhibitor and metformin even when resisting to CDK4/6 inhibitors.

Overview of Glutamine Dependency and Metabolic Rescue Protocols.

Enhanced glutaminolysis and glycolysis are the two most remarkable biochemical features of cancer cell metabolism, reflecting increased utilization of glutamine and glucose in proliferating cells. Most solid tumors often outgrow the blood supply, resulting in a tumor microenvironment characterized by the depletion of glutamine, glucose, and oxygen. Whereas mechanisms by which cancer cells sense and metabolically adapt to hypoxia have been well characterized with a variety of cancer types, mechanisms by which different types of tumor cells respond to a dynamic change of glutamine availability and the underlying importance remains to be characterized. Here we describe the protocol, which uses cultured Hep3B cells as a model in determining glutamine-dependent proliferation, metabolite rescuing, and cellular responses to glutamine depletion. These protocols may be modified to study the metabolic roles of glutamine in other types of tumor or non-tumor cells as well.

Effect of early enteral nutrition on laparoscopic common bile duct exploration with enhanced recovery after surgery protocols.

BACKGROUND/OBJECTIVES: Protocols for enhanced recovery after surgery (ERAS) provide comprehensive and evidence-based guidelines to improve perioperative care. It remains elusive whether early enteral nutrition (EEN) will play an active role in the ERAS protocols. Laparoscopic common bile duct exploration (LCBDE) is a safe and efficient method to treat patients with bile duct stones. This study aims to assess the safety, tolerability, and outcomes of EEN after LCBDE. SUBJECTS /METHODS: From January 2014 to April 2017, a total of 100 patients with postoperative LCBDE were chosen and randomly divided into control group and EEN group. Patients in the control group were treated with traditional management with regular diet when tolerated, while patients in the EEN group were fed with EEN 3 h after LCBDE. The patients' characteristics, time to first flatus, complications, hospitalization stay, and hospitalization expenses were assessed and compared between patients in these two groups. RESULTS: EEN accelerated the recovery of gastrointestinal function, being indicated by reduced time to first flatus when compared with control group (P = 0.00). In accordance, the quick recovery of gastrointestinal function resulted in shorter hospitalization stay for the EEN group (P = 0.00); however, no significant difference was shown when comparing the hospitalization expenses. On another hand, early oral feeding increased the occurrence of abdominal distension and diarrhea complications (P = 0.00 and P = 0.03). CONCLUSIONS: EEN effectively improves gastrointestinal function, but raises complications such as abdominal distension and diarrhea after LCBDE. It is recommended to implement the EEN as early as possible if the patients are reasonably expected to have high compliance.